25 - 28 MAIO

BALNEÁRIO CAMBORIÚ
SC/BRASIL

LIVRO DE

PROMOÇÃO/REALIZAÇÃO: APOIO:
 SCIENTIFIC COMMITTEE XXII BRAZILIAN CONGRESS OF TOXICOLOGY
Marcelo Dutra Arbo - Universidade Federal do Rio Grande do Sul (UFRGS) - RS - President
Aline Franco Martins - Universidade Estadual de Campinas (UNICAMP) - SP
Artur Christian Garcia da Silva - Universidade Federal de Goiás (UFG) - GO
Anderson Martino Andrade - Universidade Federal do Paraná (UFPR) - PR
Carla Brigagão Pacheco da Silva - Faculdade de Medicina de Ribeirão Preto (FMRP-USP) - SP
Eduardo Geraldo de Campos - Universidade Estadual de Campinas (UNICAMP) - SP
Eliane Dallegrave - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RS
Elisa Sauer - Instituto Geral de Perícias de Santa Catarina (IGP) - SC
Fabrício Pelição - Polícia Civil - ES
Flavia Thiesen - Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) - RS
Fávero Reisdorfer Paula - Universidade Federal do Pampa (UNIPAMPA) - RS
Flavio Manoel Rodrigues da Silva Júnior - Universidade Federal do Rio Grande (FURG) - RS
Gabriela Göethel - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RS
Isarita Martins - Universidade Federal de Alfenas (UNIFAL) - MG
José Luiz da Costa - Universidade Estadual de Campinas (UNICAMP) - SP
Juliano Smanioto Barin - Universidade Federal de Santa Maria (UFSM) - RS
Leonardo Costalonga Rodrigues - Universidade Estadual de Campinas (UNICAMP) - SP
Luciana Grazziotin Rossato-Grando - Universidade de Passo Fundo (UPF) - RS
Marina Venzon Antunes - Universidade Feevale (FEEVALE) - RS
Maurício Homem de Mello - Universidade de Brasília (UnB) - DF
Mirna Bainy Leal - Universidade Federal do Rio Grande do Sul (UFRGS) - RS
Osmar Damian Prestes - Universidade Federal de Santa Maria (UFSM) - RS
Rachel Picada Bulcão - Polícia Científica - PR
Rafael Lanaro - Universidade Estadual de Campinas (UNICAMP) - SP
Raphael Caio Tamborelli Garcia - Universidade Federal de São Paulo (UNIFESP) - SP
Sandra Manoela Dias Macedo - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RS
Sandro Cruz Chaves - Instituto Médico Legal André Roquette (IMLAR) - MG
Sarah Carobini Werner de Souza Eller - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RS
Silvya Stuchi Maria-Engler - Universidade de São Paulo (USP) - SP
Tiago Peixe - Universidade Estadual de Londrina (UEL) - PR
Vanessa Moraes de Andrade - Universidade do Extremo Sul Catarinense (UNESC) - SC

ORGANIZING COMMITTEE
José Roberto Santin - Universidade do Vale do Itajaí (UNIVALI) - SC - President
Alcíbia Helena de Azevedo Maia - Universidade Federal de Santa Catarina (UFSC) - SC
André Valle de Bairros - Universidade Federal de Santa Maria (UFSM) - RS
Ernani Pinto Junior - Universidade de São Paulo (USP) - SP
Isabel Daufenback Machado - Universidade Regional de Blumenau (FURB) - SC
Marcelo Dutra Arbo - Universidade Federal do Rio Grande do Sul (UFRGS) - RS
Marize Campos Valadares - Universidade Federal de Goiás (UFG) - GO
Tiago Franco de Oliveira - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RS

ORGANIZATION
AGeventos Assessoria

PUBLISHING
Isabel Kubaski - isabelkubaski@gmail.com
COVER CREATION
Silvio Lovato - silvio@lovatodesign.com.br
 3

SUMMARY

01  Alternative Animal Models & Emerging in vitro models............................................................................... 24

Adipose Tissue 3D: new tools in cell culture.........................................................................................................................25
Buchele, Maria Luiza Caneiro; Mora, Tamara Dal; Saleh, Najla Adel; Silva, Adny Henrique;
Monteiro, Fabíola Branco Filippin

Analysis of cytotoxicity and anti-melanogenic activity of kojic acid and glycolic acid in SK-
MEL-28 and B16F10 cells..............................................................................................................................................................26
Ribeiro, Milena Mariano; Silva, Ana Cléia Cardoso; Irioda, Ana Carolina; Oliveira, Cláudia Sirlene

Bioprinted and manual human epidermis reconstruction: a compared performance for irritation tests.....27
Bagatin, Julia de Toledo; Camarena, Denisse Esther Mallaupoma; Osaki, Luciana Harumi; Freitas,
Vanessa M.; Nold, Juliana C. Lago; Maria-Engler, Silvya Stuchi

Cellfate®Matrix: building the reality in three dimensions............................................................................................... 28

Reis, Emily Marques; Cavichion, Rafael Filipe Battisti; Colla, Guilherme; Godoi, Manuella
Machado; Koepp, Janice

Cellfate®RHE: a brazilian commercial model of reconstituted human epidermis in vitro....................................29

Reis, Emily Marques; Cavichion, Rafael Filipe Battisti; Colla, Guilherme; Koepp, Janice

Characterization and analysis of the expression of the surface marker Stro‑1 in human
dental pulp stem cells...................................................................................................................................................................30
Oliveira, Leandro Leal Rocha; Lima, Aliny Pereira; Farias, Evelyn Rayani Araújo; Gomes,
Thaisângela Rodrigues Lopes e Silva; Macedo, Larissa Matuda; Leite, Jacqueline Alves;
Valadares, Marize Campos

Characterization and applicability of a novel physiologically relevant 3D-tetraculture

bronchial model for in vitro assessment of chemical respiratory allergy................................................................. 31
Silva, Artur Christian Garcia; Mendonça, Izadora Caroline Furtado; Valadares, Marize Campos

Concomitant exposure to air particulate matter and solar radiation reduces epidermal
barrier function in a reconstructed human epidermis model.........................................................................................32
Silva, Claudia Larissa Viana; Carvalho, Larissa Anastacio da Costa; Camarena, Denisse Esther
Mallaupoma; Bagatin, Julia de Toledo; Assis, Silvia Romano; Maria-Engler, Silvya Stuchi; Barros,
Silvia Berlanga de Moraes

Creation of a biorepository from dental stem cells: as an alternative method to the use of animals...........33
Oliveira, Leandro Leal Rocha; Lima, Aliny Pereira; Farias, Evelyn Rayani Araújo; Gomes,
Thaisângela Rodrigues Lopes e Silva; Macedo, Larissa Matuda; Leite, Jacqueline Alves;
Valadares, Marize Campos
Decellularized extracellular matrix hydrogel derived from bovine cornea for application in 3D models.... 34
Santos, Jordana Andrade; Silva, Artur Christian Garcia; Dias, Wanessa Amorim; Valadares,
Marize Campos

Development of a methodology to assess key event 2 to quantify NRF2 in cutaneous allergenicity............35

Pedralli, Bruna Cristiane Oliveira; Valadares, Marize Campos

Development of an ex vivo model to evaluate pulmonary toxicity mechanisms...................................................36

Furtuoso, Marcella Miranda Siqueira; Tavares, Kvetta Pinheiro Teixeira; Pedralli, Bruna Cristiane
Oliveira; Valadares, Marize Campos

Dimethoate exposition: in vitro x in vivo study.....................................................................................................................37

Andrade, Ana Rosa Brissant; Carvalho, Deoclécio Lustosa; Souza, Asley Thalia Medeiros;
Kishishita, Juliana; Pimenta, Camila de Almeida Perez; Santana, Davi Pereira; Leal, Leila Bastos

Effects of Myrcia pubipetala Miq (Myrtaceae) extract on innate inflammatory response................................ 38

Pacassa, Pâmela; Benvenutti, Larissa; Echterhoff, Marcelo Rodrigo Franke; Lopes, Bruna
Gonçalves; Quintão, Nara Lins Meira; Debiasi, Michele Alberton; Santin, José Roberto; Machado,
Isabel Daufenback

Effects of pulmonary inhaled irritants on 3D alveolar model........................................................................................39

Furtuoso, Marcella Miranda Siqueira; Tavares, Kvetta Pinheiro Teixeira; Valadares, Marize
Campos

Efficiency of the Reconstructed Human Epidermis (RHE) in the classification of biological

product by the in vitro irritation and corrosion tests (OECD 439 and 431)............................................................... 40
Bechtold, Bruna Assunção; Cianci, Julio Cesar; Coelho, Maria Paula Mancini; Costa, Larissa
Gabrielli; Dakic, Vanja; De Vecchi, Rodrigo; Fava, Luis Paulo; Padua, Amanay Sousa; Santana,
Thatiane Nunes; Silva, Priscilla Muniz Ribeiro;Vecina, Juliana Falcato

Embryotoxicity of triclopyr in early development of zebrafish (Danio rerio)........................................................... 41

Andrade, Ítalo Bertoni Lopes; Sales, Bianca Camargo Penteado; Peixoto, Paloma Vitória Lima;
Viriato, Cristina; Pereira, Lílian Cristina

Evaluation of ocular irritation potential of raw materials presented in xampus

formulations using non-animal methodology.................................................................................................................... 42
Rocha, Daniela Barbosa; Almeida, Jéssica Azarias; Andrade, Wanessa Machado

Evaluation of the cytotoxicity and the anti-melanogenic activity of Selenium and Zinc.................................... 43
Silva, Ana Cléia Cardoso; Ribeiro, Milena Mariano; Irioda, Ana Carolina; Oliveira, Cláudia Sirlene

Galleria mellonella as an Alternative Model for the Assessment of Permeation and

Toxicity of Assets from Natural Sources................................................................................................................................ 44
Silva, Samanta de Matos; Singulani, Junya de Lacorte; Carvalho, Angélica Romão; Migliato,
Ketylin Fernanda; Giannini, Maria José Soares Mendes; Fusco-Almeida, Ana Marisa

Identification and quantification of constituents in the fetal bovine serums available in

the market used in cell culture...................................................................................................................................................45
Stival, Ana Clara Silva; Silva, Artur Christian Garcia; Valadares, Marize Campos

Implementation of SkinEthicTM Human Corneal Epithelium (HCE) in Brazil.............................................................. 46

Dakic, Vanja; Mattos, Guilherme; Rigaudeau, Anne-Sophie; Garcia, Cristina; Bouez, Charbel; De
Vecchi, Rodrigo

In silico evaluation of erythrinic alkaloids from Mulungu, Erythrina verna, in GABAA β-3
receptor complexed with benzamide......................................................................................................................................47
Bairros, André Valle; Nascimento, Marcelo Henrique Santana; Paula, Fávero Reisdorfer

In silico pharmacokinetics and pharmacodynamics prediction of resveratrol and two

glycosylated derivatives.............................................................................................................................................................. 48
Schimith, Lucia Emanueli; André-Miral, Corrine; Muccillo-Baisch, Ana Luiza; Hort, Mariana Appel

In vitro oral and topic absorption toxicity test standardization using 3D cell cultures and
microfluidic systems..................................................................................................................................................................... 49
Ganzerla, Melissa Dibbern; Indolfo, Nathalia de Carvalho; Arroteia, Kelen Fabiola; Figueira, Ana
Carolina Migliorini
In vitro strategy to assess skin irritation and sensitization potential of a commercial
formulation containing the antiseptic chlorhexidine .......................................................................................................50
Kawakami, Camila Martins; Silva, Gustavo Henrique; Moura, Kézia; Pinheiro, Ana L.T.A.;
Pinheiro, Adriano da Silva; Eberlin, Samara; Gaspar, Lorena Rigo; Fuzinaga, Thais; Vicente,
Eduardo;Facchini, Gustavo

Inflammatory alterations triggered by respiratory sensitizers in human dendritic cells:

providing evidence to support the chemical respiratory allergy mechanistic background.................................51
Mendonça, Izadora Caroline Furtado; Silva, Artur Christian Garcia; Carvalho Filho, Sérgio de
Morais; Valadares, Marize Campos

Influence of a combination containing UV filters and insect repellent IR3535 in photoinduced processes...........52
Gluzezak, Ana Júlia Pasuch; Kawakami, Camila Martins; Tavares, Renata Spagolla Napoleão;
Fuzinaga, Thais Yume Toriy; Maria-Engler, Silvya Stuchi; Gaspar, Lorena Rigo

Influence of extracellular matrix organ-specific in behavior and cellular functionality in

HEPG-2 cell line................................................................................................................................................................................53
Santos, Thaís Rosa Marques; Borges, Amanda Cecília Guimarães; Santos, Jordana Andrade;
Silva, Artur Christian Garcia; Marize Campos Valadares

Macroscopic evaluation of Score® cytotoxicity by the Allium cepa test.....................................................................54

Massucato, Lucas Eduardo; Siqueira, Gabriella Ferreira; Motomura, Larissa Tiemi Akamine; Lini,
Renata Sano; Souza-Kaneshima, Alice Maria; Mossini, Simone Aparecida Galerani

Microfibrillated cellulose and silica nanoparticles as sustainable alternatives to

developing nanotechnology products: the evaluation of skin irritation....................................................................55
Cruz, Juliana Varella; Gagosian, Viviana Costa; Magalhães, Washington; Cademartori, Pedro
Henrique Gonzalez; Oliveira, Danielle Palma; Leme, Daniela Moraes

Nasal and buccal absoption of dimethoate: in vitro x in vivo study.............................................................................56

Andrade, Ana Rosa Brissant; Carvalho, Deoclécio Lustosa; Souza, Asley Thalia Medeiros;
Kishishita, Juliana; Silva, José Wellithom Viturino; Bedor, Danilo César Galindo; Santana, Davi
Pereira; Leal, Leila Bastos

Photoprotective effect of Rapanea ferruginea bark extract-loaded nanoemulgel .............................................. 57

Cordenuzzi, Dorys Angela; Benvenutti, Larissa; Santin, José Roberto; Lucinda-Silva, Ruth Meri

Preliminary data on a newly developed biomimetic Reconstituted Human Ocular

Epithelium Model using an animal free defined medium ................................................................................................58
Bosquetti, Bruna; Catarino, Carolina Motter; Costa, Meg Cristina da Castilho; Thá, Emanoela
Lundgren; Silva, Artur Christian Garcia; Schuck, Desiree Cigaran; Canavez, Andrezza Di Pietro
Micali; Brohem, Carla Abdo; Valadares, Marize Campos

Safety evaluation of nanoemulsion and emulgel containing pomegranate peel extract

using alternative in vitro methods............................................................................................................................................59
Rudolf, Carline; Benvenutti, Larissa; Rocha, Anna Carolina Furaer; Cordenuzzi, Dorys Angela;
Santin, José Roberto; Lucinda-Silva, Ruth Meri

Spectroscopic assessment of lung mucus as a tool for toxicokinetic/toxicodynamic

assessment of chemical respiratory sensitizers............................................................................................................... 60
Mendonça, Izadora Caroline Furtado; Silva, Artur Christian Garcia; Mendanha, Sebastião
Antônio; Valadares, Marize Campos

Study of the toxicity of NPS amphetamines and cathinones using in silico tools.................................................. 61
Rodrigues, Caio Henrique Pinke; Castro, Jade Simões; Bruni, Aline Thais

Testing strategies for evaluation of eye irritation potential of agrochemical formulations

as an alternative to animal testing...........................................................................................................................................62
Choksi, Neepa; Latorre, Andreia Oliveira; Pais, Mariana Castello Novo; Murata, Rosana Zoriki
Hosomi; Catalano, Shadia M.I.; Aguilera, Mariana; Pires, Janaina Aparecida Cardoso; Ogasawara,
Maryanne; Habe, Priscila; Perjessy, Gisele; Allen, David

The endothelial compartment can actively participate in the adverse outcome pathway
(AOP) of chemical respiratory allergy .....................................................................................................................................63
Silva, Artur Christian Garcia; Carvalho Filho, Sérgio de Morais; Valadares, Marize Campos
 The Implementation of Alternative Methods to the Use of Animals for ocular toxicity:
difficulties and opportunities for innovation....................................................................................................................... 64
Gimenes, Izabela; Presgrave, Octavio; Gonzalez, Marcelo; Alves, Gutemberg

The use of NAMs to evaluate the toxicity of UV filters: emphasis on aquatic toxicity and
endocrine disrupting effects ......................................................................................................................................................65
Nonino, Elisa de Castro Wille; Leme, Daniela Morais; Pestana, Cynthia Bomfim

Toxicity features of agrochemical formulations related to eye and skin irritation potential
that are important to build weight of evidence for integrated approach of testing strategies .......................66
Latorre, Andreia Oliveira; Pepato, Ana Claudia de Andrade; Faria, Patricia Miranda; Cazarin,
Karen

Toxicological evaluation of amphetamine-likes employing in silico methodology...............................................67

Rodrigues, Caio Henrique Pinke; Bruni, Aline Thais

Toxicological evaluation of effects of bothropic venoms through the chorioallantoic

membrane assay HET-CAM ....................................................................................................................................................... 68
Braga, Jacqueline Ramos Machado; Lima, Natália Cavalcante Barbosa; Alves, Carolina
Esmeraldo Lima; Rocha, Danilo Galvão; Jorge, Roberta Jeane Bezerra; Biondi, Ilka Borges

Use of a biomolecular decellularized bovine cornea derived solution as a method of

evaluating opacity and ocular irritation..................................................................................................................................69
Santos, Jordana Andrade; Dias, Wanessa Amorim; Valadares, Marize Campos

Use of CellFate®iN for a three-dimensional in vitro model of human lung epithelium.........................................70

Santos, Jeniffer Farias; Reis, Emily Marques; Berti, Fernanda Vieira; Koepp, Janice; Nunes,
Viviane Abreu

02  Analytical Toxicology.............................................................................................................................................. 71

Analysis of cannabinoids profile in cannabis-based products by HPLC-DAD..........................................................72
Cardoso, Marilia Santoro; Oliveira, Claudete C.; Costa, José Luiz

Analysis of carbofuran and chlorpyrphos-methyl in GC-MS under different conditions of

the injection port and column oven temperatures.............................................................................................................73
Rosa, Victória Gomes; Bairros, André Valle; Ugalde, Gustavo Andrade; Chimendes, Nayomi de
Andrade; Santos, Lara Celestina; Pacheco, André Lucas Bezerra; Reginato, Fernanda Ziegler;
Berlato, Dener Gomes; Nascimento, Marcelo Henrique Santana

Analysis of cholinesterase inhibitor pesticides by GC-MS in larvae of Lucilia cuprina

(Calliphoridae) for entomotoxicological studies..................................................................................................................74
Ugalde, Gustavo Andrade; Chimendes, Nayomi de Andrade; Pacheco, André Lucas Bezerra;
Santos, Lara Celestina; Reginato, Fernanda Ziegler; Berlato, Dener Gomes; Cardoso, Leonardo
Corrêa; Nascimento, Marcelo Henrique Santana; Rosa, Victória Gomes; Santos, Rachel; Pereira,
Alessandra de Oliveira; Monteiro, Silvia Gonzalez; Bairros, André Valle

Case report of valproic acid intoxication: the relevance of chromatographic methods for
elucidating cases in hospitals..................................................................................................................................................... 75
Sampaio, Amanda Mello Kasper Vaz; Reginato, Fernanda Ziegler; Bairros, André Valle; Roehrs,
Miguel; Lovatel, Ivy Bauer; Ugalde, Gustavo Andrade

Detection of cobalt in human urine by liquid chromatography coupled with high

resolution mass spectrometry for anti-doping control purposes................................................................................76
Santos, Vanessa Farelo; Carneiro, Gabriel Reis Alves; Coelho, Matheus Campelo da Costa;
Machado, Sérgio de Paula; Pereira, Henrique Marcelo Gualberto

Determination of 5-fluorouracil in dried blood spots (DBS) by UPLC/MS-MS......................................................... 77

Silva, Laura Cé; Grando, Ana Paula; Hahn, Roberta Zilles; Linden, Rafael; Antunes, Marina Vezon

Determination of cocaine and metabolites applying dried spot oral fluid using well plate
by HPLC-MS/MS ..............................................................................................................................................................................78
Borges, Gabriela Ramos; Santos, Bruno Pereira; Gouveia, Giovanna Cristiano; Dalanhol, Carolina
Silveira; Scherer, Juliana; Eller, Sarah; Oliveira, Tiago Franco
Determination of cortisol and cortisone in urine samples from patients with Parkinson’s
disease by dispersive tip microextraction solid phase and ultra-efficient liquid
chromatography tandem-mass spectrometry....................................................................................................................79
Kakuda, Priscila; Souza, Israel Donizeti; Tumas, Vitor; Queiroz, Maria Eugênia Costa

Determination of Ethyl Glucuronide and Ethyl Sulfate in urine samples from brazilian truck drivers.......... 80
Costa, C.D.D.; Oliveira Neto, J.R.; Oliveira, N.R.L.; Alcântara, K.C.; Cunha, L.C.

Determination of pharmaceuticals and personal care products (PPCPs) in surface water

in Southern Brazil............................................................................................................................................................................ 81
Bastiani, Marcos Frank; Hahn, Roberta Zilles; Lizot, Lilian de Lima Feltraco; Bondan, Amanda
Pacheco; Castilhos, Maria Amélia; Freitas, Mariana; Favreto, Camila; Linden, Rafael

Determination of psychoactive substances in sweat samples using disposable pipette

extraction (DPX) and GC-MS...................................................................................................................................................... 82
Gomes, Nayna Cândida; De Martinis, Bruno Spinosa

Determination of synthetic cannabinoid receptor agonists (SCRAs) in infused papers and

herbal materials seized in Brazilian prisons........................................................................................................................ 83
Martins, Aline Franco; Barbosa, Ingrid Lopes; Milanez, Guilherme Paier; Costa, José Luiz

Development and validation of a method for the determination of meropenem employing

micro samples of dried blood spots obtained from capillary punctures.................................................................. 84
Busato, Maria Amelia de Castilhos; Antunes, Marina Venzon; Linden, Rafael

Development and validation of analytical method for the quantification of chemical

elements in whole blood with volumetric absorptive microsampling by ICP-MS..................................................85
Sousa Júnior, Wellington Tavares; Morais, Déborah Araújo; Oliveira, Silvana Ruella; Barbosa Jr,
Fernando

Development of a HPLC-DAD-RF method for quantification of neurotransmitters in heads

of Drosophila melanogaster ..................................................................................................................................................... 86
Carriço, Murilo Ricardo Sigal; Rodrigues, Marina Diaz; Paz, Maria Elizabeth Gomes; Molina,
Higor Severo; Nogueira, Caroline Lacerda; Denardin, Elton Luis Gasparotto; Roehrs, Rafael

Development of a method based on Green Analytical Toxicology to determine the active

components of ayahuasca in hair.............................................................................................................................................87
Santos, Fabiana Pereira; Fabris, Andre Luis; Bruno, Vitor; Yonamine, Mauricio

Development of a method for analysis of simvastatin and simvastatin hydroxyacid by

HPLC in human plasma using HF-LPME extraction........................................................................................................... 88
Vasconcelos, Mayrla Emilia Dantas; Gaitani, Cristiane Masetto; Moraes, Natália Valadares;
Demets, Mariana Barbieri Alvarez

Development of a miniaturized sample preparation method for determination of

ketamine, its metabolites and analogs in oral fluid samples using Dispersive Liquid-
Liquid Microextraction (DLLME)................................................................................................................................................ 89
Kahl, Júlia M.M.; Berlinck, Débora Zorrón; Chinaglia, Kauê de Oliveira; Rodrigues, Leonardo
Costalonga; Costa, Jose Luiz

Development of a simple and fast method for iodine determination in hair samples by
ICP-MS after alkaline solubilization at room temperature............................................................................................. 90
Morais, Déborah Araújo; Sousa Junior, Wellington Tavares; Oliveira, Silvana Ruella; Barbosa
Junior, Fernando

Development of an analytical method based on Green Analytical Toxicology employing

umbilical cord tissue for the evaluation of maternal fetal exposure to cocaine..................................................... 91
Meirelles, Gabriela de Paula; Pereira e Silva, Jefferson; Yonamine, Mauricio

Development of an extraction method for the pesticide 2,4-dichlorophenoxyacetic acid

and its metabolite 2,4-dichlorophenol from Apis mellifera............................................................................................92
Paz, Maria Elizabeth Gomes; Carriço, Murilo Ricardo Sigal; Rodrigues, Marina Diaz; Ramborger,
Bruna Piaia; Denardin, Elton Luis Gasparotto; Roehrs, Rafael

Development of dispersive solid phase microextraction (d-SPE) and HPLC-DAD for

emergency toxicological analysis of toxic drugs in urine samples..............................................................................93
Luz, Heloisa Peres; Silva, Bruna Espíndola; Santos, Claudia Regina; Marchioni, Camila
Ethanol quantification in ethanol-based hand sanitizers: alert in the COVID-19 pandemic scenario............ 94
El Haddad, Lohanna Pereira; Costa, Bruno Ruiz Brandão; Bigão, Vitor Luiz Caleffo Piva; De
Martinis, Bruno Spinosa

Evaluation of cork as a natural extraction phase for the determination of new

psychoactive substances in blood samples..........................................................................................................................95
Birk, Letícia; Eller, Sarah; Oliveira, Tiago Franco

Evaluation of the spontaneous hydrolysis of cocaine under different pH and temperature conditions.......96
Gomes, Geovana Maria de Lima; Santos, Vanessa Farelo; Carneiro, Gabriel Reis Alves; Trajano,
Christian Farias; Pereira, Henrique Marcelo Gualberto

First proof of concept of a new passive sampler for marine biotoxins......................................................................97

Fitarelli, Bruna; Fabichak, Ana; Deolindo, Carolina Turnes Pasini; Kleemann, Cristian; Hoff,
Rodrigo

In vitro metabolism studies of ADB-4en-PINACA, ADB-FUBIATA and BZO-CHMOXIZID

using human liver microsomes ................................................................................................................................................ 98
Cunha, Kelly F.; Krotulski, Alex J.; Walton, Sara E.; Papsun, Donna M.; Costa, Jose Luiz; Logan,
Barry K.

Liquid chromatography–tandem mass spectrometry method for simultaneous

quantification of neurotransmitters in rat brain tissue....................................................................................................99
Viana, Roberta Rodrigues; Pego, Ana Miguel Fonseca; Dallegrave, Eliane; Oliveira, Tiago Franco;
Eller, Sarah

Metabolism study of coca leaf tea in urine by liquid chromatography coupled with high
resolution mass spectrometry.................................................................................................................................................100
Gomes, Geovana Maria de Lima; Santos, Vanessa Farelo; Carneiro, Gabriel Reis Alves; Trajano,
Christian Farias; Pereira, Henrique Marcelo Gualberto

New tools in analytical toxicology: analysis of Paraquat in urine with a mobile phone.................................... 101
Chinaglia, Kauê de Oliveira; Lanaro, Rafael; Arantes, Ana Carolina Furiozo; Costa, José Luiz

Preliminary results of method validation for dihydropyrimidine dehydrogenase (DPD)

deficiency screening and 5-fluorouracil determination in plasma by UPLC-MS/MS..........................................102
Grando, Ana Paula; Silva, Laura Cé; Hahn, Roberta Zilles; Linden, Rafael;Antunes, Marina Vezon

Safety studies of a potential cathepsin K inhibitor: 4-methoxy chalcone and its degradation products........ 103
Benvenutti, Danyela Francine; Buzzi, Fatima de Campos; Corrêa, Rogerio; Couto, Angélica
Garcia; Wagner, Theodoro; Paula, Favero R.; Giovagnoli, Stefano; Vivani, Riccardo; Ricci,
Maurizio; Santos, Carlos Eduardo Matos; Santin, José Roberto; Bresolin, Tania Mari Bellé

Use of design of experiment tools to enhance the recovery of synthetic cannabinoids in

dried blood spots (DBS)..............................................................................................................................................................104
Berlinck, Débora Zorrón; Cunha, Kelly Francisco; Costa, José Luiz

Validation and pre-clinical evaluation of pharmacokinetic profile of antineoplasic

prototype LQMF030 in rats by LC-MS/MS...........................................................................................................................105
Pereira, I.B.; Zoghaib, I.V.J.; Gomes, S.A.; Menegatti, R.; Cunha, L.C.

Validation of a DPX-GC-MS method for simultaneous quantification of psychoactive

substances and its metabolites in human breast milk...................................................................................................106
Santos Junior, Wilson José Ramos; Gomes, Nayna Cândida; Costa, Bruno Ruiz Brandão; Bigão,
Vitor Luiz Caleffo Piva; De Martinis, Bruno Spinosa

Validation of a method for plasma vancomycin therapeutic monitoring by High

Performance Liquid Chromatography with UV detector................................................................................................107
Paula, Eliza Bianchini; Santos, Claudia Regina; Marchioni, Camila

Validation of an analytical method for the determination of psychoactive substances in

an oral fluid sample using the DPX-SCX disposable tips and the GC-MS................................................................108
Gomes, Nayna Cândida; De Martinis, Bruno Spinosa
 Validation of disposable pipette extraction method with quantification by GC-MS to
determine ethylenethiourea in human urine samples....................................................................................................109
Romoli, Jéssica Cristina Zoratto; Scanferla, Deborah Thais Palma; Aguera, Raul Gomes; Lini,
Renata Sano; Castro, Juliana Cristina; Bando, Érika; Alves, Gessé de Souza; Nerilo, Samuel
Botião; Mossini, Simone Aparecida Galerani; Marchioni, Camila;Machinski Junior, Miguel

03  Clinical and Laboratorial Toxicology................................................................................................................ 110

A simple procedure for the determination of mercury in urine by Inductively Coupled
Plasma Mass Spectrometry (ICP-MS).................................................................................................................................... 111
Sugawara, Eduardo Kinio; Silva, Graciele Machado; Paulucci, Leticia Trevisan; Ramadan, Debora
R.; Tufik, Sergio; Soares, Marcela de Oliveira

Analysis of plasmatic levels of antipsychotics through an absorptive paper-based

extraction followed by fast gas chromatography-mass spectrometry....................................................................112
Gouveia, Giovanna Cristiano; Borges, Gabriela Ramos; Santos, Bruno Pereira; Sebben, Viviane
Cristina; Arbo, Marcelo Dutra; Eller, Sarah; Oliveira, Tiago Franco

Assessment of mercury exposure and health of riverside populations affected by the

Brumadinho disaster.....................................................................................................................................................................113
Moreira, Camila Francisco; Nolasco, Daniela; Souza, Tainá Brumate; Mendes, Michele Polyana
Rocha; André, Leiliane Coelho; Paiva, Maria José Nunes

Case Report: follow-up care of a cocaine intoxication case at a Toxicological Information

and Assistance Center in Fortaleza - Ceará........................................................................................................................ 114
Salles, Gabriela Pereira; Ferreira e Silva, Hendyelle Rodrigues; Silva, Victória da Costa; Batista,
José Márcio Machado; Albuquerque, Polianna Lemos Moura Moreira; Moreira, Rhubens Levy
Rodrigues; Ferreira, Maria Augusta Drago

Case Report: follow-up care of a kerosene intoxication case at a Toxicological Information

and Assistance Center in Fortaleza - Ceará.........................................................................................................................115
Salles, Gabriela Pereira; Ferreira e Silva, Hendyelle Rodrigues; Silva, Victória da Costa;
Farias, Beatriz Valentim; Magalhães, Karla do Nascimento; Batista, José Márcio Machado;
Albuquerque, Polianna Lemos Moura Moreira; Ferreira, Maria Augusta Drago

Case Report: Phencyclidine cross-reaction investigation in immunochromatographic

tests for rapid drugs detection..................................................................................................................................................116
Cardoso, Leonardo Corrêa; Rosa, VictóriaGomes; Pacheco, André Lucas Bezerra; Santos, Rachel;
Ugalde, Gustavo Andrade; Berlato, Dener Gomes; Reginato, Fernanda Ziegler; Chimendes,
Nayomi Andrade; Santos, Lara Celestina; Nascimento, Marcelo Henrique Santana; Oliveira,
Sarah Carobini Werner de Souza Eller Franco; Oliveira, Tiago Franco; Bairros, André Valle

Clinical-epidemiological profile of elapid accidents in pediatric population registered at CIATox/SC..........117

Silva, Stephanie Soares; Messias, Nayara Casagrande; Bresolin, Nilzete Liberato; Silva, Denise
Bousfield; Santos, Claudia Regina

Determination of pesticides in blood samples from patients after suicide attempt by

micro-QuEChERS and LC-MS/MS............................................................................................................................................ 118
Godoi, Alexandre Barcia; Silva, Mariana Cristina; Costa, José Luiz

Determination of tricyclic antidepressants in whole blood by liquid chromatography with

diode diarray detector employing dispersive liquid-liquid microextraction...........................................................119
Berlato, Dener Gomes; Rosa, VictóriaGomes; Pacheco, André Lucas Bezerra; Santos, Rachel;
Ugalde, Gustavo Andrade; Cardoso, Leonardo Corrêa; Reginato, Fernanda Ziegler; Chimendes,
Nayomi Andrade; Santos, Lara Celestina; Nascimento, Marcelo Henrique Santana; Bairros,
André Valle

Development and validation of a method to measure Tenofovir concentrations in urine of

HIV-positive patients....................................................................................................................................................................120
Santos, Rafaela Knak; Linden, Rafael
Early adverse reactions to different types of anti-poison serum in the period from 2017 to
2021: a descriptive analysis........................................................................................................................................................121
Priedols, Gustavo Abud; Alves, Jonas Alher Meira; Oliveira, Jordana Meirelles; Girotto, Edmarlon;
Guidoni, Camilo Molino

Epidemiological variables and anti-poison serotherapy: a descriptive analysis of early

adverse reactions..........................................................................................................................................................................122
Alves, Jonas Alher Meira; Priedols, Gustavo Abud; Oliveira, Jordana Meirelles; Girotto, Edmarlon;
Guidoni, Camilo Molino

Estimation of hematocrit values by potassium quantification in capillary dried blood spots (DBS) ...........123
Silva, Laura Cé; Alves, Pedro Adolfo Pereira; Linden, Rafael; Antunes, Marina Vezon

Evaluation of cytotoxic potential of polyhydroxylated phenylchromones in human

osteosarcoma in vitro models..................................................................................................................................................124
Oliveira, José Miguel P. Ferreira; Proença, Carina; Santos, Raquel; Moreira, Beatriz; Rufino, Ana
T.; Freitas, Marisa; Ribeiro, Daniela; Fernandes, Eduarda

Evaluation of experimental conditions for sample preparation to identify the untargeted

metabolomic profile of plasma by GC-MS........................................................................................................................... 125
Nolasco, Daniela M.; Pires, Sumaia Araújo; Souza, Tainá Brumate; Souza, Mirna Maciel D'Auriol;
Paiva, Maria José Nunes; André, Leiliane Coelho

Exclusive therapy of N-acetylcysteine in accidental butanox intake: toxicological analysis

of methyl ethyl ketone peroxide..............................................................................................................................................126
Santos, Rachel; Bairros, André V.; Saldanha, Geovane A.; Berlato, Dener G.; Moraes, Liliana S.;
Gündel, Augusto R.; Carvalho, José A.M.; Habib, Isabela A.; De Carli, Diego M.; Oliveira, Tiago F.;
Oliveira, Sarah C.W.S.E.F.

Fast method of quantitative analysis of serum vitamin A (retinol) using LC-MS/MS......................................... 127
Soares, Marcela de Oliveira; Sugawara, Eduardo Kinio; Mazete, Fernanda Pine S.; Ramadan,
Debora R.; Tufik, Sergio

Finger-prick volumetric absorptive microsampling (VAMS) for imatinib therapeutic drug

monitoring: method development and validation ...........................................................................................................128
Guterres, Fernanda S.; Krutzmann, Maria Eduarda; Kohlrausch, Ramona; Linden, Rafael;
Antunes, Marina Vezon

Garlic burn injuries: a clinical-epidemiological profile of cases registered at CIATox/SC, Brazil....................129

Cordeiro, Gabriela Batista Cavalcanti; Arruda, Fernanda Wolff da Silva; Petry, Andrea; Resener,
Marisete Canello; Santos, Claudia Regina

Hepatotoxicity risk assessment in suicide attempts by acetaminophen with doses

between 7.5g and 10g at a reference center in Santa Catarina, Brazil......................................................................130
Fonseca, Karoline Kuhnen; Costa, Ana Carolina Conchon; Cordeiro, Gabriela Batista Cavalcanti;
Arruda, Fernanda Wolff da Silva; Resener, Marisete Canello

Liver and kidney function at the pesticide’s exposure of rural workers from Casimiro de Abreu - RJ...........131
Nunes, Rafaella Ferreira Nascimento; Poça, Kátia Soares; Cabral, Yngrid dos Santos; Aguiar,
Gilberto Santos; Siqueira, Janas; Geraldino, Barbara Rodrigues; Otero, Ubirani Barros; Martins,
Isarita; Sarpa, Márcia Sarpa de Campos

microRNAs as serum biomarker for Senecio brasiliensis poisoning in cattle .......................................................132

Winter, Evelyn; Cisilotto, Julia; Goetten, André L.F.; Veiga, Ângela; Ramos, Adriano T.; Zimermann,
Francielli C.; Reck, Carolina; Creczynski-Pasa,Tânia B.

Profile of drug-associated toxicological events in women of reproductive age...................................................133

Costa, Quezia dos Santos; Priedous, Gustavo Abud; Brunello, Giovanna Cristina Spagnuolo;
Alfieri, Daniela Frizon; Guidoni, Camilo Molino; Alves, Jonas Alher Meira; Girotto, Edmarlon

Profile of suicide attempts by self-poisoning in the Rio Grande Sul, Brazil, between 2010-
2020 and the influence of the COVID-19 pandemic..........................................................................................................134
Santos, Bruno Pereira; Eller, Sarah; Gouveia, Giovanna Cristiano; Borges, Gabriela Ramos;
Sebben, Viviane Cristina; Arbo, Marcelo Dutra; Oliveira, Tiago Franco
 Services provided by the Nucleus Applied to Toxicology (NAT) during the period 2019-2021......................... 135
Reginato, Fernanda Ziegler; Bairros, André Valle; Santos, Lara Celestina; Rosa, Victória Gomes;
Chimendes, Nayomi de Andrade; Santos, Rachel; Pacheco, André Lucas Bezerra; Cardoso,
Leonardo Corrêa; Ugalde, Gustavo Andrade

Suicide attempts in Santa Catarina, Brazil, before and during Covid-19 pandemic:
epidemiological profile................................................................................................................................................................136
Costa, Ana Carolina Conchon; Taruhn, Lillian Freitas; Resener, Marisete Canello

Suicide attempts in adolescents assisted by a toxicological information and assistance

center due to toxicological events.......................................................................................................................................... 137
Brunello, Giovanna Cristina Spagnuolo; Alves, Jonas Alher Meira; Costa, Quezia dos Santos;
Priedous, Gustavo Abud; Alfieri, Daniela Frizon; Guidoni, Camilo Molino; Girotto, Edmarlon

Variation of cholinesterasè s activity and the importance of the involved toxic agent
identification on the acute intoxication evolution by an acetylcholinesterase inhibitor – Case Report......138
Bauermann, Lauren; França, Liana Conrado; Nogueira, Janer Alves; Paula, Eliza Bianchini;
Marchioni, Camila; Santos, Claudia Regina

04  Drugs of Abuse........................................................................................................................................................139

A green dispersive liquid-liquid microextraction for quantitative analysis of synthetic
cathinones in urine and postmortem blood........................................................................................................................140
Fabris, André Luis; Yonamine, Mauricio

A novel workflow for the analysis of drugs of abuse in oral fluid samples using disposable
pipette extraction technologies (DPX-XTR)......................................................................................................................... 141
Cabrices, Oscar G.

Alcohol and alcohol combined with texting: effects on speed and braking behaviours in a
closed-course section.................................................................................................................................................................142
Costa, Bruno Ruiz Brandão; Freitas, Bruno Toledo; Bigão, Vítor Luiz Caleffo Piva; Perdoná, Gleici
da Silva Castro; De Martinis, Bruno Spinosa

Analytical evaluation of four screening devices for drug detection in Brazilian traffic enforcement...........143
Scherer, Juliana N.; Borges, Gabriela Ramos; Santos, Bruno Pereira; Dalanhol, Carolina Silveira;
Gouveia, Giovanna; Viola, Patrícia Pacheco; Carlson, Renato Romera; Govoni, Bruna; Mello,
Raissa; Vasconcelos, Mailton; Pechansky, Flavio

Assessment of possible neurotoxic effects of oxycodone in SH-SY5Y cell lineage............................................ 144

Lima, Luiza Siqueira; Costa, Nayara de Souza; Pereira, Meire Ellen; Almeida, William; Cestari,
Marta Margarete; Irioda, Ana Carolina; Oliveira, Cláudia Sirlene

Blood alcohol levels in fatal victims of traffic accidents................................................................................................145

Everton Boff; Daniel, Caroline; Sá, Clodoaldo Antônio; Brugnera, Débora Schmitz; Silva, Maria
Isabel Gonçalves; Marcon, Scheila; Corralo, Vanessa da Silva

Determination of a comprehensive set of drugs of abuse, metabolites and human

biomarkers in wastewater using passive sampling followed by UHPLC-MS/MS analysis..............................146
Hahn, Roberta Zilles; Bastiani, Marcos Frank; Lizot, Lilian de Lima Feltraco; Linden, Rafael

Determination of levamisole in cocaine samples seized in the south of Santa Catarina .................................147
Bombazar, Amanda; Martins, André Bittencourt; Steiner, Bethina Trevisol; Ávila, Ricardo Andrez
Machado

Developing a drug checking service in Brazil and the potential of harm reduction
strategies to reduce intoxication risks................................................................................................................................. 148
Maluf, Ana Cristhina Sampaio; Costa, José Luiz

Development and validation of a highly sensitive method for detection of Synthetic

Cannabinoid JWH-018, AB-CHMINACA, and their metabolites in hair samples by LC-MS/MS........................149
Paulo, Breno F. Pereira; Zauli, Danielle Alves Gomes Breno
 Epidemiological aspects of suicide in the State of São Paulo.......................................................................................150
Souza, Karla Aparecida de Oliveira; Gianvecchio, Victor Alexandre Percinio; Gianvecchio, Daniele
Muñoz; Jorge, Maria Helena Prado de Mello; Costa, José Luiz

Exogenous poisoning by drug abuse in Brazil: before and during the COVID-19 pandemic...............................151
Baccule, Nicole Santos; Scanferla, Deborah Thais Palma; Lini, Renata Sano; Marchioni, Camila;
Mossini, Simone Aparecida Galerani

Identification of synthetic drugs seized in the region of Balneário Camboriú using ATR-FTIR....................... 152
Costa, Karina Oliveira; Bagio, Jéssica

In silico evaluation of biological, therapeutical, and toxicological properties for five

groups of NPS: amphetamines, cathinones, benzodiazepines, synthetic opioids, and
synthetic cannabinoids............................................................................................................................................................... 153
Santos, Christiano; Bruni, Aline Thais

In silico parameters and chemometrics applied to the study of NBOMes and

amphetamine-type stimulants................................................................................................................................................154
Mariotto, Lívia Salviano; Rodrigues, Caio Henrique Pinke; Bruni, Aline Thais

Intoxication case series by N-ethylpentylone (N-ethylnorpentylone or ephylone)........................................... 155

Denkena, Isadora Locilento; Cunha, Kelly Francisco; Lanaro, Rafael; Cunha, Ricardo Leal; Costa,
Jose Luiz

MDA in silico toxicity assessment suggests low hepatotoxicity and calls for further pre-
clinical investigations...................................................................................................................................................................156
Oliveira, Arthur Lima; Miranda, Raul Ghiraldelli; Dorta, Daniel Junqueira

MDMA in silico toxicity analysis reinforces need of mimicking consumption patterns

towards translational reliability.............................................................................................................................................. 157
Oliveira, Arthur Lima; Miranda, Raul Ghiraldelli; Dorta, Daniel Junqueira

Profile of new psychoactive substances (NPS) in seized materials analyzed in the Rio de
Janeiro State during the COVID-19 pandemic .....................................................................................................................158
Oliveira, Adriana Sousa; Almeida, Fernando G.; Bhering, Cecília de A.; Antonio, Ananda da S.;
Wurzler, Gleicielle T.; Costa, Gabriela Vanini

Risk factors associated with deaths by multiple drugs of abuse intoxications....................................................159

Messias, Nayara Casagrande; Silva, Stephanie Soares; Resener, Marisete Canello; Albino,
Danielle Bibas Legat; Barotto, Adriana Mello; Santos, Claudia Regina; Marchioni, Camila

Study of NBOMes using MetadrugTM and Chemometrics...............................................................................................160

Mariotto, Lívia Salviano; Bruni, Aline Thais

Study of synthetic cannabinoids regarding their metabolites: an in silico approach...........................................161

Castro, Jade Simões; Rodrigues, Caio Henrique Pinke; Bruni, Aline Thais

Tracking methamphetamine surges in the Joinville region (Santa Catarina) from 2014 to 2021....................162
José, Gustavo Pinheiro Coelho; Pericolo, Suellen; Parabocz, Gisele Chibinski; Costa, Karina
Oliveira; Marchioni, Camila; Ramos, Silvia Aparecida

Use of psychedelics in the treatment of anxiety and depression disorder - literature review.......................163
Silva, Luíza Madureira; Holanda, Wiron Pimentel; Honório Júnior, José Eduardo Ribeiro

05  Environmental Safety and Health.....................................................................................................................164

Acute toxicity of Diuron, DCA and DCPMU in the early life and adultohood of the zebrafish
(Danio rerio) ....................................................................................................................................................................................165
Sales, Bianca Camargo Penteado; Andrade, Ítalo Bertoni Lopes; Peixoto, Paloma Vitória Lima;
Camargo, João Lauro Viana; Pereira, Lilian Cristina

Blood total mercury levels of preschool children from Sao Paulo, Brazil, and associated risk factors ......166
Olympio, Kelly Polido Kaneshiro; Salles, Fernanda Junqueira; Pereira, Elizeu Chiodi; Oliveira,
Allan Santos; Costa, Eric Augusto Caravaggio; Costa, Brenda Natasha Souza; Pereira, João Paulo
 Goes; Jesus, Iracina Maura; Queiroz, Thais Karolina Lisboa; Lima, Marcelo de Oliveira; Silva,
Agnes Soares; Cardoso, Maria Regina Alves

Determination of biomarkers of exposure to pythroid pesticides through wastewater-

based epidemiology...................................................................................................................................................................... 167
Lizot, Lilian de Lima Feltraco; Bastiani, Marcos Frank; Hahn, Roberta Zilles; Linden, Rafael

Embryonic exposure to genistein induces persistent anxiolytic and antisocial behaviors in zebrafish..........168
Freddo, Natália; Menegasso, Aloma Santin; Soares, Suelen Mendonça; Fortuna, Milena; Maffi,
Victoria Costa; Mozzato, Mateus Timbola; Barcellos, Leonardo José Gil; Rossato-Grando,
Luciana Grazziotin

Energetic metabolism and antioxidant system modulation after fish chronic exposure to
glyphosate and imidacloprid ....................................................................................................................................................169
Sobjak, Thaís Maylin; Zazula, Matheus Felipe; Macarini, Leanna Camila; Rizzo, Elizete;
Guimarães, Ana Tereza Bittencourt⁴

Ethnic-racial disparity in exogenous intoxications in Brazil.........................................................................................170

Moraes, Niely Galeão da Rosa; Silva Júnior, Flavio Manoel Rodrigues

Evaluating the levels of toxic elements and toxicity testing in water samples of
Paraopeba River after the Brumadinho dam rupture.......................................................................................................171
Devóz, Paula Pícoli; Rocha, Cecília Cristina de Souza; Barbosa Jr, Fernando

Evaluation of the effect of a nutrient solution on the phytoremediation potential of a blue

dye by the aquatic macrophyte Salvinia auriculata......................................................................................................... 172
Schmitt, Maria Laura Videiro; Carriço, Murilo Ricardo Sigal; Nogueira, Caroline Lacerda;
Rodrigues, Marina Diaz; Molina, Higor Severo; Denardin, Elton Luis Gasparotto; Roehrs, Rafael

New approaches to periphytic diatoms as bioindicators of contamination by iron ore

tailings from the collapse of the Fundão dam in the Doce River (Brazil)................................................................. 173
Souza, Luiz Claudio Cindra; Reis, Luciane Ayres Castro; Rangel, Lara Luiza Pimenta; Barroso,
Gilberto Fonseca

Periphytic community in experimental rivers as a tool for the assessment of

anthropogenic stressors in fluvial ecosystems.................................................................................................................174
Reis, Luciane Ayres Castro; Almeida, Stefano Zorzal; Barroso, Gilberto Fonseca

Urinary lead levels and factors associated with their increase in schoolchildren living in a
coal mining area in the extreme south of Brazil................................................................................................................ 175
Brum, Rodrigo de Lima; Baisch, Ana Luíza Muccillo; Silva Júnior, Flavio Manoel Rodrigues

Water quality assessment of the Rio do Peixe: a preliminary study.......................................................................... 176

Gemelli, Bianca Aparecida Martins; Summy, Maria Julia; Cesaro, Humberto Luis; Alves, Rômulo
Couto; Saraiva, Illyushin Zaak; Remor, Aline Pertille; Baú, Morgana; Lima, Daina; Almeida,
Eduardo Alves; Müller, Gabrielle do Amaral e Silva

06  Experimental Toxicology..................................................................................................................................... 177

Acute toxicity of 2,4-dichlorophenoxyacetic acid in the early life of the zebrafish (Danio rerio)...................178
Viriato, Cristina; Andrade, Ítalo B.L.; Peixoto, Paloma V.L.; Pereira, Lílian C.

Alterations in the activity of antioxidant enzymes and NA+K+-ATPase in lead poisoning in

the cerebral cortex and hippocampus of rats.................................................................................................................... 179
Ferrazza-Heitich, Magda; Lima, Daniela Delwing; Borgmann, Gabriela; Plautz, Katherine

Carvacrol and its anti-inflammatory effect under cigarette smoke-induced acute lung injury......................180
Moura, Maria Joana Nogueira; Silva, Aline Gabrielle Gomes, Santos, Caio Cesar Araújo; Pereira,
Artemia Kelly Holanda; Felix, Renata Gleysiane de Sousa; Lima, Crystiane Calado, Golçalves,
A.P.; Melo, Paolo Oliveira; Silva, Gerlane Modesto; Feitosa, Emanuel Kenedy; Borges, Cibele dos
Santos

Chronic exposure to methylphenidate during juvenile period alters zebrafish behavior in adulthood....... 181
Nardi, Jessica; Freddo, Natália; Biazus, Inara Carbonera; Oliveira, Ana Paula; Soares, Suelen
Mendonça; Fortuna, Milena; Pompermeier, Aline; Siqueira, Lisiane; Cole, Amanda Carolina;
 Tamagno, Wagner; Amaral, Francieli Ubirajara India; Costa, Vitoria Cadore; Mozzato, Mateus
Timbola; Barcellos, Leonardo José Gil; Grando, Luciana Grazziotin Rossato

Chronic plus binge ethanol feeding synergistically induces abdominal aorta dysfunction
and gut dysbiosis in mice...........................................................................................................................................................182
Silva, Carla Brigagão Pacheco; Rodrigues, Vanessa Fernandes; Costa, Bruno Ruiz Brandão;
Sartori, Daniela Carlos; De Martinis, Bruno Spinosa; Tostes, Rita C.

Commercial 2,4-D and its isolated compounds, 2,4-dichlorophenoxyacetic acid and 2,4-D
dimethylamine, induces changes in mitochondrial function parameters...............................................................183
Polido, Lucas Roberto Ferreira; Rizzi, Joyce Santana; Pereira, Lílian Cristina

Could Eugenol promote the reduction of the inflammatory process in acute injuries to the
lung of rats caused by cigarette smoke?............................................................................................................................. 184
Silva, Aline Gabrielle Gomes; Moura, Maria Joana Nogueira; Santos, Caio Cesar Araújo; Pereira,
Artemia Kelly Holanda; Felix, Renata Gleysiane de Sousa; Araujo, Bruno Vinicios Silva; Barbosa,
Maria Clara de Oliveira; Melo, Paolo Oliveira; Feitosa, Emanuel Kenedy; Borges, Cibele dos
Santos

Cytotoxicity assessment of potential antitumor 4-methyl coumarins....................................................................185

Göethel, Gabriela; Souza, João Pedro Silveira; Neves, Gustavo Machado; Kagami, Luciano Porto;
Peruzzi, Caroline Portela; Cattani, Shanda; Garcia, Solange Cristina; Battastini, Ana Maria
Oliveira; Figueiró, Fabrício; Eifler-Lima, Vera Lucia

Cytotoxicity evaluation in human hepatic cells HepG2 induced by emerging contaminants

Decametilcyclopentasiloxane and Triclopyr.......................................................................................................................186
Kohori, Natália Akemi; Teodoro, João Soeiro; Palmeira, Carlos Manuel Marques; Pereira, Lilian
Cristina

Different diets and physical exercises: assessment of the impact on the female
reproductive system after 90 days.........................................................................................................................................187
Moura, Maria Joana Nogueira; Silva, Aline Gabrielle Gomes; Santos, Caio Cesar Araújo; Gomes,
Francisca Tayná da Silva; Pereira, Artemia Kelly Holanda; Felix, Renata Gleysiane de Sousa;
Fonseca, Ivana Alice Teixeira; Borges, Cibele dos Santos

Discreet alterations on sperm quality promoted by Aripiprazole ............................................................................. 188

Moura, Maria Joana Nogueira; Felix, Renata Gleysiane de Sousa; Pereira, Artemia Kelly Holanda;
Angelo, Ana Beatriz Silva; Santos, Caio Cesar Araújo; Silva, Aline Gabrielle Gomes;Silva, Ana
Lucelha dos Santos; Freire, Livia Horrana Forte; Dantas, Joao Artur Diogenes; Silva, Mateus
Limerio Carlos; Borges, Cibele dos Santos

Diuron herbicide-induced toxicity on Caenorhabditis elegans ...........................................................................189

Lima, Thania Rios Rossi;Martins Jr., Airton Cunha; Pereira, Lílian Cristina; Aschner, Michael

Effect of inorganic selenium on triple-negative breast cancer cell lines.................................................................190

Costa, Nayara de Souza; Lima, Luiza Siqueira; Oliveira, Franciele Aparecida Mendes; Galiciolli,
Maria Eduarda de Andrade; Oliveira, Cláudia Sirlene; Irioda, Ana Carolina

Evaluation of irritability of Myrcia pubipetala miq. by HET-CAM methodology.....................................................191

Perini, Camila Maria; Kohler, Cristofer José Weege; Wagner, Eduardo José; Machado, Isabel
Daufenback

Evaluation of metabolic activity of HepG2 cells co-exposed to micro or nanoplastics and

bisphenol A or S..............................................................................................................................................................................192
Rocha, Cecília Cristina de Souza; Devoz, Paula Pícoli; Antunes, Lusania Maria Greggi; Barbosa Jr,
Fernando

Evaluation of the female wistar rats reproductive system submitted to different diet
formulations and activity patterns during 180 days........................................................................................................193
Dantas, Joao Artur Diogenes; Silva, Aline Gabrielle Gomes; Moura, Maria Joana Nogueira;
Santos, Caio Cesar Araújo; Gomes, Francisca Tayná da Silva; Pereira, Artemia Kelly Holanda;
Felix, Renata Gleysiane de Sousa; Fonseca, Ivana Alice Teixeira; Borges, Cibele dos Santos
Evaluation of the oil toxicity of Astrocaryum vulgare Mart. through renal and hepatic
indicators of Wistar rats.............................................................................................................................................................194
Reginato, Fernanda Ziegler; Guex, Camille Gaube; Figueredo, Kássia Caroline; Graiczik, James
Ramires Penteado; Cassanego, Gabriela Buzatti; Pappis, Lauren; Heck, Amanda Szymansky;
Bauermann, Liliane de Freitas

evaluation of toxicity of extracts from Myrcia tijucensis in Artemia salina leach model .................................195
Kohler, Cristofer José Weege; Perini, Camila Maria; Alberton, Michele Debiasi

Exposure of adult female rats to different doses of the antipsychotic Aripiprazole: a look
at the reproductive system........................................................................................................................................................196
Silva, Aline Gabrielle Gomes;Moura, Maria Joana Nogueira; Pereira, Artemia Kelly Holanda;
Felix, Renata Gleysiane de Sousa; Angelo, Ana Beatriz Silva; Santos, Caio Cesar Araújo; Silva,
Ana Lucelha dos Santos; Freire, Livia Horrana Forte; Dantas, Joao Artur Diogenes; Silva, Mateus
Limerio Carlos; Borges, Cibele dos Santos

Imidacloprid-based commercial pesticide causes behavioral and biochemical

impairments in Wistar rats........................................................................................................................................................ 197
Tonietto, Bruna Ducatti; Laurentino, Ana Olívia Martins; Costa-Valle, Marina Tuerlinckx;
Cestonaro, Larissa Vivan; Antunes, Bibiana Pereira; Manfio, Cleofas Sates; Santos, Nícolas
Guimarães; Dallegrave, Eliane; Garcia, Solange Cristina; Leal, Mirna Bainy; Arbo, Marcelo Dutra

Immediate toxicological impairment of Lisdexamfetamine Dimesylate in male rats

treated during juvenile period and periuberty....................................................................................................................198
Stein, Julia; Jorge, Bárbara Campos; Reis, Ana Carolina Casali; Nagaoka, Lívia Trippe; Manoel,
Beatriz de Matos; Valente, Letícia Cardoso; Arena, Arielle Cristina

Investigation of the lipid-lowering and antiatherogenic effects of Plinia cauliflora bark

extract in an experimental model of atherosclerosis......................................................................................................199
Dalmagro, Mariana; Donadel, Guilherme; Pinc, Mariana Moraes; Berta, João Antonio; Borba,
Maria Eduarda; Gasparotto Junior, Arquimedes; Jacomassi, Eliza; Zardeto, Giuliana; Hoscheid,
Jaqueline; Ceranto, Daniela de Cassia Faglioni Boleta; Lourenço, Emerson Luiz Botelho

Levamisole, a cocaine adulterant, promotes acute and subchronic toxic effects in wistar rats ..................200
Laurentino, Ana Olívia Martins; Salomón, Janaína; Sebben, Viviane; Tonietto, Bruna Ducatti;
Cestonaro, Larissa Vivan; Dallegrave, Eliane; Garcia, Solange; Arbo, Marcelo Dutra; Leal, Mirna
Bainy

Maternal exposure to a phthalate combination associated with prostate lesions and

cancer in F1 and F2 offspring.....................................................................................................................................................201
Aquino, Ariana Musa; Costa, Luiz Guilherme Alonso; Santos, Sérgio Alexandre Alcantara;
Magosso, Natália; Souza, Patrick Vieira; Rocha, Vanessa Aguiar; Oliveira, Marcos Antônio
Fernandes; Barbisan, Luis Fernando; Justulin Junior, Luís Antonio; Flaws, Jodi A.; Scarano,
Wellerson Rodrigo

Methylmercury toxicity induces structural and cellular damage in cardiac ventricles.....................................202

Krüger, Nathália Ronconi Zilli; Pinheiro, Jacqueline; Simioni, Carmen; Nazari, Evelise Maria

Mitochondrial injury induced by Diuron and its metabolites in the rat liver..........................................................203
Seloto Danielle Gabriel; Lima Thania Rossi Rios Rossi; Camargo João Lauro Viana;Pereira Lilian
Cristina

Paternal Origins of Health and Disease: BaP exposure via paternal germ cells causes
negative reproductive consequences in female offspring (F2) in rats ....................................................................204
Jorge, Bárbara Campos; Stein, Julia; Reis, Ana Carolina Casali; Nogueira, Jéssica Bueno;
Paschoalini, Beatriz Rizzo; Moreira, Suyane da Silva; Manoel, Beatriz de Matos; Kassuya,
Cândida Aparecida Leite; Arena, Arielle Cristina

Pathological findings arising from continuous exposure to trace concentrations of

organochlorine DDT residues in multigeneration of wistar rats................................................................................ 205
Guimarães, Ana Tereza Bittencourt; Silva, Fernanda Coleraus; Quiozini, Nathaly de Matos;
Simon, Jaqueline; Pavlak, Jaíne Luana; Azevedo, Camilla de Marchi Sanches; Rangel, Ana Lúcia
Carrinho Ayroza
Production and in vitro toxicological studies of natural dyes......................................................................................206
Yli-Öyrä, Johanna; Herrala, Mikko; Albuquerque, Anjaina Fernandes; Farias, Natália Oliveira;
Morales, Daniel Alexandre; Räisänen, Riikka; Freeman, Harold S.; Umbuzeiro, Gisela de Aragão;
Rysä, Jaana

Reproductive repercussions in adulthood of male rats exposed to Lisdexamfetamine

Dimesylate on peripuberty ...................................................................................................................................................... 207
Stein, Julia; Jorge, Bárbara Campos; Reis, Ana Carolina Casali; Nagaoka, Lívia Trippe; Manoel,
Beatriz de Matos; Valente, Letícia Cardoso; Pupo, André Sampaio; Arena, Arielle Cristina

Skeletal abnormalities caused by L-mimosine: development toxicity study in rats..........................................208

Almeida, Elaine Renata Motta; Pereira, Edimar Cristiano; Hueza, Isis Machado

Study of the toxicological effects of thimerosal and aluminum hydroxide on Danio rerio’s kidney.............209
Galiciolli, Maria Eduarda A.; Silva, Juliana F.; Guiloski, Izonete C.; Oliveira, Cláudia S.

Systemic toxicity and testicular damage produced by exposure to benzo(a)pyrene in low-

dose from juvenile to peripuberty in male rats..................................................................................................................210
Jorge, Bárbara Campos; Reis, Ana Carolina Casali; Stein, Julia; Paschoalini, Beatriz Rizzo;
Nogueira, Jéssica Bueno; Arena, Arielle Cristina

Teratogenic and toxic effects induced by the herbicide 2,4-D (DMA® 806) in zebrafish
(Danio rerio) embryos...................................................................................................................................................................211
Viriato, Cristina; Andrade, Ítalo B.L.; Peixoto, Paloma V.L.; Pereira, Lílian C.

The relationship between oxidative system and sperm motility in wistar rats
continuously exposed to organochlorine DDT residues.................................................................................................212
Silva, Fernanda Coleraus; Quiozini, Nathaly de Matos; Simon, Jaqueline; Schwengber, Heloysa
Talia; Lesniewski, Isabela Ramos; Azevedo, Camilla de Marchi Sanches; Itinose, Ana Maria;
Marek, Carla Brugin; Guimarães, Ana Tereza Bittencourt

The white blood cells behavior in multigenerations of wistar rats exposed to DDT
organochlorine residues.............................................................................................................................................................213
Silva, Fernanda Coleraus; Quiozini, Nathaly de Matos; Simon, Jaqueline; Azevedo, Camilla
de Marchi Sanches; Vieira, Bruna Todeschini; Marek, Carla Brugin; Guimarães, Ana Tereza
Bittencourt

Toxicological effects of Color Index Disperse Red 1 textile azo dye in sperm of mice orally exposed..........214
Tanamachi, Amanda Rodrigues; Fernandes, Fábio Henrique; Lima, Geovana Cristina Ribeiro;
Mariani, Noemia Aparecida Partelli; Silva, Alan Andrew dos Santos; Silva, Erick José Ramo;
Salvadori, Daisy Maria Fávero

Toxicological safety studies of plant extract of Eugenia uniflora L. at different safety doses........................ 215
Donadel, Guilherme; Dalmagro, Mariana; Pinc, Mariana Moraes; Berta, João Antonio; Borba,
Maria Eduarda; Gasparotto Junior, Arquimedes; Zardeto, Giuliana; Hoscheid, Jaqueline;
Lourenço, Emerson Luiz Botelho

Transgenerational reproductive effects (F2) in male offspring exposed to benzo(a)pyrene

by paternal germ cells in rats ..................................................................................................................................................216
Jorge, Bárbara Campos; Reis, Ana Carolina Casali; Stein, Julia; Paschoalini, Beatriz Rizzo;
Nogueira, Jéssica Bueno; Moreira, Suyane da Silva; Manoel, Beatriz de Matos; Arena, Arielle
Cristina

Triclopyr induces changes in mitochondrial parameters............................................................................................... 217

Rizzi, Joyce Santana; Seloto, Danielle Gabriel; Pereira, Lílian Cristina

Use of zebrafish as animal model for research for neurotoxicological substances: a literature review.........218
Silva, Luíza Madureira; Pinheiro, Dávylla Rennia Saldanha; Braga, Ádrya Lariela Lima; Holanda,
Wiron Pimentel; Honório Júnior, José Eduardo Ribeiro

Whole-mount skeletal staining in rabbits fetuses for teratogenicity evaluation (OECD 414).........................219
Matsui, Andresa;Alves, Paula Daniela Sabino de Freitas; Santana, Thatiane Nunes; Vecina,
Juliana Falcato; Bechtold, Bruna Assunção; Fava, Luis Paulo
07  Food Safety............................................................................................................................................................. 220
Aluminum levels in fruits and in fruit and vegetable juices..........................................................................................221
Pinelli, Juliana Junqueira; Custódio, Flávia Beatriz

Analysis of physical chemical parameters and cyanogen residues in macaxeira flour

(Manihost esculenta Crantz), produced in a municipality of Paraense................................................................... 222
Araújo, Maria Eduarda Lima; Estumano, Saulo Braga; Oliveira, Sidney Julio Vieira; Santiago,
Mayla Andra de Andrade; Costa, Eliene dos Santos da Silva; Oliveira, Cláudia Simone Baltazar

Assessment of the incidence and concentration of polycyclic aromatic hydrocarbons in

cocoa-derived products............................................................................................................................................................. 223
Silva Júnior, Clovis Reis; Almeida, Gabriela de Oliveira; Moretti, Gabriela; Jager, Alessandra
Vincenzi

Caffeine content in pre-workout supplements and energy drinks marketed in Brazil: are
the actual levels safe and in accordance with the labels? ..........................................................................................224
Costa, Bruno Ruiz Brandão; El Haddad, Lohanna Pereira; Freitas, Bruno Toledo; Marinho, Pablo
Alves; De Martinis, Bruno Spinosa

Cytotoxic activity of green banana (Musa sinensis) flour by Allium cepa test..................................................... 225
Tadeu, Vitória Costa; Roehrs, Rafael; Farias, Fabiane Moreira; Kieling, Ketelin Monique
Cavalheiro; Nogueira, Caroline Lacerda; Denardin, Elton Luis Gasparotto

Design of Experiments as a tool for HS-SPME-GC-MS method development applied to

qualitative analysis of volatile alcohol congeners in seized whiskeys.................................................................... 226
Bigão, Vítor Luiz Caleffo Piva; Costa, Bruno Ruiz Brandão; Gomes, Nayna Cândida; Marinho,
Pablo Alves; Silva, Jonas Joaquim Mangabeira; De Martinis, Bruno Spinosa

Development of a new quantitative field-testing assay for the analysis of histamine in

seafood samples.......................................................................................................................................................................... 227
Cabrices, Oscar G.

Food Safety: regulatory and toxicological aspects of food packaging.....................................................................228

Almeida, Giulia Forni; Pinheiro, Fabriciano

Ozone processing of peanut “milk”: degradation of aflatoxins, reduction of allergenic

proteins and impact on lipid oxidation................................................................................................................................. 229
Romero, Alessandra de Cássia; Sartori, Alan Giovanini de Oliveira; Caetano-Silva, Maria Elisa;
Alencar, Severino Matias; Calori-Domingues, Maria Antonia; Augusto, Pedro Esteves Duarte

Using R language to study worldwide Lead contamination distribution on food using the
GEMSFOODS(WHO) database during 1995-2020.............................................................................................................230
Silva, Fabiano; Couto, Nilton; Almeida, Mariana

08 Forensic....................................................................................................................................................................231
An overview of NPS in northeast Brazil: NMR-based identification and analysis of ecstasy
tablets by GC-MS.......................................................................................................................................................................... 232
Cunha, Ricardo Leal; Oliveira, Celinalva da Silva Lima; Oliveira, Aline Lima; Maldaner, Adriano
Otávio; Pereira, Pedro Afonso de Paula

Analysis of diglycolic acid in victims intoxicated by the consumption of beer containing

diethylene glycol........................................................................................................................................................................... 233
Goulart, Cristiano O.L.; Bordoni, Leonardo S.; Nascentes, Clésia C.; Costa, Letícia M.

Determination of phosphatidyl ethanol in dried blood spots by LC-MS/MS.........................................................234

Guterres, Fernanda S.; Tegner, Mariane; Ott, Isabela Ritter; Linden, Rafael; Antunes, Marina
Vezon

First Report of national database on toxicological criminal information (ToxCrim system)........................... 235
Costa, Rony Anderson Rezende; Costa, Jose Luiz
Forensic entomotoxicology: application of studies in the development and rearing of
Lucilia cuprina flies (Diptera: Calliphoridae)...................................................................................................................... 236
Chimendes, Nayomi de Andrade; Bairros, André Valle; Ugalde, Gustavo Andrade; Santos, Lara
Celestina; Pacheco, André Lucas Bezerra; Reginato, Fernanda Ziegler; Berlato, Dener Gomes;
Rosa, Victória Gomes; Nascimento, Marcelo Henrique Santana; Pereira, Alessandra de Oliveira;
Monteiro, Silvia Gonzalez

Interference of larvae matrix of flies Lucilia cuprina in the determination of Azamethiphós

and Paraoxon ethyl using dispersive solid phase extraction (dSPE)........................................................................ 237
Pacheco, André L.B.; Ugalde, Gustavo A.; Chimendes, Nayomi A.; Santos, Lara C.; Berlato,
Dener G.; Nascimento, Marcelo H.S.; Reginato, Fernanda Z.; Rosa, Victória Gomes; Santos,
Rachel; Cardoso, Leonardo Correa; Monteiro, Silvia Gonzalez; Stainki, Daniel Roulim; Pereira,
Alessandra de Oliveira; Bairros, André V.

Nortriptyline overdose and quantitative analysis in post mortem vitreous humor – a case study.............238
Cunha, Ricardo Leal; Dalbosco, Juliana Santos; Brito, Maria Carolina Santos Ribeiro; Costa, José
Luiz

Optimization of extraction procedure for benzodiazepines and antidepressants analysis

in vitreous humor using cork sheet....................................................................................................................................... 239
Ossanes, Daniela Souza; Birk, Letícia; Eller, Sarah; Oliveira, Tiago Franco

Prevalence of volatile organic compounds in forensic toxicology reports of São Paulo State......................240
Rodrigues, Taís B.; Medeiros, Elvis A.; Chinaglia, Kauê O.; Gianvecchio, Victor A.P.; Costa, Jose Luiz

Profile of synthetic drugs seized between January 2019 and December 2021 analyzed by
the Forensic Toxicology Center of the Forensic Expertise in the State of Ceará (PEFOCE) ...............................241
Oliveira, Juliana Ribeiro Ibiapina Leitão; Magalhães, Danielle de Paula; Martins, Mayane
Emanuella Melo Lopes; Almeida, Vivian Romero Santiago; Saboya, Andrea Luiza Rocha; Holanda
Júnior, Wanderley Pinheiro

Profile of toxicological analysis of suicide cases reported by the Santa Catarina Scientific
Police in the last 5 years............................................................................................................................................................242
Silva, Bruna Espíndola; Boff, Bruna de Souza; Silveira Filho, Jair; Schroeder, Samilla Driessen;
Rezin, Kéttulin Zomer; Marchioni, Camila

Profile of victims of traffic accidents with blood samples collected for alcohol tests and
analyzed by the Forensic Expertise of the State of Ceará (PEFOCE) from January 2020 to
December 2021..............................................................................................................................................................................243
Magalhaes, Danielle de Paula; Holanda Júnior, Wanderley Pinheiro; Saboya, Andréa Luiza
Rocha; Oliveira, Juliana Ribeiro Ibiapina Leitão; Martins, Mayane Emanuela Melo Lopes;
Santiago, Vivian Romero

Quantifying ethanol content in alcoholic beverages by HS-GC/FID..........................................................................244

Bigão, Vítor Luiz Caleffo Piva; Costa, Bruno Ruiz Brandão; Santos Júnior, Wilson José Ramos; De
Martinis, Bruno Spinosa

Selective determination of 4-aminopyrine in seized cocaine samples by a reduced

graphene oxide/Prussian blue nanocomposite............................................................................................................... 245
Reis, Karsonn B.; Borges, Pedro H.S.; Rocha, Raquel G.; Ramos, David L.O.; Muñoz, Rodrigo A.A.;
Richter, Eduardo M.; Nossol, Edson

Sociodemographic profile of authors involved in illicit drug arrest in the city of Betim, Minas Gerais.......246
Costa, Anna Carolina de Moura; Oliveira, Lara Luiza Freitas; Sales, Thais Lorenna Souza;
Sanches, Cristina; Chequer, Farah Maria Drumond

Survey of drugs/adulterants found concomitantly with cocaine, in victims of violent

death, in the state of Minas Gerais, in the year 2021....................................................................................................... 247
Corrêa, Brunna F.; Bordoni, Leonardo S.; Couto, Tauer J.G.; Goulart , Christiane G.L.; Goulart,
Cristiano O.L.

The increase in the number of incarcerations in Brazil involving drug crimes and the main
drugs seized over ten years (2009-2019)...........................................................................................................................248
Bonfioli, Maysa Guilherme; Coelho, Júlia França Figueredo; Melo, Saulo Nascimento; Belo,
Vinícius Silva; Chequer, Farah Maria Drumond
 The mystery behind the apprehensions of the selective agonist to cannabinoid type 2
receptor BZO-HEXOXIZID (MDA-19) as a drug of abuse.................................................................................................249
Araujo, Karen Rafaela Gonçalves; Fabris, André Luis; Neves Júnior, Luiz F.; Ponce, Júlio de
Carvalho; Costa, José Luiz; Yonamine, Mauricio

Toxicological findings in cases attended by Division of Postmortem Inspection of Porto Alegre in 2021...... 250
Birk, Letícia; Barbosa, Fábio de Souza; Petry, Adriana Ubirajara Silva; Menezes, Francisco Paz;
Gonzaga, Alexsandro Pinto; Eller, Sarah; Oliveira, Tiago Franco

Validation of methods for the determination of cocaine, benzoylecgonine, and

cocaethylene in dried blood using the hemapen microsampling device................................................................. 251
Smidt, Mariana; Bastiani, Marcos Frank; Hahn, Roberta Zilles; Linden, Rafael

09  Genotoxicity and Carcinogenesis..................................................................................................................... 252

Cellular spheroids as a platform for toxicogenomic assays in cancer.................................................................... 253
Queijo, Rodrigo Gonçalves; Carvalho, Larissa Anastacio da Costa; Maria-Engler, Silvya Stuchi

Collaborative analysis of complex nitrosamines............................................................................................................. 254

Avila, Carolina Martins; Ponting, David. J.; Werner, Anne-Laure D.; Tennant, Rachael E.; Oliveira,
Antonio Anax F.; Heghes, Crina

Copper (II) complex as a potential treatment to overcome BRAF inhibitor resistance and
aggressive NRAS point-mutation........................................................................................................................................... 255
Moraes Junior, Manoel Oliveira; Carvalho, Larissa Anastácio da Costa; Nunes, Cleia Justino;
Ferreira, Ana Maria da Costa; Maria-Engler, Silvya Stuchi

Evaluation of photomutagenic potential of UV filters and IR3535 combination.................................................. 256

Fuzinaga, Thaís Y.T.; Gluzezak, Ana J.P.; Tavares, Renata S.N.; Kawakami, Camila M.; Abe, Flávia
R.; Oliveira, Danielle P.; Maria-Engler, Silvya S.; Gaspar, Lorena R.

Gliotoxin and its potential cytotoxicity against NRAS-mutated melanoma.......................................................... 257

Noma, Isabella Harumi Yonehara; Carvalho, Larissa Anastacio da Costa; Maria-Engler, Silvya
Stuchi

Strategies for overcoming toxicity and resistance in melanoma progression and in

adaptative resistance to braf inhibitors using ADK modulation................................................................................. 258
Silva, Julia Rezende; Oliveira, Érica Aparecida; Carvalho, Larissa Anastacio da Costa;
Maria‑Engler, Silvya Stuchi

The peroxiredoxin mimetic gliotoxin overcomes heterogeneity, intrinsic and acquired

resistance to BRAF inhibitor in metastatic melanoma................................................................................................... 259
Carvalho, Larissa A.C.; Noma, Isabella H.Y.; Uhera, Adriana H.; Siena, Ádamo D.D.; Harumi,
Luciana; Mori, Matheus P.; Goding, Colin; Pinto, Nadja C. Souza; Freitas, Vanessa M.; Silva-Jr,
Wilson A.; Smalley, Keiran S.M.; Maria-Engler, Silvya S.

10 Immunotoxicology................................................................................................................................................ 260
Analyzes of clove essential oil immunotoxicity based on in vitro and in silico studies......................................261
Etcheverry, Bibiana Frasson; Gomes, Gabriela Cristiane Mendes; Rios, Nathália Vieira; Chaves,
Pamella Eduardha Espindola; Sotelo, Êmily Clori; Zuravski, Luísa; Machado, Michel Mansur

Cigarette smoke and heat-not-burn tobacco vapor exposures alter granulopoiesis and
neutrophil functions during experimental arthritis......................................................................................................... 262
Scharf, Pablo; Heluany, Cíntia; Sandri, Silvana; Schneider, Ayda Henriques; Barbim, Paula
Donate; Fock, Ricardo; Cunha, Fernando; Farsky, Sandra

Cytotoxicity of Marinobufagenin in Glial Cells Chalenged Whith LPS....................................................................... 263

Farias, Evelyn Rayani Araújo; Silva, Rivia Regina Lopes; Souza, Natacha Medeiros; Marques, Ana
Maria; Scavone, Cristoforo; Quintas, Luís E.M.; Leite, Jacqueline Alves
 In vitro and in silico analysis of the cytotoxicity of Foeniculum vulgare essential oil in
human lymphocytes....................................................................................................................................................................264
Gomes, Gabriela Cristiane Mendes; Etcheverry, Bibiana Frasson; Chaves, Pamella Eduardha
Espindola; Rios, Nathalia Vieira; Campos, Dyene Nascimento; Zuravski, Luísa; Machado, Michel
Mansur

Lymphotoxicity of Brazil mint essential oil: an in vitro and in silico study.............................................................. 265
Chaves, Pamella Eduardha Espindola; Rios, Nathália Vieira; Gomes, Gabriela Cristiane Mendes;
Etcheverry, Bibiana Frasson; Campos, Dyene Nascimento; Zuravski, Luísa; Machado, Michel
Mansur

Mitochondria as a target for imidacloprid toxicity on RAW 264.7 cells .................................................................. 266
Cestonaro, Larissa V.; Schmitz, Felipe; Ferreira, Fernanda S.; Conte, Fernanda M.; Piton, Yasmin
V.; Wyse, Angela T.S.; Garcia, Solange C.; Arbo, Marcelo D.

New pyrazoline compounds: effects on neutrophils and macrophages functions............................................ 267

Goldoni, Fernanda C.; Benvenutti, Larissa; Vaz, Milena Menegazzo; Carlos Rafael; Buzzi, Fátima
C.; Quintão, Nara L.M.; Santin, José Roberto

Patchouli (Pogostemon cablin) essential oil: safe or a hidden risk? .......................................................................268

Rios, Nathália Vieira; Chaves, Pamella Eduardha Espindola; Gomes, Gabriela Cristiane Mendes;
Etcheverry, Bibiana Frasson; Sotelo, Êmily Clori; Zuravski, Luísa; Machado, Michel Mansur

Trilobolide-6-O-isobutyrate shows in silico and in vitro potential for antitumor activity of

A549 and NCI-H460 lung cancer cell lines.......................................................................................................................... 269
Miranda-Sapla, Milena Menegazzo; Concato, Virginia Marcia; Gonçalves, Manoela Daiele;
Matos, Ricardo Luiz Nascimento; Conchon-Costa, Ivete; Arakawa, Nilton Syogo; Pavanelli,
Wander Rogério

11 Nanotoxicology....................................................................................................................................................... 270
Cytotoxicity and effectiveness in macrophage polarization of Annexin A1-surface-
functionalized metal-complex multi-wall lipid core nanocapsule............................................................................ 271
Broering, Milena F.; Leão, Matheus C.; Scharf, Pablo; Sandri, Silvana; Uchiyama, Mayara K.; Araki,
Koiti; Guterres, Silvia S.; Pohlmann, Adriana R.; Farsky, Sandra H.P.

Preclinical evaluation of short-term gold nanoparticles toxicity.............................................................................. 272

Plautz, Katherine; Gastaldi, Alessandra Betina; Maia, Thayná Patachini; Pereira, Eduardo
Manoel; Ferreira, Gabriela Kozuchovski; Borgmann, Gabriela; Cabral, Heloisi; Delwing-Dal
Magro, Débora; Delwing-De Lima, Daniela

Predictive analysis of ocular and lung damage in cells exposed by zinc oxide or cerium
dioxide nanoparticles using electrical impedance compared to MTT test............................................................. 273
Alexandre, Angela de Oliveira; Souza, Wanderson; Dal-Cheri, Beatriz Kopke de Assis; Granjeiro,
Jose Mauro; Pereira, Leonardo da Cunha Boldrini

Study of the acute and chronic toxicity of chrome III oxide nanoparticles on the marine
microcrustacean Mysidopsis juniae (Silva, 1979)............................................................................................................. 274
Plautz, Katherine; Fugazza, Jonas; Gastaldi, Alessandra Betina; Kleine, Tamila; Matias, William
Gerson; Oliveira, Therezinha Maria Novais

Study of the interaction between a potential labeled nano-enabled pesticide and aquatic plant............... 275
Forini, Mariana M.L.; Antunes, Débora R.; Cavalcante, Luiz A.F.; Pontes, Montcharles S.; Santiago,
Etenaldo F.; Sanches, Alex O.; Martins, Aline R.; Grillo, Renato

Survival rate of Drosophila melanogaster exposed to nanoparticles containing

Naringenin and in vitro determination of inhibition kinetics against cholinergic enzymes............................. 276
Cunha, Viviane Augusta de Medeiros Garcia; Rossi, Bruna Franzon; Leimann, Fernanda Vitória;
Ineu, Rafael Porto; Foleis, Vanessa Kaplum; Gonçalves, Odinei Hess

Survival rate of Drosophila melanogaster exposed to Silymarin-loaded nanoparticles and

the ex vivo inhibition of cholinergic enzymes.....................................................................................................................277
Souza, Daniela Cristina; Rossi, Bruna Franzon; Leimann, Fernanda Vitória; Gonçalves, Odinei
Hess; Appelt, Patrícia; Ineu, Rafael Porto
12  Risk Assessment.................................................................................................................................................... 278
Animal-Free Safety Assessment of Cosmetics: a global education and training program.............................. 279
Marigliani, Bianca; Willett, Catherine

Biomonitoring of occupational exposure to triazoles and the disruptive effects of

steroidogenesis on androstenedione biosynthesis in humans..................................................................................280
Marciano, Luiz Paulo de Aguiar; Costa, Luiz Filipe; Freire, Josiane Oliveira; Feltrim, Fernando;
Machado, Simone Caetani; Silvério, Alessandra Cristina Pupin; Martins, Isarita

Buccal micronucleus cytome assay as a biomarker of genotoxic effect rural workers

exposed to triazole fungicides.................................................................................................................................................281
Costa, Luiz Filipe; Marciano, Luiz Paulo de Aguiar; Freire, Josiane Oliveira; Feltrim, Fernando;
Silvério, Alessandra Cristina Pupin; Martins, Isarita

Changes in biomarkers of exposure and potential harms in adult smokers following 180
days of gloTM tobacco heating product use.........................................................................................................................282
Esteves, Iuri; Zebele, Patricia; Gale, Nathan; Hardie, George; McEwan, Michael; Gaca, Marianna;
Goodall, Sharon

COVID-19 and susceptibility of firefighters exposed to smoke: A scope review ..................................................283

Silva, Rafael Araújo; Marciano, Luiz Paulo de Aguiar;Costa, Luiz Filipe; Nunes, Rafaella Ferreira
Nascimento; Sakakibara, Isarita Martins

Determination of urinary triazoles as reliable biological indicator for the application in coffee growers.....284
Marciano, Luiz Paulo de Aguiar; Costa, Luiz Filipe; Freire, Josiane Oliveira; Feltrim, Fernando;
Machado, Simone Caetani; Silvério, Alessandra Cristina Pupin; Martins, Isarita

Dispersive liquid-liquid microextraction to determinate parabens in body cream by liquid

chromatography........................................................................................................................................................................... 285
Ramos, Thalita da Silva; Barbosa, Alyne Maria da Costa; Rath, Susanne;Martins, Isarita

Evaluating the cytotoxicity of quantum dots using two different keratonocyte-cell based systems.........286
Souza, Isisdoris Rodrigues; Cruz, Juliana Varella; Thá, Emanoela Lundgren; Gagosian, Viviana
Stephanie Costa; Araujo-Souza, Patrícia Savio; Cestari, Marta Margarete; Faustman, Elaine M.;
Leme, Daniela Morais

Exposure driven data generation strategies for dietary and non-dietary risk evaluation of
crop protection products to inform safety and minimise unnecessary animal testing.................................... 287
Catalano, Shadia M.I.; Pais, Mariana Castello Novo; Latorre, Andreia Oliveira; Faria, Patricia
Miranda; Soares, Daniel; Freeman, Elaine

GHS classification of chemicals frequently used in laboratories: A risk assessment........................................288

Silvério, Kérolyn Aparecida; Pinheiro, Fabriciano

How can nitrosamine risk assessments be supported by in silico systems and data
sharing initiatives?.......................................................................................................................................................................289
Waechter, Fernanda; Kocks, Grace; Avila, Carolina Martins; Burns, Michael J.; Ponting, David J.;
Tennant, Rachael E.; Oliveira, Antonio Anax F.; Heghes, Crina

Human health risks assessment by iron mining dam failure (VALE S.A) in Brumadinho-MG.........................290
Domingos, Líllian Maria Borges; Castilhos, Zuleica Carmen

Occurrence and dietary exposure to histamine by the Brazilian population..........................................................291

Diniz, Fabiana Barbosa; Braga, Douglas Evangelista; Custódio, Flávia Beatriz; Gloria, Maria
Beatriz Abreu

Occurrence and exposure to aspartame from soft drinks by the Brazilian population.................................... 292
Sousa, Roberto Cesar Santos; Custódio, Flávia Beatriz; Gloria, Maria Beatriz A.

Permitted Daily Exposure (PDE) from preclinical studies of ginkgo biloba dry extract .................................... 293
Lamb, Liliane Weber Bolfe; Rodrigues, Gabriela Zimmermann Prado; Saraiva, Thalia
Emmanoella Sebulsqui; Souza, Douglas; Berna, Gabriel da Costa; Garcia, Ana Letícia Hilário;
Bertoldi, Fernando; Kayser, Juliana Machado; Ferreira, Julia Gabriele de Jesus, Veiverberg,
Andriele; Marco, Mariana; Gehlen, Günter; Betti, Andresa Heemann;Mattos, Cristiane Bastos
 Thyroid and sex hormonal profile and manganese exposure in pregnant women from a
Brazilian birth cohort: preliminary results..........................................................................................................................294
Santos, Nathália Ribeiro; Bah, Homegnon Antonin Ferréol; Gomes-Júnior, Erival Amorim;
Martinez, Victor Otero; Costa, Daisy Oliveira; Menezes-Filho, José Antonio

Total and inorganic arsenic in fish and exposure to inorganic arsenic by fish consumption in Brazil......... 295
Ramos, Vitor Serrão; Vasconcelos Neto, Milton Cabral; Custódio, Flávia Beatriz

13  Toxicologic Hazard in the Workplace............................................................................................................... 296

Assessment of serum levels of DDT and metabolites in workers in malaria control
campaigns in the state of Pará, Amazon, Brazil............................................................................................................... 297
Rocha, Thiago L.; Rocha, Cássia C.S.; Miranda, Antônio M.M.; Mendes, Rosivaldo de A.

Case Studies: challenges on implementing the GHS classification of pesticides in the

reevaluation of active substances.........................................................................................................................................298
Aguiar, Larissa Muratori; Braz, Juliana Machado; Moreira, Camila Queiroz; Santos, Thiago
Santana; Freitas, Daniel Roberto Coradi

Clinical and laboratorial evidenced organic damage due to the incorrect use of PPE in
rural producers of tomato and onion crops in the contestado region - Santa Catarina................................... 299
Boff, Everton; Kampmann, Micheli Gabardo; Benvenutti, Régis Carlos

Evaluation of proteins and genes expression in workers exposed to crystalline silica in

Southern Brazil..............................................................................................................................................................................300
Peruzzi, Caroline Portela; Göethel, Gabriela; Flesch, Ingrid; Nascimento, Sabrina; Nardi, Jessica;
Arbo, Marcelo Dutra; Garcia, Solange

Evaluation of residual contamination in anticancer drug packaging using UHPLC-MS/MS............................301

Silva, Luciana Stein; Silva, Laura Cé; Machado, Cibele da Silva Barbosa; Capp, Edison; Linden,
Rafael; Ness, Sandro Luis Ribeiro; Antunes, Marina Vezon

Green tobacco sickness occurrence and evaluation of life quality among tobacco workers
in Paraná countryside.................................................................................................................................................................302
Bortoli, Stella; Schamne, Tatiane; Kalv, Danielle Crystiane; Pedroso, Bruno; Vellosa, Jose Carlos
Rebuglio

Pesticide poisoning in banana cultivation in Corupá city, Santa Catarina.............................................................. 303

Borgmann, Gabriela; Plautz, Katherine; Zimmath, Michel, Tenfen, Adrielli

Skin exposure to KrCl excimer lamp emitting at 222 nm cause tissue and cellular
alterations of concern................................................................................................................................................................304
Tavares, Renata Spagolla Napoleão; Girassole, Alessandra; Adamoski, Douglas; Caznok, Ana
Clara; Domingues, Romênia; Leme, Adriana Paes; Carvalho, Murilo; Dias, Sandra Martha Gomes

The influence of occupational exposure to cyanide on metahemoglobinemia in cassava

flour producers.............................................................................................................................................................................. 305
Estumano, Saulo Braga; Oliveira, Cláudia Simone Baltazar; Araújo, Maria Eduarda Lima; Costa,
Eliene dos Santos da Silva; Chisté, Renan Campos; Lameira, Christian Neri; Amaro, Beatriz
Oliveira; Pinheiro, Maria da Conceição Nascimento

Toxic metal backgrounds among waste pickers in hair sampling: a cross-sectional study
in Brazil (Federal District)..........................................................................................................................................................306
Gonçalves, Michelly Rodrigues; Verpaele, Steven; Marques, Carla Pintas; Bashash, Morteza;
Cruvinel, Vanessa Resende Nogueira; Santos, Vivian da Silva

14 Other......................................................................................................................................................................... 307
Attempted suicide: temporal series and agents involved before and during the COVID-19
pandemic in Brazil........................................................................................................................................................................308
Holanda Júnior, Wanderley Pinheiro; Magalhães, Danielle de Paula; Oliveira, Juliana Ribeiro
Ibiapina Leitão
 Brazil now prioritizes animal-free safety assessment of school supplies............................................................309
Marigliani, Bianca

Early exposure to toxic metals and child neurodevelopment: proposal of a theoretical

model based on Bronfenbrenner’s bioecological theory................................................................................................310
Bah, Homègnon Antonin Ferréol; Santos, Nathália Ribeiro; Costa, Daisy Oliveira; Carvalho,
Chrissie Ferreira; Martínez, Victor Otero; Gomes-Júnior, Erival Amorim; Menezes-Filho, José
Antonio

Junkie – Teaching project in comic book for the development of learning in toxicology.....................................311
Bairros, André Valle; Reginato, Fernanda Ziegler; Ugalde, Gustavo Andrade; Berlato, Dener
Gomes; Pacheco, André Lucas Bezerra; Nascimento, Marcelo Henrique Santana; Oliveira, Letícia
Cimó; Santos, Lara Celestina; Chimendes, Nayomi de Andrade; Rosa, Victória Gomes; Dalmazzo,
André Kusser; Maia, Affonso Enriques Montagner

The use of paracetamol as cause for male infertility......................................................................................................312

Silva, Luíza Madureira; Holanda, Wiron Pimentel; Capelo, Melissa Figueiredo; Honório Júnior,
José Eduardo Ribeiro

Index of Authors..............................................................................................................................................................313
 01 
ALTERNATIVE ANIMAL
MODELS & EMERGING
IN VITRO MODELS
 25

Adipose Tissue 3D: new tools in cell culture

Buchele, Maria Luiza Caneiro1; Mora, Tamara Dal1; Saleh, Najla Adel1;
Silva, Adny Henrique1; Monteiro, Fabíola Branco Filippin1,2
1
Programa de Pós Graduação em Farmácia, Centro de Ciências da Saúde, Universidade Federal
de Santa Catarina, Florianópolis, SC, Brasil; 2 Departamento de Análises Clínicas, Centro de
Ciências da Saúde, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil.

Obesity is characterized by the excessive accumulation
of white adipose tissue resulting from the imbalance
between energy consumption and expenditure.
It is considered a complex, multifactorial and
heterogeneous disease with genetic, environmental,
behavioural and socioeconomic origins. Worldwide,
obesity has become a main public health issue
responsible for 2.8 million deaths per year, according
to the World Health Organization (WHO). Besides,
obesity increases considerably the risk for type 2
diabetes, cardiovascular diseases and some types
of cancer. The involvement of the adipose tissue,
considered a complex endocrine organ, raises the
importance of a better understanding of its metabolic
state at a cellular level is essential. Thus, three-
dimensional (3D) in vitro models could represent a
new alternative to the classic models and provide
new insights on the study of obesity and related
diseases. Therefore, this study aimed to develop a 3D
co-culture model using three different cell types that
are constituents of the native adipose tissue. The cell
lines used were 3T3-L1 (murine pre-adipocyte), NIH-
3T3 (murine embryo fibroblasts) and J774 (murine
macrophages). The 3D model was obtained through
a non-adhesive 96 well plate coated with agarose
2% seeded with 1.2 × 104 cells/well (1:1:1). After 4
days of culture in standard conditions, the spheroids
were submitted to adipogenic differentiation medium
(MDI), containing DMEM supplemented with 10%
of fetal bovine serum (FBS), 1 µM dexamethasone,
0.5 mM IBMX, and 1.67µM insulin for 72h. After that,
50% of the supernatant was replaced by DMEM
supplemented with 10% FBS and 1.67µM insulin
changed every 48h until 11 days of culture. The control
medium was prepared by supplementing DMEM with
10% calf serum (CS). Finally, the spheroids were
stained with Nile Red, a selective fluorescent stain for
intracellular lipid droplets. Spheroids submitted to the
MDI medium were compared with spheroids formed in
the control medium. According to the results obtained
by trypan blue assay, the cell viability for both types
of spheroids, formed in MDI or control medium, was
around 60%. Spheroid diameter and morphology were
analyzed by photomicroscopy and scanning electron
microscopy (SEM), respectively. The differentiation of
pre-adipocytes into adipocytes was analyzed by flow
cytometry and confocal microscopy. Interestingly, the
results of flow cytometry and confocal microscopy
showed that spheroids formed in the control
medium also presented adipogenic differentiation. In
conclusion, it was possible to develop a 3D co-culture
spheroid model of adipose tissue that can be used to
study new metabolic pathways and new therapeutic
targets, as well, toxicity effects of new drugs for the
therapy of obesity and obesity-related diseases.
 26

Analysis of cytotoxicity and anti-melanogenic

activity of kojic acid and glycolic acid
in SK-MEL-28 and B16F10 cells
Ribeiro, Milena Mariano1; Silva, Ana Cléia Cardoso2; Irioda, Ana Carolina2; Oliveira, Cláudia Sirlene2
1
Curso de Farmácia, Faculdades Pequeno Príncipe, Curitiba, Paraná, Brazil;
2
Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, Paraná, Brazil.

Background: Melasma is known as a hypermelanosis
caused by a dysfunction in the melanogenesis process.
The main factors that stimulate this dysfunction
is exposure to the sun, causing an increase in the
production of melanin in melanocytes, which will
be deposited in keratinocytes. There are several
compounds used to treat melasma. Among them
are kojic and glycolic acids. Kojic acid is used as a
depigmenting agent, causing a melanin reduction. Also,
kojic acid has antioxidant potential and is compatible
with non-ionic bases, so it can be associated with
glycolic acid, which results in an exfoliative and
antioxidant effect. Although these compounds are
already used in the treatment of melasma in isolation,
it is of scientific relevance to analyze the cytotoxicity
of these compounds, as well as their anti-melanogenic
potential, isolated and combined. Objective: To
evaluate the cytotoxicity and anti-melanogenic effect
of kojic and glycolic acid, isolated or combined in SK-
MEL-28 and B16F10 cells. Methods: The research was
carried out with melanoma cells (SK-MEL-28 and
B16F10). The cytotoxicity of the compounds kojic acid
and glycolic acid in the two lines was investigated by
the MTT assay. This assay was carried out in a 48-
well plate, where cells were seeded (2.5x104 cells/
well). In the SK-MEL-28 cell, the compounds were
tested isolated or combined at concentrations of 0, 1,
10, 30, 100, 300 and 1000 µM and in B16F10 only at a
concentration of 1000 µM. The exposure period was
48h. To analyze the anti-melanogenic activity, the
melanin content of the B16F10 cell was measured, in
the presence or absence of the alpha-MSH hormone
(200 nM) and the compounds kojic acid and glycolic
acid (1000 µM) isolated and combined. For this,
the cells were cultivated in a 24-well plate, with a
concentration of 4.5x104 cells/well. The exposure
period was 48h. The results were analyzed by
one-way ANOVA followed by Tukey’s pos hoc. The
results were considered significant when p<0.05.
The program used was GraphPad Prisma 6. Results:
One-way ANOVA revealed no effect of kojic acid and
glycolic acid, isolated and combined, on cell viability.
In the presence of the compounds, in the SK-MEL-28
lineage, there was no reduction in cell viability
at any of the concentrations tested. From these
results, a concentration of 1000 µM was selected to
assess the melanin content and cell viability in the
presence of acids, isolated or combined, in the cells.
It was observed that the compounds did not show
cytotoxicity to the B16F10 cells. By obtaining the
results of melanin content, one-way ANOVA revealed
that both kojic acid and glycolic acid, in the presence
or absence of alpha-MSH hormone, decreased the
melanin content. Conclusion: It is concluded that the
acids, isolated or combined, did not cause cytotoxic
effect, in the concentrations and cells tested. Also,
regarding the anti-melanogenic activity, the acids
at 1000 µM concentration, in combined, obtained a
more significant reduction in the melanin content
when compared to them isolated. Thus, kojic acid and
glycolic acid can be used in future experiments at any
concentration tested in this work. Further test will be
carried out to clarify the effects that the compounds
have on their anti-melanogenic activity.
 27

Bioprinted and manual human

epidermis reconstruction: a compared
performance for irritation tests
Bagatin, Julia de Toledo1; Camarena, Denisse Esther Mallaupoma1; Osaki, Luciana Harumi2;
Freitas, Vanessa M.2; Nold, Juliana C. Lago3; Maria-Engler, Silvya Stuchi1
1
School of Pharmaceutical Sciences - University of São Paulo; 2 Biomedical Sciences
Institute – University of São Paulo; 3 Natura & Co - São Paulo, Brazil.

Introduction: The progress of in vitro skin models
relies on increasing its human relevance and
reliability as platforms to evaluate the safety of
consumable products. 3D bioprinting allows the
precise spatial control in the deposition of biological
materials to achieve more complex and accurate skin
mimetic models. To evaluate skin models for safety
assessment, the Organization for Economic Co-
operation and Development (OECD) guideline 439 (TG
439) provides in vitro hazard identification of irritant
chemicals using validated human reconstructed
epidermis (RHE) models as an alternative method
for the Draize test. This guideline also describes
detailed quality control and performance standards
required for the validation of similar RHE models for
skin irritation testing. Objective: This study aimed
to compare the performance of a bioprinted human
epidermis (B-RHE) to a manually reconstructed
human epidermis (M-RHE) at the in vitro skin
irritation test described in the OECD TG 439. Methods:
To reconstruct the RHEs primary isolated human
epidermal keratinocytes were seeded in transwell
inserts previously coated with collagen using manual
techniques or a BioEnder 3 extrusion bioprinter
(BioEdTech, Brazil). The models were evaluated
following OECD guidelines TG 439. Tissue morphology
was verified by histology and immunofluorescence.
The quality of the barrier function was determined
by the inhibitory concentration that reduces viability
by 50% (IC50) after 18h of exposure to SDS using the
MTT method. Skin irritation testing was assessed by
the exposure of the tissues to reference chemicals (3
irritants and 3 non-irritants) using the MTT viability
assay. Results: RHEs developed using manual or
bioprinted methodologies exhibited high quality
morphology consistent with the quality control
described by the OECD TG 439. Both models exhibited
a well-differentiated and multiple layered epidermis
with a viable corneal layer. The epidermal barrier
function performance of both models also achieved the
performance standards as the validated RHE models
described in TG 439, with no significative differences
within the methodologies. After the achievement
of quality control standards for morphology and
barrier function, the RHEs performance for the in vitro
irritation was evaluated. Both RHE models correctly
discriminated the reference substances classified as
irritant or non-irritant according to tissue viability
threshold value (50%). No significative differences
between the methodologies were observed, except
for the higher viability of potassium hydroxide
applied to bioprinted RHE. Discussion/Conclusion:
3D bioprinting is an emerging technology issued
to achieve more reliable human mimetics tissues,
but methodological comparison to the classical
manual techniques is scarce in literature. The semi-
permeable function of the corneal layer determines
the pathological response against the exposure to
irritant chemicals. Both epidermal models presented
excellent morphology and barrier function. Therefore,
the models were capable of distinguishing reference
irritant chemicals from non-irritant chemicals
when compared to the validated models described
in TG 439. The similarity of the performance of
the methodologies for the in vitro skin irritation
test indicates that bioprinting can be of great
contribution for the automation of reconstruction of
epidermal models. Therefore, bioprinting can provide
great assistance for cosmetic and pharmaceutical
industries to increase the production of epidermal
mimetics aiming to perform large scale in vitro
trials for safety assessments of consumable topical
products. Acknowledgments: This work was
supported by FAPESP [grant number #2019/14527-7
and # 2017/04926-6] and CAPES [scholarship grant
number #88887.363766/2019-00].

Recent technologies in three-dimensional (3D) cell
culture have called much attention in the fields
of cancer study and drug development due to the
capacity of 3D cell culture mimicking aspects of in
vivo microenvironments. 3D cell culture systems
can provide cells with ideal structures that are much
closer to tumors in vivo conditions in terms of the
organization and formation of biological, histological
and molecular microenvironment, when compared
to standard 2D cultures. The latter offers them a
homogeneous concentration of nutrients, growth
factors and cytokines present in the culture media.
This affects intracellular signaling and phenotypic
fate, leading to unnatural interactions with soluble
factors, in contrast to in vivo circumstances. Great
efforts have been made to develop 3D cell culture
models that have strong potential to overcome the
deficiencies of 2D cell cultures. However, most of
them are studies carried out at universities and
research centers and are not applied in industries
and companies that have screening tests routine for
new drugs, for example, representing a big barrier
and a challenge when it comes to changing the 2D cell
culture to the 3D techniques in Brazil. The problems
encountered in this routine change are due to the
lack of availability of robust commercial 3D matrices,
without interference, with an affordable price and
defined protocols. In addition, training staff members
to start using 3D tests in their screening routines
is necessary, as there is no doubt that 3D culture
faithfully mimics the in vivo environment, leading to
much more robust data results than those found in
2D. Biocelltis became the first Brazilian company to
produce and sell matrices for cell culture in 3D - the
3D CellFate® matrix. 3D CellFate® matrix is a hydrogel
biomaterial made up by natural polymeric nanofibers
28
Cellfate®Matrix: building the
reality in three dimensions
Reis, Emily Marques; Cavichion, Rafael Filipe Battisti; Colla,
Guilherme; Godoi, Manuella Machado; Koepp, Janice
Biocelltis Biotecnologia S.A., Santa Catarina, Brazil.
from 50 to 100 nm in diameter, similar to human
collagen, biocompatible, sterile and ready-for-use,
ideal for growing human and animal cells in research
labs. This study aims to pre-validate an alternative
cytotoxicity methodology using the 3D CellFate®
Matrix, a candidate for replacement of traditional
methods of cytotoxicity assays in a 2D environment.
Cell concentration, seeding volume, adhesion time,
culture time, exposure time, morphology and cell
viability were evaluated parameters in some tumoral
cell lines. The results obtained showed that cells in
3D CellFate® culture showed a completely different
behavior and morphology when compared to 2D
culture. In addition, these results corroborate with
drug resistance presented by cells in 3D culture in
relation to cytotoxicity found in 2D culture. Together
the results herein obtained suggest that a protocol
using 3D CellFate® Matrix is a great candidate for an
alternative to 2D methods in vitro. The 3D CellFate®
matrix proved to mimic the in vivo microenvironment,
influencing cell survival, shape, migration,
proliferation and differentiation in the presence
of cytotoxic agents, thus leading cells to have its
morphology and physiology similar to in vivo behavior.
Besides that, we have developed a robust and easy - to
- use protocol that can be used in high demands for in
vitro assays, showing the potential use of a 3D matrix
in industry. In this context, the use of 3D CellFate®
matrix could avoid the over or underestimation
of a specific drug in cases of drug sensitivity and
resistance assays, as well as its dosage, leading to
a reduction in the number of animals in subsequent
phases of drug screening, making the process more
exact and resources consuming. Keywords: 3dmodel,
cellfate, cytotoxicityassay

Cellfate®RHE: a brazilian commercial model
of reconstituted human epidermis in vitro
Reis, Emily Marques; Cavichion, Rafael Filipe Battisti; Colla, Guilherme; Koepp, Janice
In 2019, normative resolution No. 18/2014 of
the National Council for the Control of Animal
Experimentation - CONCEA came into force in Brazil,
which made the implementation of alternative
methods to the use of animals mandatory, imposing on
manufacturers of products that pose a risk to human
health to cease the use of animals in their safety trials,
especially corrosion and irritation tests, proposing as
an alternative the use of in vitro reconstituted human
skin (RHE). The majority of RHE models are produced by
European and American countries, and its importation
is not feasible due to the nature of the product. In
fact, in Brazil, there is the commercialization of
just one validated RHE model, where all the inputs
for the construction of the model are imported,
including the cells, making the physiology of the skin
very different from the Brazilian one. Based on this,
Biocelltis a national biomaterials industry, has been
developing a commercial model of RHE, using its 3D
CellFate® matrix and adapting it to the anatomical
and physiological functions of Brazilian skin. Here, we
present CellFate®RHE Model a solution for generating
3D epidermal skin models composed of normal human
primary epidermal keratinocytes derived from skin.
Numerous performance standards were evaluated,
growth media and supplements, cell seeding density,
passage number, as well as stratification conditions.
29
Biocelltis Biotecnologia S.A., Santa Catarina, Brazil;
The results show that seeding neonatal keratinocytes
in the 3D CellFate® matrix and cultured in chemically
defined medium resulted in the consistent generation
of 3D epidermal skin equivalents. The models
produced using the optimized protocol exhibit
physiologically relevant morphology, displaying a
comparable number of cell layers and stratification
to the human epidermis. The CellFate®RHE model
developed presents a well-differentiated epidermis
similar to Validated Reference Methods (VRM) and
native human epidermis. Quality parameters, ie,
negative control optical density, tissue integrity, and
barrier function, were similar to VRM, making it ideal
for evaluating preclinical and R&D effects of topical
compounds on human skin. Biocelltis has developed a
low-cost commercial model of reconstructed human
epidermis that mimics the barrier function of the
human stratum corneum as well as a viable complete
epidermis. The model meets a national demand,
overcoming of technical barriers and promoting the
country’s technological autonomy in alternative
methods to animal experimentation. Still, for the
availability of the product to the national territory,
intra- and inter-laboratory validation tests are
undergoing to meet the OECD regulations. Keywords:
reconstituted human epidermis in vitro, rhe, cellfate,
3Dmodel, invitromodel.
 30

Characterization and analysis of the

expression of the surface marker Stro‑1
in human dental pulp stem cells
Oliveira, Leandro Leal Rocha; Lima, Aliny Pereira; Farias, Evelyn Rayani Araújo; Gomes, Thaisângela
Rodrigues Lopes e Silva; Macedo, Larissa Matuda; Leite, Jacqueline Alves; Valadares, Marize Campos

Universidade Federal de Goiás - UFG.

Introduction: Dental pulp stem cells (DPSCs) are a
promising source of cells to be applied in regenerative
medicine. DPSCs are stem cells which can originate
from the neural crest exhibiting neuro-ectodermal
features and mesenchymal (MSC) properties. DPSCs
express MSC cell surface markers, such as CD90,
CD105, CD146 and Stro-1, in addition several studies
have provided evidence of the differentiation capacity
of these cells and they can be used in the repair of
bone defects, treatment of neural tissue injuries and
degenerative diseases. Traditionally, rodents are used
for toxicological studies. However, the differences
between primates and rodents make it difficult for
rodent models to accurately or even recap human
physiology and clinic. In this way, the technogolia
of dental stem cells emerges as a possibility for the
development of fully humanized models to better
understand the action of toxicants. Objective: to
characterize the culture of DPSCs, as well as the
expression of Stro-1 during serial passages in the
routine cell culture system, as well as the application
of this technology to the development of human
models for the toxicological study of new drugs.
Concerning that, the naive DPSCs were obtained
from the biorepository of dental stem cells of the
Laboratory of Teaching and Research in Toxicology
In Vitro-Tox In- from Faculdade de Farmácia at UFG
(University of Góias). Cells were cultured in DMEM/
F12 medium supplemented with fetal bovine serum
(10%), penicillin/streptomycin (1%). After the second
and fifth passage, the cells were trypsinized and
their viability determined. The morphology as well
as the expression of the surface marker Stro-1 were
evaluated by means of flow cytometry. Results:
The partial results demonstrate that DPSC cells
showed proliferative capacity after thawing, reaching
confluence after 7 days in culture. We interestingly
observed a reduction in Stro-1 expression with
passages. Conclusion: In this way, we can partially
conclude that the passages alter the expression
of the MSC Stro-1 marker, which may restrict the
differentiation capacity of DPSCs, however, further
assays need to be performed to better understand
the effect of passages on the differentiation capacity
of these cells. Furthermore, the standardization of
the model by our group will allow the development
of human models for the toxicological study of new
drugs. Keywords: stem cells, dental pulp, cell culture
 31

Characterization and applicability of a novel

physiologically relevant 3D-tetraculture
bronchial model for in vitro assessment
of chemical respiratory allergy
Silva, Artur Christian Garcia; Mendonça, Izadora Caroline Furtado; Valadares, Marize Campos

Laboratory of Research and Education in In vitro Toxicology (Tox In),

Faculty of Pharmacy, Federal University of Goiás.

Introduction: Chemical Respiratory Allergy (CRA)
consists of pathological conditions characterized by
clinical manifestations that encompass asthma and
rhinitis, which can be correlated with exposure to Low
Molecular Weight (LMW) sensitizers, mainly regarding
an occupational context. From a risk/toxicity assessment
point of view, there are still no validated methods
for evaluating the respiratory sensitization potential
of chemicals once the in vivo-based models usually
employed for inhalation toxicity addressment do not
comprise allergenicity endpoints specifically. Besides,
most of the classic cell culture-based in vitro techniques
reside in submerged culture conditions, which fail to
emulate toxicant exposure’s toxicokinetics within the
airways. Summed up, these issues justify the urgent
need for developing physiologically relevant methods
that resemble the respiratory system complexity, allow
the mimicking of airways toxicant exposure conditions,
and the establishment of quantifiable endpoints that
correlate with the allergenicity potential of chemicals.
Objective: In this work, we developed, characterized,
and evaluated the applicability of a 3D-tetraculture
bronchial model reconstructed with the main airway cell
types. Furthermore, we performed the tissue exposure
to aerosols of two LMW respiratory sensitizers at an
air-liquid interface, following the measurement of
tissue viability and the activation of dendritic cells
within the model. Methods: The co-culture model was
reconstructed using four different cell lines, including
bronchial epithelial cells (BEAS-2B), lung fibroblasts
(MRC-5), endothelial cells (EA.hy926), and monocytic
cells (THP-1). Briefly, the endothelial cells were cultivated
in the basolateral side of 24-well semipermeable
transwell inserts for 4 hours. Then, a collagen type I
hydrogel containing lung fibroblasts was polymerized
into the apical surface of the system. After 24 hours of
cultivation, BEAS-2B epithelial cells were added upon
the collagen matrix and drifted to an air-liquid interface.
On the fifth day, the models were exposed to different
concentrations of Chloramine-T and Maleic Anhydride
aerosols, generated using the VitroCell Cloud 12 chamber
and cultivated for additional 24 hours in the presence
of THP-1 monocytic cells in the basal compartment.
Tissue viability was assessed using MTT reduction
assay and dendritic cells activation/maturation
biomarkers CD86 and HLA-DR were evaluated using
flow cytometry. Results: The co-culture model was
successfully reconstructed with the four respiratory
system cell types. The morphological characterization
demonstrated the presence of a continuous epithelial
cell layer distributed upon a collagen matrix containing
homogeneously dispersed lung fibroblasts. The bronchial
epithelial cells also regularly expressed the biomarkers
cytokeratin, E-cadherin and MUC-1, demonstrating
suitable cohesion and function of epithelial cells.
Moreover, endothelial cells constituted a continuous
layer that remained integrally attached until the sixth
day of cultivation in the basolateral side of the transwell
inserts. The LMW sensitizers aerosols promoted a
concentration-dependent decrease of tissue viability
upon the 3D bronchial model, and further exposures
to the CV80 (80% tissue viability concentration) led to
an increase of CD86 and HLA-DR surface biomarkers
expression by THP-1 cells in the basal compartment,
demonstrating activation/maturation of dendritic
cells led by the toxicant permeation, as well as the
crosstalk between the different cell types. Discussion/
Conclusion: Taken together, our results demonstrated
that the 3D-tetraculture bronchial model presents
hallmarks that can be correlated with the structure and
function of human airways. Moreover, the integration of
immune system cells to the epithelial model allowed the
measurement of quantifiable endpoints that represent
the proposed mode of action of LMW respiratory
sensitizers, considering a physiologically-relevant air-
liquid interface exposure to aerosols that mimics the
human airway susceptibility to chemicals. Thus, the
model is a promising tool for assessing respiratory
toxicity/chemical respiratory allergy potential of
inhaled toxicants. Aknowledgements: Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) e Financiadora de Estudos e Projetos
(FINEP).
 32

Concomitant exposure to air particulate

matter and solar radiation reduces
epidermal barrier function in a
reconstructed human epidermis model
Silva, Claudia Larissa Viana; Carvalho, Larissa Anastacio da Costa; Camarena, Denisse Esther Mallaupoma;
Bagatin, Julia de Toledo; Assis, Silvia Romano; Maria-Engler, Silvya Stuchi; Barros, Silvia Berlanga de Moraes

Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical

Sciences, University of Sao Paulo, São Paulo, SP, Brazil.

Exposure to airborne particulate matter (PM) has
been associated with premature skin aging and the
aggravation of several skin diseases such as atopic
dermatitis. Solar radiation initiates the inflammatory
process in the skin and promotes DNA damage. The
consequences of concomitant exposure of the skin
to air PM and solar radiation as observed in the daily
life are poorly understood. Reconstructed human
epidermis (RHE) model was used to investigate
the alterations induced by the exposure to sun
light emitted by a solar simulator (5 J/cm2), and a
standardized air particulate matter – NIST SRM 1648a
(8,9 µg/cm2), alone or concomitantly. Histological
parameters, cell viability, epidermal barrier function,
inflammatory cytokines IL1-α and IL8- secretion and
epidermal proteins loricrin (LOR), cytokeratin 10 (CK10),
aquaporin 3 (AQP3) and NOTCH1 were evaluated.
Reduction in viability in radiation-only exposed RHEs
was observed when compared to non-exposed group.
Interestingly, epidermis barrier function was increased
in the irradiated-only group when compared to all
other treatments, while the double exposure with PM
reversed this alteration. There were no changes in
the secretion of IL1-α in all treatments, whereas IL-8
showed a significant reduction in expression in the
double exposed group when compared to the all other
groups. Immunohistochemical observations indicated
no noteworthy differences. CK10 and NOCTH1 protein
expression were increased in the double exposure
compared to irradiated-only group followed by a
significant reduction in LOR expression compared
to the control and irradiated-only group. Double
exposure to PM and solar radiation induced a decrease
in AQP3 expression when compared to the irradiated-
only group. Our findings reiterate the role of NOTCH1
in the signaling of barrier function epidermal proteins
AQP3, CK10 and LOR. These observations highlight
the importance of further studies with concomitant
exposure for tightly controlling the release of
pollutants and diminishing solar exposure to prevent
more serious diseases. Supported by CNPq (Process:
130103/2019-5) Claudia L.V.da Silva MSc fellowship at
the MSc Program of Pharmacy- Physiopathology and
Toxicology and FAPESP 16/19963-1.
 33

Creation of a biorepository from

dental stem cells: as an alternative
method to the use of animals
Oliveira, Leandro Leal Rocha1; Lima, Aliny Pereira1; Farias, Evelyn Rayani Araújo2; Gomes, Thaisângela
Rodrigues Lopes e Silva3; Macedo, Larissa Matuda2; Leite, Jacqueline Alves2; Valadares, Marize Campos1
1
Laboratório de Ensino e Pesquisa em Toxicologia In vitro, Faculdade de
Farmácia, Universidade Federal de Goiás - UFG; 2 Instituto de Ciências Biológicas,
Universidade Federal de Goiás-UFG; 3 Centro universitário UNIFASAM.

Introduction: Preclinical studies to screen for new
drugs involve the use of animals, which is very
expensive and time-consuming and, depending on the
research hypothesis, may have scientific limitations,
as well as little relevance to human biology. According
to this, alternative models have been used to replace
animals in scientific research, representing a social
and economic improvement. Stem cells are generally
defined as clonogenic cells capable of self-renewal
and differentiation into multiple lineages, and may be
of embryonic or postnatal origin. Isolating postnatal
stem cells from available high-quality sources has
been an important target for toxicological research
into new drugs. Dental stem cells (DSCs) which are
originate from the neural crest have been gaining
prominence, exhibiting neuro-ectodermal features.
Recent studies have shown a broad spectrum of
differentiation of DSCs in osteoblasts, adipocytes,
chondrocytes, endothelial cells, muscle tissue, neural
cells, among others, thus demonstrating a potential in
the use of these cells for toxicological investigation,
as well as drug discovery. Objective: isolation of stem
cells from dental pulp to maintain a biorepository.
Methodology: first step: to divide the teeth into three
areas: pulp, papilla and periodontal ligament. second
step: separating the cells from the selected parts
using sterile materials. third step: through the enzyme
collagenase, there is digestion of the extracellular
matrix. fourth step: centrifugation and insertion
of the explant into the cell culture vial. Results: we
seek to quantify how many cells it is possible to have
from a single tooth. Acknowledgments: The authors
gratefully acknowledge the financial support CNPq,
CAPES, FAPEG, FINEP.
 34

Decellularized extracellular matrix

hydrogel derived from bovine cornea
for application in 3D models
Santos, Jordana Andrade; Silva, Artur Christian Garcia; Dias, Wanessa Amorim; Valadares, Marize Campos

Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, Goiás, Brasil.

The use of 3D models has been growing in recent years,
due to the demand for new methodological approaches
to toxicology that can mimic microarchitecture,
physiology and biochemistry of biological tissues in a
better way than 2D models. The development of these
models is based on the use of cells and scaffold similar
to the extracellular matrix, which provides an ideal
environment for cell proliferation, differentiation and
functioning. The extracellular matrix is an​​ essential
component for the construction of the 3D model and
is usually composed of biocompatible biomaterials
from synthetic or natural sources. As it is more
abundant in the extracellular matrix of mammals,
the collagen type 1 is widely used. Of the commercial
sources of collagen type 1, rat tail tendon stands out,
which has disadvantages such as high cost, ethical
issues and for being an isolated component, which
does not completely mimic the original complexity
of the matrix. The use of Decellularized Extracellular
Matrix hydrogel from industrial waste may be a
more sustainable, low-cost alternative with greater
biometric potential. Based on this, the present study
aims to propose obtaining a hydrogel from a natural
source (bovine cornea extracellular matrix) for
application in 3D corneal models. Bovine eyes were
collected in a slaughterhouse in Goiania, Goias, Brazil
and the corneas were isolated in the laboratory. To
obtain the hydrogel, the cornea samples underwent
a decellularization process with Sodium Dodecyl
Sulfate (SDS), lyophilization, enzymatic digestion,
neutralization and for polymerization the hydrogels
were incubated at 37ºC. The decellularization process
in the decellularized extracellular matrix (dECM) and
in the dECM hydrogel were confirmed by histology
in Hematoxylin and Eosin (HE) and by Hoechst
staining. The preservation of matrix components
was qualitatively evaluated by histology with Azan
Blue stains for collagen and Alcian Blue stain for
glycosaminoglycans. The structure of collagen fibers
was observed in scanning electron microscopy (SEM).
To evaluate the amount of proteins, the BCA method
was used. The applicability of the hydrogel in cell
culture was evaluated in culture with HaCat cells
(human keratinocyte), compared to a 3D corneal
model developed in the TOXIN/UFG laboratory by
Da Silva (2019), in which murine collagen type 1
was used. Cultures were maintained for 21 days and
subsequently evaluated for viability using the MTT
reduction method and histological evaluation with HE
staining.Histological characterization showed that
decellularization was effective, due to the absence
of cells stained with HE and Hoechst in the dECM and
hydrogel models. Collagen was preserved, however
there was a slight loss of glycosaminoglycans. The
collagen fibers were presented in a randomized and
similar way between the native tissue, the dECM and
hydrogel, confirming the presence of collagen and
other proteins in large quantities. The hydrogel was
able to support cell culture, and previous results
showed that the culture remained stable until the
twenty-first day of culture and offered conditions
for cell proliferation, as observed for the 3D corneal
model based on collagen type 1. The bovine cornea
dECM hydrogel can be a natural biomaterial that can
be used for cell culture and 3D cornea models making,
and can contribute to the national production of
research inputs, reduction of animal experimentation
and to the growth of the area tissue engineering in the
country.
 35

Development of a methodology to
assess key event 2 to quantify NRF2
in cutaneous allergenicity
Pedralli, Bruna Cristiane Oliveira; Valadares, Marize Campos

Laboratory of Research and Education in In vitro Toxicology (ToxIn) –

Faculty of Pharmacy – Federal University of Goiás (UFG).

Introduction: when it comes to discuss about quantification. Therefore, the development of

regulation, modern toxicology aims to develop
several alternative methods based on human
physiology in order to generate more relevant data
about chemical substances when in contact with the
skin. Despite the countless discoveries already made
in the field of dermal sensitization being evaluated
with animal tests, the data obtained with in vitro
methodologies have allowed a greater recognition
of the 3R’s policy (reduce, replace and refine), and
consequently a significant evolution in the change
of guidelines in order to analyze chemical products
and substances. In addition, alternative methods
may effectively complement the analysis of potential
allergenicity, generating reliable data for safety and
risk assessment. The mechanistic understanding of
the AOP was of paramount importance for assessing
skin sensitization in vitro and for the development
of new OECD guidelines. This pathway is based on
four key events: formation of immunogenic hapten-
protein complexes or molecular initiation event;
activation of keratinocytes; dendritic cell activation
and T lymphocyte activation and proliferation.
Among the guidelines used to assess key events,
the KeratinoSensTM assay principle is the only OECD
validated test guide to assess the Keap1-Nrf2-Are
regulatory pathway, which is commonly activated
by contact allergens. Despite being a technique
validated by the OECD, the high costs can make
the method’s accessibility difficult, as it requires
the HaCaT strain containing the luciferase enzyme
gene and specific equipment for luminescence
non-animal methodologies which assess skin
allergenicity in a safe, effective and accessible way
are essential. Objective: The work aims to develop
a new methodology which evaluates key event 2 to
quantify the transcription factor Nrf2 involved in
the potential for skin allergenicity. Methodology:
Substances categorized as dermal sensitizers and
non-sensitizers according to the OECD were selected
to assess cytotoxicity and NRF2 activation. The
human keratinocyte line HaCaT without the luciferase
enzyme gene was also used. To assess cytotoxicity,
day 1 was chosen for plating at a density of 3x105
cells/ml and stored in a culture oven with controlled
humidity and temperature for 24 hours. On day 2,
exposure was performed at different concentrations.
On day 3, the results were read by flow cytometry
using the propidium iodide marker and the CV75 of
each analyzed substance was found. To assess the
activation of Nrf2, the same protocol for cytotoxicity
was performed, however, using the Nrf2 antibody.
Discussion/Conclusion: Preliminary results suggest
that flow cytometry showed satisfactory results
after exposure of substances in HaCaT cells through
quantitative measurement by detection of emitted
fluorescence. Non-sensitizing glycerol and lactic acid
were not statistically significant, whereas sensitizing
PPD and DNCB were statistically significant. This
condition was verified when ‘’p’’ was lower than 0.05.
All the analyzes were performed using the Graphpad
Prism 8.0 software. Acknowledgments: CAPES, CNPq,
Toxin e FINEP.
 36

Development of an ex vivo model to

evaluate pulmonary toxicity mechanisms
Furtuoso, Marcella Miranda Siqueira; Tavares, Kvetta Pinheiro Teixeira;
Pedralli, Bruna Cristiane Oliveira; Valadares, Marize Campos

Laboratory of Education and Research in In Vitro Toxicology, Tox In, Faculty

of Pharmacy, Federal University of Goiás, Goiânia, Goiás, Brazil.

Introduction: The main function of the respiratory
system is to promote gas exchange through
respiration, however, several toxic substances can
be inhaled causing irritation, inflammation, and
sensitization of the respiratory tract. Currently, safety
tests validated by international regulatory bodies to
assess inhalation toxicity are performed on animals,
demanding the development of innovative techniques
that can mimic the respiratory system for the
replacement, reduction, and refinement of the use of
animals. In vitro studies using monolayer A549, BEAS-
2B and Calu-3 cell lines are under development to
assess the toxicity of these substances. Furthermore,
three-dimensional (3D) models of human respiratory
epithelium are even better than the 2D ones, however,
these approaches are limited by not presenting
the complex architecture present in the human
respiratory tract for mechanistic studies. On the
other hand, human ex vivo models of Precision Cut
Lung Slices (PCLS) contain functional cells of the
lungs, extracellular matrix and mimic the cellular
biological environment, however, there is limited
human tissue availability, making its application
difficult. Regarding this, the porcine lung, a residue
of the food industry, has anatomical, physiological,
and cellular features like the human, presenting a
high potential of ex vivo model in PCLS for preliminary
tests in the evaluation of toxicity. Objective: To
develop and characterize an ex vivo porcine model
in PCLS to assess pulmonary mechanisms of toxicity.
Methods: The porcine lungs were donated by the
regional industry. The PCLS were obtained using the
Tissue Slicer DTK-3000W (Vibratome) equipment, at
a thickness of approximately 300µm to 500µm and
were cultivated in an air-liquid interface for twenty
days. To analyze the tissue viability, the tetrazolium
reduction method (MTT) was performed. Tissues were
processed and stained with hematoxylin-eosin for
histological analysis. Tissue exposure to aerosolized
paraformaldehyde by nebulization (Vitrocell® Cloud
12 exposure chamber) was performed on the fifth day
of cultivation. The presence of ROS was measured by
DCFH-DA staining and assessment of mitochondrial
activity was also measured by MITOTRACKER® labeling.
In addition, using indirect immunofluorescence,
caspase was evaluated. Results: Preliminary results
suggest that the ex vivo porcine model of PCLS in the
histological and MTT assays-maintained cell viability
and alveolar morphology for eight days, without signs
of tissue degeneration. Tissue exposure to aerosol of
paraformaldehyde, followed by assessment of ROS,
mitochondrial activity, and caspase were performed
to determine toxicity mechanisms. Discussion/
Conclusion: The ex vivo porcine model in PCLS can be
an important tool for the assessment of pulmonary
toxicity. Acknowledgments: CAPES, CNPq and FINEP.
 37

Dimethoate exposition: in vitro x in vivo study

Andrade, Ana Rosa Brissant; Carvalho, Deoclécio Lustosa; Souza, Asley Thalia Medeiros; Kishishita,
Juliana; Pimenta, Camila de Almeida Perez; Santana, Davi Pereira; Leal, Leila Bastos

Universidade Federal de Pernambuco (NUDFAC-UFPE), Recife, PE, Brazil.

Introduction: In developing countries, such as Brazil,
family farming is responsible for the income of more
than 70% of Brazilians employed in rural areas. This
type of agriculture is transferred between generations
and is seen by workers as safe and harmless, so that
often, in addition to failing to comply with health and
safety standards (indiscriminate use of pesticides,
without adequate protection measures), the extensive
contact of workers with different classes of pesticides
exposes them to greater risk. This fact is evidenced
by the records in the Notifiable Diseases Information
System (Sinan), where 84,206 cases of pesticide
poisoning were reported in Brazil between 2007 and
2015. Objective: In view of these aspects, the objective
of this study was to evaluate dermal absorption of
dimethoate, using in vitro and in vivo approaches,
considering the environmental conditions of work
in family agriculture. Methods: In order to evaluate
dermal absorption under typical working conditions,
dimethoate on clothing and stratum corneum (SC) was
measured in seven rural workers during application,
1 resident and 1 researcher who monitored all
applications. The evaluation was made by using eight
cotton patches fixed in specific places on workers’
clothing, and SC was collected in three different areas
by tape stripping. The in vivo dermal exposure and SC
penetration factor (PF) of dimethoate were calculated
according to the OECD guideline No.9 (1997) modified.
To correlate the results, the in vitro permeation test
(IVPT) was performed by using rat, pig and human
skin in Franz vertical diffusion cells with receptor
medium of 6.0 mL, stirring at 32 ± 1°C and 1.77 cm2 of
permeation area. 36 µL of the Dimexion® (600 µg/mL)
was applied and at 2, 4, 6, 8, 10, 12, and 24 hours, 1
mL of the receptor fluid was collected and replaced.
After 6 hours, the surfaces of the skins were cleaned
without stopping the test. The retention was analysed
in SC and in the rest of skin. The in vivo study and
use of human skin for IVPT was approved by CEP/
UFPE 5.007.282 as well as the use of rat skin (CEUA/
UFPE: 007/2021). The dimethoate was analysed by
validated LC-MS/MS method. Results: In this IVPT
study, excluding all tapes, the calculated absorption
data were 6.49 ± 0.04; 3.49 ± 0.29 and 1.79 ± 0.19 µg/
cm2 from rat, pig, and human skin respectively for
diluted product. The calculated absorption through
the human skin was 14,75%. However, according
to EFSA (2017), the default value to be used in the
absence of experimental data for this commercial
product is 70%. The mass balance was between 95
and 105%, demonstrating the reproducibility of the
methodology. The dimethoate extracted from cotton
patches in different regions of body was between
0.38 and 318,155.14 ng/cm2. The calculated dermal
exposure was between 0.27 and 1,353.04mg/body
region and the PF (%) was between 0.06 and 25.24
in the forearms and between 0.44 and 14.44 in the
back of the neck. Discussion/Conclusion: Except for
the researcher, none of the workers in the study wore
the appropriate clothing as described in the product
package insert. These data reveal great concern about
the family farm workers who have been routinely
working without the proper use and care of safety
equipment specifically for “pesticide applicators”.
Further studies performed with other pesticides
with different characteristics will contribute to the
understanding of their transport through the skin.
Acknowledged: CNPq and FACEPE.
 38

Effects of Myrcia pubipetala Miq (Myrtaceae)

extract on innate inflammatory response
Pacassa, Pâmela1; Benvenutti, Larissa2; Echterhoff, Marcelo Rodrigo Franke3; Lopes, Bruna Gonçalves3;
Quintão, Nara Lins Meira2; Debiasi, Michele Alberton1; Santin, José Roberto2; Machado, Isabel Daufenback1
1
Postgraduate Program in Biodiversity, Fundação Universidade Regional de
Blumenau, Blumenau/SC, Brazil; 2 Postgraduate Program in Pharmaceutical Science,
Universidade do Vale do Itajaí, Itajaí/SC, Brazil; 3 Postgraduate Program in Chemistry,
Fundação Universidade Regional de Blumenau, Blumenau/SC, Brazil.

Background: Myrcia pubipetala, an endemic plant
present in Brazil, which has few studies, is part of
the Myrtaceae family, a large group with important
specimens in popular use, such as “jaboticaba”,
“goiaba” and “eucalipto” and others that have reported
anti-inflammatory activity. Studies on M. pubipetala
are scarce, and this work is the first to report the
biological activity of the M. pubipetala extract.
Objective: Evaluate the innate immune response
involved in the in vivo and in vitro anti-inflammatory
activity of M. pubipetala Crude Hydroalcoholic Extract
(CHE-MP). Methods: The CHE-MP was obtained from
the maceration of dried leaves in 70% alcohol, and
dried so that the biological activity could be described.
The in vivo anti-inflammatory activity was evaluated
by the air pouch method, which was approved by
the ethics committee of Universidade Regional de
Blumenau under protocol 009/20. The exudate
was quantified as well as the total and differential
leukocytes, nitric oxide and cytokines (interleukin
– IL-1β, IL-6 and tumor necrosis factor – TNF) and a
histological analysis of the tissue that lines the pouch
was performed. The in vitro part used RAW 264.7
cells where nitric oxide and cytokines (IL-1β, IL-6 and
TNF) were measured in the culture supernatant and
cell viability and expression of adhesion molecules
(CD62L [L- Selectin] and CD18 [β2-Integrin]) on the
cell surface. For the toxicological analysis was
performed MTT and HET-CAM assay. Results: CHE-
MP inhibited leukocyte migration as well as reduced
polymorphonuclear migration at all doses (3, 30 and
300 mg/kg v.o.), which was confirmed by histology.
At doses of 30 and 300 mg/kg, the reduction in
exudate volume and nitric oxide concentration was
highlighted. As for immunomodulation, there was
a reduction in the expression of IL-1β, IL-6 and TNF.
Cell viability in vitro demonstrated that CHE-MP does
not show cytotoxicity at the doses tested (1, 10 and
100 µg/mL). CHE-MP inhibited the increase in nitric
oxide at all doses tested, and the dose of 100 µg/mL
showed a statistical difference compared to the dose
of 10 µg/mL. There was a reduction of IL-1β, IL-6 and
TNF in the culture supernatant, confirming the results
obtained in vivo. There was lower expression in CD18
in cells treated with doses of 10 and 100 µg/mL. In
the both toxicological assays, CHE-MP preserve the
cell viability and preserve the integrity of the blood
vessel in HET-CAM. Conclusion: The results obtained
confirm that M. pubipetala has immunomodulatory
activity, as well as anti-inflammatory activity in vivo
and in vitro and the extract provides security to use.
Acknowledgments: Fundação Universidade Regional
de Blumenau – FURB and Fundação de Amparo à
Pesquisa e Inovação do Estado de Santa Catarina –
FAPESC.
 39

Effects of pulmonary inhaled

irritants on 3D alveolar model
Furtuoso, Marcella Miranda Siqueira; Tavares, Kvetta Pinheiro Teixeira; Valadares, Marize Campos

Laboratory of Education and Research in In Vitro Toxicology, Tox In, Faculty

of Pharmacy, Federal University of Goiás, Goiânia, Goiás, Brazil.

Introduction: Innovation on pharmaceutical fields has
been developing a series of new products concerning
the most diverse purposes that demand toxicological
investigations. One of the main routes of exposure to
chemicals and/or particles is the human respiratory
tract. The vulnerability of the lung occurs due to its
external connection susceptible to environmental
exposure, along with systemic connection, with
proximity to the brain and heart. Regulatory agencies
seek to expand existing information about inhalation
toxicology of products in the global market. Currently,
all inhalation toxicity investigations are performed
through in vivo tests. With the advancement of science
and the New Approach Methodologies (NAMs), which
mimics regions of the human respiratory system
have been developed. According to this, our group
developed an in-house 3D model, using the A549
human lung alveolar cells, that can mimic human
exposure through cultivation at an air-liquid interface
for toxicity assessments. Objective: To investigate the
effects of exposing lung cells to pulmonary irritants
directly on the tissue, using the in-house developed
lung model. Methods: The lung model was developed
in-house through the cultivation of the A549 cell
in an air-liquid interface. The test substances were
prepared according to the stablished concentrations
and physicochemical properties of each compound.
The toxicants from different categories of the
Globally Harmonized System of Classification and
Labelling of Chemicals (GHS) were added directly on
the tissue, and PBS was used as a negative control.
The tissues were exposed for 3 hours to toxicants
and incubated at 37°C in a humidified atmosphere
of 5% CO2, after which they were evaluated by the
MTT assay. Morphological evaluation of the tissue
developed in-house was also carried out through
histological processing. Results: The results showed
that using the in-house pulmonary model developed
was possible to categorize the pulmonary irritant
toxicants used according to the GHS classification and
also, allowed perform prediction for inhalatory LC50
concentrations in animals. Discussion: The model
showed to be useful to investigate inhaled toxicants
like the commercially available models. In addition,
the model can predict the LC50 value in animals for
inhalation toxicity tests, it can also drastically reduce
the number of these tests in 80%, and it may reach
100% for tests with corrosive substances. Conclusion:
Based on the results obtained, the in-house developed
pulmonary biomimetic model has the potential to be
a useful tool in acute inhalation toxicity tests and
highlights the importance of NAMs instead of using
models concerning animals. Acknowledgments:
CAPES, CNPq and FINEP.
 40

Efficiency of the Reconstructed Human

Epidermis (RHE) in the classification of
biological product by the in vitro irritation
and corrosion tests (OECD 439 and 431)
Bechtold, Bruna Assunção1; Cianci, Julio Cesar1; Coelho, Maria Paula Mancini1; Costa, Larissa
Gabrielli1; Dakic, Vanja2; De Vecchi, Rodrigo2; Fava, Luis Paulo1; Padua, Amanay Sousa1;
Santana, Thatiane Nunes1; Silva, Priscilla Muniz Ribeiro1; Vecina, Juliana Falcato1
1
Mérieux NutriScience / Bioagri Laboratórios Ltda, Piracicaba-
SP, Brazil; 2 Episkin Academy, Rio de Janeiro-RJ, Brazil

Introduction/Objective: Following the global trend
and especially Art 6 of RDC 294 (July 29, 2019), in which
alternative methods must be presented for regulatory
purposes, we evaluated the in vitro irritation (OECD
439) and corrosion (OECD 431) methods to classify
biological products, using reconstructed human
epidermis (RHE, SkinEthicTM) provided by Episkin
Brasil. This model represents the in vitro target organ,
that consists of normal human keratinocytes cultured
to maturity of the epidermis. For this reason, it has a
histological morphology comparable to in vivo tissue.
Some care must be taken when handling the biological
product in order to avoid cross contamination or loss
of effectiveness of the product. Methods: A biological
product has been tested according to OECD 439 and
OECD 431. For the Irritation test (OECD 439) the tissues
were firstly exposed to 16 mg or 16 µL of each item,
according its physical characteristics, concurrently
to negative (NC=PBS) and positive (PC=SDS, 5%)
controls for 42 minutes, then washed and incubated
for an additional 42 hours at 37 °C, recovery period.
Following this post incubation period, the tissues
were incubated with a MTT (1 mg/mL) solution for 3
hours. For the Corrosion Test (OECD 431), the tissues
were firstly exposed to 20 mg or 40 µL of each item,
according its physical characteristics, concurrently
to negative (NC= sterile water) and positive (PC=
KOH, 8N) controls for 60 and 3 minutes, then washed
and incubated with a MTT (1 mg/mL) solution for
3 hours. After 3 hours of incubation with MTT, the
formed formazan crystals were solubilized with
isopropanol. The optical density was evaluated at 570
nm. Results/Discussion: Viability results obtained in
vitro (mean ± SD) were NC: 100 ± 3.27; PC: 1.92 ± 0.07;
Biological Product: 104.05 ± 7.92 for the irritation test
(OECD 439). The viability obtained were NC: 100 ±
7.90; Biological Product: 99.62 ± 1.19 for 3 minutes of
exposure and NC: 100 ± 1.65; PC: 2.75 ± 0.13; Biological
Product: 94.63 ± 0.46 for 60 minutes of exposure for
the Corrosion test (OECD 431). Our results were within
the acceptance criteria of the guides and our historical
data. As expected, the positive control demonstrated
a significant relative cell viability reduction when
compared to the negative in both tests. This fact
is explained by the cytotoxic action of SDS and
KOH. In addition, according to the results obtained,
the Biological Product was classified according
to UN GHS Category as “No Category” and “Non-
Corrosive”, corroborating with in vivo classification
tests (Pubchem, 2021; Meleney and Johnson, 1949).
Conclusion: In view of the results, the use of RHE for
classification of biological products is valid, as long as
proper precautions are taken to obtain quality results.
Acknowledgments: -Episkin, Rio de Janeiro-RJ, Brazil;
-Bioagri Laboratórios Ltda (Merieux NutriSciences),
Piracicaba-SP, Brazil.
 41

Embryotoxicity of triclopyr in early

development of zebrafish (Danio rerio)
Andrade, Ítalo Bertoni Lopes1,2; Sales, Bianca Camargo Penteado1,2; Peixoto,
Paloma Vitória Lima1,2; Viriato, Cristina2,3; Pereira, Lílian Cristina2,4
1
São Paulo State University (Unesp), Medical School, Botucatu; 2 Center for Evaluation of
Environmental Impact on Human Health (TOXICAM); 3 São Paulo State University (Unesp), Institute
of Biosciences, Botucatu; 4São Paulo State University (Unesp), School of Agriculture, Botucatu.

Background: Triclopyr is an auxin-like herbicide used
to control a wide range of weeds in food crops and
pastures. Triclopyr is known to pollute the aquatic
ecosystems. Its potential effects on the aquatic
environment and human health, and the lack of
monitoring of triclopyr in environmental compartments
are an emerging concern. In this study, we used
zebrafish as a biomarker of ecotoxicity to assess the
effects induced by triclopyr at concentrations already
detected in aquatic systems. Objective: The aim of
the current work was to evaluate the mechanisms of
the acute toxicity of triclopyr in early development
of zebrafish. Methods: For all tests, we used six
environmentally realistic concentrations of triclopyr:
0.5, 1, 5, 10, 50 and 100 µM. The embryotoxicity
assessment was based on the Fish Embryo Acute
Toxicity (FET) test (OECD TG236), extended up to 144
hours. We determined the LC50, EC50 and teratogenic
index for triclopyr, based on the lethal and sublethal
effects induced in embryos and larvae within 96
hours of exposure. Acetylcholinesterase activity,
based on Ellman’s essay adapted to 96-well plate,
was used as a neurotoxicity biomarker for triclopyr.
The results were evaluated by analysis of variance
(ANOVA) followed by the Dunnett test. Results with
p=<0.05 were considered statistically significant.
Results: In the FET test triclopyr significantly induced
lethal effects (coagulated embryos and lack of
heartbeat) in zebrafish embryos at all concentrations
tested. The LC50 50.07 µM and LC50 47.74 µM for
triclopyr was defined at 96 and 144 h, respectively.
In addition, it was observed sublethal effects such as
pericardium and yolk sac edema, non-hatched eggs,
and uninflated swim bladder in zebrafish embryos
exposed to at all non-lethal concentrations tested of
triclopyr (0.5-50 µM), up to 144 hours. At 10 and 50
µM of triclopyr was observed teratogenic effects such
as scoliosis and malformation of the head and eyes. A
significant decrease in larval length was observed for
all tested concentrations of triclopyr, when compared
to the negative control. The EC50 38.77 µM at 96 h
was defined based the effects observed and the
teratogenicity index of 1.29 was defined for triclopyr.
Acetylcholinesterase activity did not change after
exposure to triclopyr. Discussion/Conclusion: Even
at relatively low concentrations, the present study has
shown that triclopyr can cause embryotoxicity in early
development of zebrafish by inducing both lethal and
sublethal effects. Furthermore, triclopyr has proven
to be a teratogenic compound for zebrafish, which
can directly and indirectly impact the survival and
maintenance of the fish population. Also, triclopyr
showed no neurotoxic activity at the sub-lethal
concentrations tested. Although further studies are
needed, taken together and from an ecotoxicological
perspective, our results suggest the impact on
the health of other aquatic organisms. Keywords:
Triclopyr; herbicide; contaminant of emerging
concern; toxicity; fish. Acknowledgments: We thank
the São Paulo Research Foundation (Fundação de
Amparo à Pesquisa do Estado de São Paulo – FAPESP:
Processes No. 18/00229-1, São Paulo, Brazil) for the
financial support.
 42

Evaluation of ocular irritation potential

of raw materials presented in xampus
formulations using non-animal methodology
Rocha, Daniela Barbosa; Almeida, Jéssica Azarias; Andrade, Wanessa Machado

Pontifícia Universidade Católica de Goiás - PUC GOIAS.

Introduction: Cosmetic products must be safe
under normal conditions or have predictable risks,
remaining within an adequate safety margin. As they
are freely accessible, cosmetics can accidentally
permeate barriers, being partially or totally absorbed
and causing possible signs of irritation such as
eye irritation, so it is necessary to guarantee their
safety when in contact with the eyes, and must be
approved in tests of eye irritation. In the 80’s the
development of alternative experimental models
for the cosmetic area was started, replacing the use
of laboratory animals. Among these methods, the
BCOP test is an in vitro test using corneas isolated
from the eyes of cattle obtained as a by-product
from slaughterhouses to assess the potential ocular
irritation of a test substance. Objective: This work
aims to support the development of alternatives to
animal experimentation by assessing the potential
for ocular irritation. Methods: The substances used in
this work were selected according to their frequency
of occurrence on shampoo labels marked in Brazil.
The four substances most commonly found in the
composition of the different shampoos evaluated
were cataloged according to their pharmacotechnical
characteristics in shampoos, usual concentration
and ocular toxicological profile. The BCOP assay was
performed according to OECD 437. Results: Raw
materials EDTA 20%, Cocoamidopropyl betaine 20%,
Cocoamidopropyl betaine 6% and Sodium lauryl
ether sulfate 30% were classified as No prediction
can be made, because IVIS was higher than 3, IVIS
10.6; 5.8 and 10.5 respectively. While EDTA 0.1%,
Silicone DC 200/350 100%, Silicone DC 200/350
40% and Sodium Lauryl ether sulfate 10% were
classified as No Category because they presented
IVIS less than 3, IVIS 1.1; 0.7; 2.0 and 1.2 respectively.
Discussion/Conclusion: The various limitations
of in vivo methods in force, such as ethical issues,
animal management, biological variability and the
worldwide trend to replace the use of animals in
experimentation demonstrates the current need for
the development and implementation of alternative
in vitro methodologies to enhance performing tests
with greater accuracy, deadlines and lower costs
than tests performed on animals. From the studies
and technologies developed for the cosmetic tests
and worldwide mobilization for the development of
alternative methodologies, it is possible to envisage
the substitution or reduction of the use of animals
in scientific experimentation. Acknowledgments:
This research was supported by the Laboratory of
Teaching and Research in Toxicology In Vitro (Tox In)
of the Faculty of Pharmacy of the Federal University
of Goiás (UFG) and by the Faculty of Pharmacy of the
Pontifical Catholic University of Goiás (PUC-GO).
 43

Evaluation of the cytotoxicity and the anti-

melanogenic activity of Selenium and Zinc
Silva, Ana Cléia Cardoso; Ribeiro, Milena Mariano; Irioda, Ana Carolina; Oliveira, Cláudia Sirlene

Programa de Pós-graduação Strictu sensu Aplicada à Saúde da Criança e do Adolescente. Instituto de

Pesquisa Pelé Pequeno Príncipe, Curitiba, PR, Brazil; Faculdades Pequeno Príncipe, Curitiba, PR, Brazil

Introduction: Melasma is characterized by dark spots
on the skin, resulting from increased melanogenic
activity that alters epidermal pigmentation. The cause
of melasma is multifactorial, the most common being
exposure to the sun, pregnancy, and birth control pills.
There are several compounds used in the treatment
of melasma, such as kojic acid and hydroquinone,
which can cause side effects. In this context, the
trace elements, zinc (Zn) and selenium (Se) must be
explored. Zn is a component of superoxide dismutase
and metallothionein, both antioxidant molecules, in
addition to displacing more dangerous metal ions,
which cause the formation of free radicals. Se is a
component of important selenoproteins, for example,
glutathione peroxidase, which has antioxidant
activity. Based on the above data, it is of scientific
relevance to analyze the influence of Zn and Se on the
pathophysiology of melasma. Method: The research
was carried out with SK-MEL-28 (human melanoma)
and B16F10 (mouse melanoma) cells. The cytotoxicity of
zinc chloride, sodium selenite and diphenyl diselenide
was evaluated by colorimetric MTT assay. About
2.5x10⁴ cells per well were seeded in 48-well plates;
then, the cells were exposed to zinc chloride (1-1000
µM), to sodium selenite (1-1000 µM) and to diphenyl
diselenide (1-1000 µM) for 48h. After exposure, 10
µL of MTT (5 mg/mL) were added to the cells and
incubated for 3h at 37ºC. After the supernatant was
discarded and 100 μL of DMSO was added. Cells were
kept shaking for 30 minutes at room temperature.
Absorbance was measured at 570 nm. The melanin
content of the B16F10 cell line was also verified. Cells
(2 x 104/well) were plated in 24-well plates for 24h.
Afterwards, the cells were incubated in the presence
of α-MSH hormone (200 nM) and the compounds
sodium selenite (5 µM), diphenyl diselenite (1 µM),
and zinc chloride (100 µM) for 48h. After incubation,
the cells will be washed with phosphate saline buffer
(PBS), dissolved in 200 µL, then 500 uL of NaOH with
10% DMSO was added, incubated at 80 °C for 1h, then
the absorbance was reading was at 490 nm. Statistical
analyzes were performed on GraphPad Prism 6
(version 6.01, GraphPad Software, Inc., USA). Result:
One-way ANOVA revealed effect of sodium selenite,
zinc chloride and diphenyl diselenide on SK-MEL-28
cell viability. Sodium selenite caused a decrease in
cell viability from the concentration of 10 µM. Zinc
chloride reduced cell viability from the concentration
of 300 µM; and, diphenyl diselenide caused a decrease
in cell viability from the concentration of 100 µM.
Based on the results observed in the SK-MEL-28 cells,
concentrations of 5 µM for sodium selenite, 1 µM for
diphenyl diselenide and 100 µM for zinc chloride were
selected to verify the absence of cell cytotoxicity in
the B16F10 cells (which will be used for testing anti-
melanogenic activity). One-way ANOVA revealed no
effect of sodium selenite and zinc chloride, at the
concentrations tested, on cell viability of the B16F10
cells; however, it revealed an effect of diphenyl
diselenide. At a concentration of 1 µM, diphenyl
diselenide caused a slight increase in cell viability
(~18%). For the melanin content, one-way Anova
revealed treatment effect. Cells exposed to zinc
chloride showed a significant 18% decrease in melanin
content when compared to the control. Conclusion:
Among the compounds tested, zinc chloride was the
most promising as an anti-melanogenic compound.
It is likely that it is inhibiting the tyrosinase enzyme,
further tests will be carried out to elucidate its
mechanism of action.
 44

Galleria mellonella as an Alternative Model

for the Assessment of Permeation and
Toxicity of Assets from Natural Sources
Silva, Samanta de Matos1,2; Singulani, Junya de Lacorte1; Carvalho, Angélica Romão1,2; Migliato,
Ketylin Fernanda3; Giannini, Maria José Soares Mendes1,2; Fusco-Almeida, Ana Marisa1,2
1
Department of Clinical Analysis, Laboratory of Mycology, School of Pharmaceutical Sciences,
São Paulo State University – UNESP, Araraquara, São Paulo, Brazil; 2 Community Service
Center – NAC - Laboratory of Alternative Methods for Bioproducts - FCF UNESP Araraquara
SP, Brazil; 3 Central Paulista University Center – UNICEP, São Carlos, São Paulo, Brazil.

Introduction Recent advances in legislation focus on
alternative methods to guarantee efficacy and safety,
following the 3Rs principles (reduce, replace, and
refine) of animal experimentation. Galleria mellonella
is an alternative widely used and established safety
and efficacy research model. This animal model is
mostly studied in the injectable route in studies of
pathogens-host interaction and chemical compounds
toxicity. Although it is a promising methodology due
to its cuticle composition of three layers, like human
skin, mostly composed of lipids, proteins, and chitin,
there is a lack of data that assess other administration
routes, such as topical that could be used as an in vivo
alternative animal model for absorption and toxicity
assays. Objective To remedy this lack and contribute
to the validation of G. mellonella as a model in
cutaneous absorption and toxicity studies, this study
aims to evaluate the absorption of gallic acid (GA), a
secondary plant metabolite with several biological
activities. Method The study was conducted using an
in vitro and in vivo toxicity testing platform, using three
alternative assays. The first uses GA concentrations
in human keratinocyte (HaCat), human dermal
fibroblasts (HDFa), and human liver (HepG2) cell lines
in the concentration range between 7.81 to 1000 µg/
ml. The second trial evaluated the toxicity of GA in C.
elegans in the same concentration ranges. Finally, the
third in G. mellonella using the injectable and topical
routes according to the OECD recommendations (404,
2002), by the “Acute dermal irritation/corrosion in
vivo method”, in concentrations of 261 to 920mg /kg.
Results The results of the in vitro assays showed that
HaCaT cells were more sensitive to GA (IC50 of 138.8µg/
ml), followed by HepG2 cells ( IC50 of 207.8 µg/ml),
and HDFa (IC50 of 247 µg/ml). For in vivo assays, GA
showed greater toxicity to C. elegans with LC50 values​​
of 16.08 µg/ml. For G. mellonella, the data showed
that this alternative animal has a high tolerance to
GA with LD50 values greater
​​ than 920mg/kg both by
the topical and injectable route, however, alterations
were observed in the humoral immune response of the
larvae, represented by an increase in melanization in
concentrations of 463 to 920mg/kg for the injectable
route and 695 to 920mg/kg for the topical route. In
addition, characteristic histopathological findings
were presented at the same concentrations for both
evaluated routes of administration. Discussion/
conclusion According to the literature, the tests
carried out on G. mellonella showed results equivalent
to those of mammals according to studies carried out
in mice and rats by the same routes of administration.
The literature does not point to physiological and
biochemical correlations between alternative and
conventional methodologies that could equate to
toxicity results. However, alternative animals are
proven methodologies for evaluating the toxicity of
chemical substances, as well as being predictive of
toxicity in mammals. In this way, the combination
of more than one model guarantees an increase in
the reliability of the safety of natural compounds,
promoting a reduction in research costs and an
improvement in ethical conduct in the world.
 45

Identification and quantification of

constituents in the fetal bovine serums
available in the market used in cell culture
Stival, Ana Clara Silva; Silva, Artur Christian Garcia; Valadares, Marize Campos

Laboratório de Ensino e Pesquisa em Toxicologia in vitro,

Universidade Federal de Goiás, Goiânia-GO, Brasil.

Introduction: Fetal Bovine Serum (SFB) is a complex
mixture of biomolecules in distinct concentration
and composition. The variation imposed on the
product is a result of the complexity involved from
its obtention, seasonality, dietary characteristics to
which the animals are exposed. The animal product
is the main source of amino acids, vitamins, trace
elements, hormones and factors for cell growth
and maintenance in vitro. The product can present
contamination, composition and concentration varied
in each batch and/or brand available, ethical factors
related to animal suffering and genetic characteristics
affecting cell differentiation. The variation found
and not routinely tracked, imposes an inconsistency
in cell culture with unreproductive results in
experimental studies. The lack of reproducibility of
already validated methods and the disparity between
the results described in the literature regarding in
vitro methodologies, has been related to the use of
SBF from different sources. Objective: Identify and
quantify the constituents of FBS available on the
market applied to cell culture. Methodology: Forty-
seven biochemical constituents estimated in the
composition of FBS were quantitatively evaluated.
Tests performed were enzymatic, colorimetric,
bromocresol biuret, chemiluminescence, UPLC, UV
kinetic and HPLC MS/MS to quantify SBF constituents.
Samples from each batch were analyzed in triplicate
and the results compared with those described in the
literature. Results: The composition of the SFB was
estimated based on literature data according to their
characteristics correspond to: 69% of proteins, 10% of
lipids, 10% of ions, 3% of sugars and 3% of vitamins.
The preliminary quantification of the SFB components
indicated a variation higher than 15%, when related to
the theoretical reference. For triglycerides, albumin,
and T4, the values ​​reached 17%, 148% and 25% of
difference in relation to the literature. The results
showed difference between the reference values ​​and
those found in the preliminary analyses. Discussion:
It is already described in the literature that inter-lot
factors alter the concentration of FBS constituents. The
identification and quantification of the components of
FBS can demonstrate that a biological product with no
standardization of the constitutions, such as FBS, can
interfere in cell growth and, consequently in the cell
culture results, with high impact in the reproducibility
of in vitro assays. Acknowledgment: CAPES, FUNAPE,
FAPEG, FINEP and CNPq
 46

Implementation of SkinEthicTM Human

Corneal Epithelium (HCE) in Brazil
Dakic, Vanja1,3; Mattos, Guilherme1,3; Rigaudeau, Anne-Sophie3; Garcia,
Cristina3; Bouez, Charbel4; De Vecchi, Rodrigo1,3
1
Episkin, Rio de Janeiro, Brazil; 2 Episkin, Lyon, France; 3 Ĺ Oréal Research and
Innovation, Rio de Janeiro, Brazil; 4 Ĺ Oréal Research and Innovation, Clark, USA.

Eye irritation data are essential for safety and
efficacy evaluation of topically applied products and
chemicals. In Europe, the full ban of animal testing
was implemented in 2013 and validated methods like
Eye irritation test (EIT, adopted into Test Guideline 492
of OECD) are highly used for chemicals classification.
Brazil and South America is going along with this trend
step by step, and the urge for robust alternative models
is increasing in the last few years. This work describes
the implementation of validated SkinEthicTM Human
Corneal Epithelium (HCE) model, that closely mimics
the histological, morphological, and physiological
properties of the human corneal epithelium. 15
chemicals were tested and correctly classified using
tissues produced in Brazil. The highly reproducible and
consistent results confirm the quality and robustness
of HCE model and methods. Moreover, recent
publications described the development of Time-to-
Toxicity (TTL) approach based on this model, showing
that the EIT is capable to distinguish chemicals that
do not require classification for serious eye damage/
eye irritation (no cat.), from chemicals that require
classification for eye irritation (Cat. 2), and serious
eye damage (cat. 1). With this update EIT replaces the
animal model (Draize test), increasing its usefulness
and making its availability a big priority in Brazil and
Sought American countries.
 47

In silico evaluation of erythrinic alkaloids

from Mulungu, Erythrina verna, in GABAA
β-3 receptor complexed with benzamide
Bairros, André Valle1; Nascimento, Marcelo Henrique Santana2; Paula, Fávero Reisdorfer3
1
Nucleus Applied to Toxicology (NAT) Federal University of Santa Maria
(UFSM); 2 NAT – UFSM; 3 Federal University of Pampa (UNIPAMPA).

Introduction: The mulungu, Erythrina verna, has
its bark used in folk medicine as a tea to treat mild
emotional disorders such as anxiety, agitation,
depression, panic attacks, epilepsy, and compulsion
due to its anxiolytic and hypnotic actions. Objective:
Evaluate the interactions of erythrinic alkaloids
present in mulungu tea with the amino acid residues
of the active site of the GABAA β-3 receptor and
prediction of toxicity through computer modeling.
Methods: The alkaloids selected for the study were
erythravine, erythrartine, 11-α-hydroxy erythravine,
11-α-hydroxy erysothrin, 11-hydroxy erythratidinone,
erisorthine-n-oxide, erysortrine, erythradine,
erythrartine-n-oxide and erythratidinone. For the in-
silico evaluation, molecular docking methodologies
were adopted using Spartan’08® software and
quantum chemistry methodologies, AM1 followed by
DFT B3LYP / with 6-311G data base in gas phase, for
geometry optimization and conformational analysis
of the erythrinic alkaloids. The iGemDOCK® software
was used for molecular docking, using the 3D model
of the benzamide-complexed GABAA β-3 receptor,
PDB ID 4COF. The alkaloids from the group mentioned
above and the diazepam molecule, used as a control,
were submitted individually for docking in the BEN
cavity of the 4COF model. For the prediction of
toxicological risks based on chemical structure, OSIRIS
Property Explorer® software was used. Parameters of
mutagenic, carcinogenic, irritant potential, effects
on reproduction, drug likeness and drug-score based
on molecular structure were evaluated. Results and
Discussion: In the prediction of toxicity risks, none of
the molecules presented a potential risk, in addition,
the drug likeness and drug-score parameters obtained
high ranking values, indicating a good potential of
the molecules as a drug. In the docking process, the
molecules were evaluated at the central molecular
site of interaction BEN, using the slow calculation
method with ten individual conformational poses. The
pharmacological interactions of hydrogen bonding,
Van der Waals and electrostatics, alkaloids and
diazepam were evaluated. The interactions occur
at the main amino acid residues of the receptor,
the main interaction is Van der Waals. All alkaloids
studied showed good interaction with the site of
interaction, with energy values -109.44 kcal for
erythrartine and -100.56 kcal for diazepam, the other
molecules showed values between -83.09 and -94.14.
Unlike the alkaloids that also showed hydrogen
interactions, diazepam only has Van der Waals
interactions. Conclusion: The in-silico evaluation
corroborates the benzodiazepine-like action of the
alkaloids, having interactions with the main amino
acid residues of the GABAA β-3 receptor like those
that occur with diazepam, justifying its anxiolytic and
hypnotic effects. In addition, the molecules present
in mulungu tea demonstrate the safety regarding the
toxicological risks of the molecules according to the
evaluated methodology.
 48

In silico pharmacokinetics and

pharmacodynamics prediction of resveratrol
and two glycosylated derivatives
Schimith, Lucia Emanueli1; André-Miral, Corrine2; Muccillo-Baisch, Ana Luiza1; Hort, Mariana Appel1
1
Programa de Pós-graduação em Ciências da Saúde, Faculdade de Medicina, Universidade
Federal do Rio Grande – FURG, Rio Grande, RS, Brazil; 2 Nantes Université, Unité en
Sciences Biologiques et Biotechnologies (US2B), UMR 6286, Nantes, France.

Background/Introduction: Resveratrol (3,4’,5- time presented by resveratrol was approximately 1.5

trihydroxy-trans-stilbene) is a natural stilbene found
mainly in grapes, peanuts, and wines. It is known for
having different biological activities as antioxidant,
anti-inflammatory, antibacterial, and, neuroprotective,
although their poor water-solubility and limited
therapeutic efficacy. Structural optimization via
glycosylation of molecules appears as an alternative
for this issue. Pharmacokinetic properties may be one
of the main causes of new drug candidates’ failures in
clinical trials. In silico prediction methodologies may
impact the drug development process, becoming a
crucial part of the drug and related target discovery
progress. Objective: We aimed to evaluate the
pharmacokinetics and pharmacodynamics profile of
resveratrol and two glycosylated derivatives through
in silico methods. Methods: Swiss ADME, ADMETlab,
and SwissTargetPrediction online platforms were used
to predict pharmacokinetics and pharmacodynamics
properties of resveratrol, polydatin (resveratrol-
3-O-β-ᴅ-glucoside), and resveratrol 3-α-glucoside
(resveratrol-3-O-α-ᴅ-glucoside). Results: The results
obtained from in silico analysis indicated that the three
compounds are highly absorbed in the gastrointestinal
tract when orally administered and showed a
bioavailability score of 55%. Additionally, no one of
them acts as a P-glycoprotein substrate, responsible
to affect the bioavailability of drugs. Resveratrol is
able to cross the blood-brain barrier (BBB), however,
its glycosylated forms showed no BBB permeability
limiting its central nervous system use. The half-
hours, slightly longer than polydatin and resveratrol
3-α-glucoside that exhibited a half-time of about 1.1
hours. Glycosylated forms showed no interaction with
cytochrome P450 enzymes, different from resveratrol
which can act as CYP1A2, CYP2C9, and CYP3A4 inhibitors
and as a substrate for CYP2A9 and CYP2D6 isoforms.
Druglikeness analysis was used to recognize how
similar the substances are to drugs already approved.
Resveratrol exhibited no violations of Lipinski’s Rule
of Five, in contrast to both glycosylated derivatives
that showed the number of hydrogen bond donors
exceeded as a violation. A range of possible molecular
targets was identified for resveratrol (69 targets),
polydatin (11 targets), and resveratrol 3-α-glucoside
(10 targets), including enzymes, receptors, and
transporters, being able to modulate several cell
signaling pathways. Discussion/Conclusion: Finally,
through in silico approaches, we obtained information
about the pharmacokinetics and pharmacodynamics
profile of resveratrol, polydatin, and resveratrol
3-α-glucoside. Thus, our study may guide future
investigations regarding the possible mechanisms of
action of resveratrol and its glycosylated derivatives
in order to contribute to diseases treatments.
Acknowledgements: This study was supported by the
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior – Brasil (CAPES) [Finance Code 001]; and the
Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) [research grant 423028/2018-9].
 49

In vitro oral and topic absorption toxicity

test standardization using 3D cell
cultures and microfluidic systems
Ganzerla, Melissa Dibbern1; Indolfo, Nathalia de Carvalho2; Arroteia,
Kelen Fabiola2; Figueira, Ana Carolina Migliorini1
1
Laboratório Nacional De Biociencias, CNPEM, Campinas, Brasil; 2 Natura Co, São Paulo, Brazil.

Organ-on Chip is a result of tissue engineer and
microfluidic convergency, acting as an effective
solution to pursue new methodologies for drug
discovery and personalized disease treatments. The
high cost of drug development demands the need to
develop more predictive tissue models using human
cells to determine drug efficacy and safety in advance
of clinical testing. However, a more predictive model
requires the integration of different tissues, dynamic
cell environments and cellular communication to
the expression of high-fidelity organ function. In
this context, the microfluidic technology is poised to
fill the gaps in drug screening by offering predictive
human tissue models with methods of sophisticated
tissue assembly. Here we propose a junction of
three different 3D tissue engineered cultures (skin,
intestine and liver) in a 3-organ-on chip microfluidic
device to propose a methodology to verify topic or
oral drug administration and liver toxicity. For this,
we developed models of human reconstituted skin,
intestinal barrier and liver spheroids, which were
deep characterized in terms of histology, morphology
and functionality. Our results show that our models
is functional and mimetites functions of the real
organs. The Chip integration of all the tissues on the
chip was well succeeded and improved viability of
the 3D cultures. After treatment with known toxic
substances and, we observed absorption of drugs,
which caused liver injury, as expected. In conclusion,
here we present a new methodology to screen liver
toxicity before animal testes in two contexts, oral and
topic administration of drugs. Acknowledgments –
CNPEM, NATURA, MCTI
 50

In vitro strategy to assess skin

irritation and sensitization potential
of a commercial formulation containing
the antiseptic chlorhexidine
Kawakami, Camila Martins1; Silva, Gustavo Henrique1; Moura, Kézia1; Pinheiro, Ana L.T.A.1; Pinheiro, Adriano
da Silva1; Eberlin, Samara1; Gaspar, Lorena Rigo2; Fuzinaga, Thais2; Vicente, Eduardo3; Facchini, Gustavo1

1
Kosmoscience Group, Valinhos, SP, Brazil; 2 School of Pharmaceutical Sciences
of Ribeirão Preto, University of Sao Paulo, Brazil; 3 School of Science and
Engineering/Chemistry Institute, Sao Paulo State University, Brazil.

Introduction: The recent scenario of COVID-19 has
resulted in an increase in hand cleansing products such
as soaps, synthetic detergents, antiseptic handwashes
and alcohol-based hand sanitizers. Consequently,
it has been observed an emerging of several skin
conditions including irritant contact (ICD) and
allergic contact dermatitis (ACD) due to the excessive
personal hygiene measures. These skin problems
may result from a cascade of events which involve
cell damage, inflammatory responses, cytokines
release and activation of dendritic and T-cells; the
latter being a key biological event underlying skin
sensitization. Chlorhexidine is an effective antiseptic
agent which is widely used in cosmetic, hospital
products and medicines. Although clinical studies for
chlorhexidine are common, the in vitro assessment
and the molecular mechanisms involved in the irritant
and allergic contact dermatitis are rarely reported.
Objective: Evaluate the skin irritation and skin
sensitization potential of a commercial formulation
containing the antiseptic chlorhexidine (1%) by in
vitro methods. Methods: The skin irritation potential
was evaluated according to OECD TG 439 through
the determination of cell viability in ex vivo human
skin fragments using the vital dye MTT. Additionally,
the inflammatory cytokine IL-1α was quantified.
For skin sensitization assessment, the in chemico
direct peptide reactive assay (DPRA) was performed
following OECD TG 442C, which evaluates the
cysteine- or lysine-containing peptide depletion using
high-performance liquid chromatography (HPLC). The
skin sensitization was also evaluated using the human
cell line activation test (h-CLAT, OECD TG 442E), which
quantifies changes of cell surface marker expression
(CD86 and CD54) on a human monocytic leukemia cell
line (THP-1 cells), using flow cytometry. Results: The
results of skin irritation (OECD TG 439) demonstrated
that the positive control LSS (5%) was considered
irritant since it reduced more than 50% in cell viability
when compared to non-treated control. On the other
hand, the product containing chlorhexidine (1%)
was classified as non-irritant, since they presented
cell viability of approximately 73.55%. In addition,
treatment with the product assessed did not promote
a significant increase in the inflammatory cytokine
IL1-α release when compared to baseline control.
The in chemico DPRA assay demonstrated that, as
expected, the positive control dinitrochlorobenzene
(DNCB) presented positive prediction for skin
sensitization. The ingredient and the product
assessed also presented positive prediction for skin
sensitization (low reactivity) according to OECD TG
442C. Finally, the results from h-CLAT assay showed
that the positive control DNCB presented relative
fluorescence intensities (RFI) ≥ 150 for CD86 and
≥ 200 for CD54, which classifies this substance as
sensitizing, according to OECD TG 442E. Moreover, the
product containing chlorhexidine (1%) also presented
sensitizing potential since RFI for CD86 were above
the acceptable value for non-sensitizing substances
(on going experiment). Conclusion: The results
showed that the product containing chlorhexidine
(1%) did not present potential for skin irritation. In
addition, it demonstrated positive prediction for skin
sensitization potential, for both in chemico DPRA and
in vitro h-CLAT assays. Further studies should be
conducted with this hand hygiene products category
to better understand the molecular mechanisms
involved in the irritant and allergic contact dermatitis.
 51

Inflammatory alterations triggered by

respiratory sensitizers in human dendritic cells:
providing evidence to support the chemical
respiratory allergy mechanistic background
Mendonça, Izadora Caroline Furtado; Silva, Artur Christian Garcia;
Carvalho Filho, Sérgio de Morais; Valadares, Marize Campos1

Laboratory of Research and Education in In vitro Toxicology (Tox In),

Faculty of Pharmacy, Federal University of Goiás.

Introduction: Chemically-induced respiratory TNF-α) in both supernatant and lysate samples

sensitization is an immune-mediated response
caused by repetitive exposures to low molecular
weight (LMW) allergens and consequently the
development of clinical symptoms comprising allergic
asthma and rhinitis. However, there are no in vivo
or in vitro methods for predicting pre-clinically the
respiratory sensitization potential of LMW chemicals.
One of the challenges concerning this scenario is
the lack of knowledge related to the immunological
mechanisms involved in respiratory sensitization.
Regarding contact dermatitis, for instance, it is
known that dendritic cells (DCs) induce a Th1-type
response mediated by T lymphocytes. On the other
hand, although it is well documented that chemical
respiratory allergens can trigger a Th2 reaction even
after dermal exposure, the mechanisms underlying
this immunological outcome are still not fully
understood. Given the mandatory role of DCs in linking
the innate and adaptative immune responses, we
investigated the inflammatory alterations triggered
by LMW respiratory allergens in this cell type, aiming
to subsidize the development of in vitro methods for
predicting chemical respiratory allergy potential.
Objective: We proposed using the THP-1 monocytic
dendritic cells model to investigate the inflammatory
profile, maturation, and activation of DCs after
exposure to respiratory sensitizers. Methods: In
the first stage, we evaluated the cytotoxicity of
seven known respiratory sensitizers (Chloramine-T,
Piperazine, Maleic Anhydride, Cyanuric Chloride,
Trimellitic Anhydride Chloride, Trimellitic Anhydride,
and Glutaraldehyde) using the MTT reduction assay
24h after exposure for further determination of the
80% cell viability concentration (CV80). Thus, cells
were exposed to respiratory sensitizers CV80 for 24
hours, following the quantification of inflammatory
cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12p70, and
using the Cytometric Bead Array (CBA) technique.
Moreover, THP-1 dendritic cells underwent flow
cytometry analysis to verify the expression of surface
activation biomarkers (CD86, HLA-DR and CD11c)
subsequently the respiratory allergens exposure.
Results: The results demonstrated that most LMW
respiratory sensitizers increased the CD86 and HLA-
DR activation/maturation biomarkers expression by
THP-1 cell line. Moreover, the surface integrin CD11c
was upregulated by all evaluated test chemicals.
Regarding the inflammatory profile, five of the seven
allergens triggered a significant production increment
of IL-6 in both cell lysate and supernatant, meanwhile
increased IL-8 expression was observed just for three
evaluated chemicals. Discussion/Conclusion: The
obtained results support the hypothesis that exposure
to respiratory allergens can promote alterations in
DCs maturation/activation status, as well as trigger
pro-inflammatory changes in these cells, highlighting
the increased expression of CD11c surface biomarker
and IL-6. The literature previously reported that the
increase in IL-6 production by pulmonary CD11c+
DCs is linked to a polarization of the immune
response towards a Th2 profile, suppressing the
Th1 response that is regularly observed considering
dermal sensitizers exposure and contact dermatitis
progression. Therefore, evidence was found to
support the elucidation of chemical respiratory allergy
pathogenesis, as well as give mechanistic background
to allow the differentiation between dermal and
respiratory sensitizers regarding the seeking of Th2
and Th1 responses, respectively. Aknowledgements:
Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) e
Financiadora de Estudos e Projetos (FINEP).
 52

Influence of a combination containing

UV filters and insect repellent IR3535
in photoinduced processes
Gluzezak, Ana Júlia Pasuch1; Kawakami, Camila Martins1; Tavares, Renata Spagolla Napoleão1;
Fuzinaga, Thais Yume Toriy1; Maria-Engler, Silvya Stuchi2; Gaspar, Lorena Rigo1
1
School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São
Paulo, Brazil; 2 School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.

Despite the fact that UV filters are considered safe to
use, there are few reports about their skin sensitizing
and photosensitizing potential. The majority of tests
are performed in monolayers or through photopatch
test reactions. Recently, OECD published TG 498
addressing in vitro phototoxicity (photoirritation)
using a reconstructed skin model, but there are still
no validated assays using the 3D model to assess skin
sensitization and photosensitization. In recent years,
the concomitant use of insect repellents, mainly DEET
and IR3535, with UV filters has increased due to the
high incidence of UV radiation and a higher occurrence
of mosquito-borne diseases such as dengue, Zika and
Chikungunya. Therefore, the purpose of this study
was to evaluate the phototoxic and skin sensitizing/
photosensitizing potential of the combination of UV
filters (avobenzone, ethylhexyl methoxycinnamate
and octocrylene) with the insect repellent IR3535 (ethyl
butylacetylaminopropionate), using a reconstructed
human skin model. This combination was submitted to
phototoxicity tests, according to OECD TG 498 (OECD,
2021) and to skin sensitization and photosensitization
tests (IL-18 analysis) in a reconstructed human skin
model, exposed or not to 6 J/cm2 of UVA radiation.
Among the results obtained, the combination
containing UV filters and IR3535 proved to be non-
phototoxic, with lower reduction in cell viability
(Δ: 3.5%) when compared to non-irradiated tissue
treated with the same concentrations of substances,
following the international recommendations for the
assay and OECD TG 498, which indicate a cut-off of
30%. Furthermore, the combination did not present
photosensitizing potential, since it did not promote a
statistically significant reduction in IL-18 production
after irradiation and presented a low stimulation
index, IL-18 SI (Irr/non-irr): 0.9, which according to
Galbiati et al. (doi.org/10.1016/j.tiv.2013.06.008) is not
considered a photoallergen (cut-off: SI > 1.3). According
to the ECHA (European Chemicals Agency) and HSDB
(Hazardous Substances Data Bank) platforms, IR3535
has no evidence of sensitizing or photosensitizing
potential, while avobenzone, alongside with other UV
filters, continued to be the most common allergens
that elicited photopatch test reactions. To conclude,
the 3D model used in this study proved to be promising
in the evaluation of phototoxicity and sensitization/
photosensitization of the combination containing UV
filters and insect repellent IR3535, which so far has
proven to be safe for use. Acknowledgments: This
work was developed within the framework “Fundação
de Amparo à Pesquisa do Estado de São Paulo”
(FAPESP, Brazil), Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES, Brazil) and
“Conselho Nacional de Pesquisa” (CNPq, Brazil).
 53

Influence of extracellular matrix

organ-specific in behavior and cellular
functionality in HEPG-2 cell line
Santos, Thaís Rosa Marques1; Borges, Amanda Cecília Guimarães1; Santos, Jordana
Andrade1; Silva, Artur Christian Garcia1; Marize Campos Valadares2
1
Laboratory of Education and Research in In Vitro Toxicology, Faculty of Pharmacy, Universidade
Federal de Goiás, Goiânia, Goiás State, Brazil; 2 Laboratory of Education and Research in In Vitro
Toxicology, Faculty of Pharmacy, Universidade Federal de Goiás, Goiânia, Goiás State, Brazil

Introduction: The number of molecules applying
for new drugs discarded in large drug development
studies justifies the efforts of the scientific community
to develop more predictive models for liver toxicity
assessment. The lack of biosimilar models capable of
predicting these damages has led to the development
of models on different fronts, such as bioengineering
to recreate the hepatic parenchyma’s complex
organization or even using liver-on a-chip to simulate
the elimination flow of newly metabolized xenobiotics.
As an additional strategy, the use of an organ-
specific matrix to construct more predictive liver
models is a strategy to stimulate the differentiation
of cellular phenotype and the functionality of already
established liver lines. Objective: The aims to evaluate
the influence of organ-specific extracellular matrix
and bioengineering resources on the functionality of
HepG2 liver cells, creating a model for evaluating liver
metabolism in vitro closer to the relevant functional
subsets in liver biology using, for this, the complex
extracellular matrix from the porcine liver of spoil
from the food industry. Methods: The complex
hepatic extracellular matrix was derived from the
decellularization of swine liver and the chemical
digestion that followed. The resulting product was
characterized by biochemical and kinetic endpoints
using histological and biochemical methods. The
proposed model was used using the sandwich
configuration and the hepatic lineage was used HepG2.
Increased liver metabolism due to the organ-specific
microenvironment, cell morphology, formation of
liver canaliculi, presence of MRP-2 receptors, and
cytochrome p-450 activity were assessed. Results:
The obtained product and the biochemical parameters
defined show that obtaining the hepatic extracellular
matrix derived from the porcine liver is feasible, as
well as suitable for cell culture, and can be used as
a scaffold for models of bioconstructed liver tissue.
The cell morphology of the HepG2 line cultivated in
a complex organ-specific matrix exhibits polygonal
form, clear cytoplasm, and structures like stable
canaliculi, constating to HepG2 line cultivated without
an extracellular matrix. Discussion/Conclusion:
Changings on the cellular morphology of the HepG2
scanner, when grown in an organ-specific matrix, can
indicate an increase in metabolic activity compared
to single-layer culture. The presence of canaliculi
and elimination of specific substrate via MRP-2
are indicators of biliary activity, fundamental for
metabolic activity. Furthermore, the acquisition and
use of the swine liver-derived extracellular matrix is
consistent with the 3R principles. Acknowledgments:
CAPES, CNPq, FINEP, FUNAPE, and FAPEG.
 54

Macroscopic evaluation of Score®

cytotoxicity by the Allium cepa test
Massucato, Lucas Eduardo1; Siqueira, Gabriella Ferreira1; Motomura, Larissa Tiemi Akamine1;
Lini, Renata Sano2; Souza-Kaneshima, Alice Maria3; Mossini, Simone Aparecida Galerani1,2
1
Laboratory of Toxicology, Department of Basic Health Sciences, State University of
Maringá, Paraná, Brazil; 2 Postgraduate Program in Bioscience and Physiopathology,
State University of Maringá, Paraná, Brazil; 3 Laboratory of Pathology, Department
of Basic Health Sciences, State University of Maringá, Paraná, Brazil.

Background: The growing and excessive use of
pesticides has made the Brazilian agricultural
production process increasingly dependent on these
products to increase food production. According to
IBAMA, in 2019, 583,864.50 tons of active ingredients
of pesticides were used and in 2020 there was an
increase of 8.65% in commercialization in the country,
totaling 634,372.03 tons. When comparing data from
2010 we can see an increase in sales of 86.82% in
ten years. Constant exposure to these toxic agents
can cause chronic effects such as difficulty sleeping,
forgetfulness, changes in the functioning of some
organs, such as the liver, kidneys, and abortions.
In addition, studies point to groups of pesticides
as potential carcinogens. Fungicides are a class of
pesticides used to combat various fungal groups
that invade plantations, they can be classified as
organic, inorganic, and systemic. The most exposed
population are rural workers who handle, apply these
fungicides, harvest, transport, store, and often reside
on the property next to the crops. Objective: To assess
the cytotoxic effect of Score® by Allium cepa test.
Methodology: The test with Allium cepa described in
the literature was used, with some modifications to
adapt the method to our laboratory. This test allowed
the evaluation of the cytotoxicity from the growth
of Allium cepa roots. For the evaluation, onions of
the Allium cepa species were chosen and a systemic
fungicide that contains difenoconazole as active
ingredient and has as a trade name Score®, was acquired
from a grape producer. Two concentrations of Score®
recommended by the Agência de Defesa Agropecuária
do Paraná were prepared. Being concentration 1
(C1)= 0.08µl/mL and concentration 2 (C2)= 0.12µl/
mL. As a positive control (C+) a concentration 4 times
higher than C2 was used. For the negative control (C-)
distilled water was used. The tests were performed
in quadruplicate. The onions were left with the bulb
immersed in water for 48 hours. After this time, the
onions of C1, C2, and C+ were removed from the water
and submerged in the respective concentrations of
pesticide. The onions were left for another 72 hours
with the bulb immersed, totaling 120 hours of the
test. Throughout the test, the negative control was
kept in distilled water. After this period, the onions
were removed from the solutions and the size of the
roots was measured. Result: C1 presented an average
growth of 0.5 cm, C2 an average growth of 0.4 cm,
C+ average growth size of 0.3 cm and in C- there was
an average growth of 2 cm. Discussion/Conclusion:
From the macroscopic analysis it was possible to
observe the low growth of the roots that were exposed
to the fungicide compared to the negative control.
This low growth may be related to the cytotoxic
effect of the Score® fungicide. Comparing the growth
of Allium cepa roots, between the concentrations
of the fungicide and the negative control, there was
a difference in growth, which may be related to the
cytotoxic effects of Score®. In the future, we intend to
analyze nuclear alterations in dividing cells of these
same roots to observe the genotoxic effects of the
fungicide Score®. Keywords: Score®, Allium cepa test,
cytotoxicity. Acknowledgments: The State University
of Maringá, Postgraduate Program in Biosciences and
Pathophysiology (PBF/UEM), CAPES e CNPq.
 55

Microfibrillated cellulose and silica

nanoparticles as sustainable alternatives
to developing nanotechnology products:
the evaluation of skin irritation
Cruz, Juliana Varella1,2; Gagosian, Viviana Costa1; Magalhães, Washington3; Cademartori,
Pedro Henrique Gonzalez4; Oliveira, Danielle Palma2; Leme, Daniela Moraes1
1
Department of Genetics, Federal University of Paraná, Curitiba, PR, Brazil; 2 School of
Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP,
Brazil; 3 Embrapa Florestas, Colombo, PR, Brazil; 4 Graduate Program in Engineering and
Science of Materials – PIPE, Federal University of Paraná, Curitiba, PR, Brazil.

Introduction: Microfibrilated cellulose (MFC) and
silica nanoparticles (SiO2NP) has been proposed
for several applications in industry. MFC presents
a thickener property and allows interactions with
polymers and nanoparticles. SiO2NP can be used
to increase the time of action of chemical products,
working as a controlling delivery system. Those MFC
and SiO2NP properties are being used to develop
a more sustainable and renewable alcohol-based
hand rub and a long-term disinfectant. Considering
the SARS-CoV-2 pandemic, disinfection of hands and
surfaces became an important nonpharmaceutical
intervention, and the increase in the usage of
disinfectants and alcohol led to a lack in the source
of materials to produce hygiene and disinfection
products. Therefore, new alternatives are being
implemented with a sustainable and renewable
feature, predicting that the use of alcohol-based hand
rubs and disinfectants will now become a habit to
avoid other public health outbreaks. Furthermore, it
is important to consider the use of more sustainable
and renewable source materials for other types of
nanotechnology products. However, few studies
have evaluated the skin irritation potential of these
nanomaterials. Objective: This work aims to verify
whether MFC and SiO2NP are skin irritants after acute
and repeated exposure, using a reconstructed human
epidermis (RHE). Methods: The skin irritation test
was performed accordingly with OECD TG 439 using
an in-house RHE model constructed from neonatal
keratinocytes. The model used was within the quality
control, presenting the four epidermis layers, verified
by hematoxylin and eosin histology. The acute
exposure of MFC (1%) and SiO2NP (0.5%) followed the
SkinEthic protocol. Repeated exposure was performed
from an adaptation of the exposure conditions
described by the same protocol. For negative controls,
RHE was exposed to phosphate buffered saline and
ultrapure water, which were also used as a vehicle.
For the positive control, RHE was exposed to sodium
dodecyl sulfate at 1%. Cell viability was measured by
enzymatic conversion of the 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye
into formazan salt. Results: According to OECD TG
439, an irritant response is defined by a threshold
of cell viability lower than 50% compared to the
negative control. Our results showed that MFC and
SiO2NP are not potential irritants in both exposure
conditions (acute and repeated exposure). Previous
studies obtained similar findings; Rashad et al., 2019
reported that after 3 days of exposure to in vitro U937
cells, MFC did not induce inflammatory chemokine
expression, and 4 days after exposure on in vivo
model, a higher expression of anti-inflamatory IL-
1Ra was observed. Park et al., 2012 showed that
SiO2NP did not cause acute skin irritation. Although
our work confirm previous findings, there is no data
regarding MFC and SiO2NP repeated exposure in RHE
model. Conclusions: This study showed that MFC and
SiO2NP are not skin irritants even after in a condition
of continuous exposure. Overall, we believe that
MFC and SiO2NP may be sustainable alternatives to
be used in developing nanotechnology products,
such as products proposed for control SARS-CoV-2
transmission. Acknowledgments: CAPES and CNPq
for the financial support.
 56

Nasal and buccal absoption of

dimethoate: in vitro x in vivo study
Andrade, Ana Rosa Brissant; Carvalho, Deoclécio Lustosa; Souza, Asley Thalia Medeiros; Kishishita, Juliana;
Silva, José Wellithom Viturino; Bedor, Danilo César Galindo; Santana, Davi Pereira; Leal, Leila Bastos

Universidade Federal de Pernambuco (NUDFAC-UFPE), Recife, PE, Brazil.

Introduction: The impact of pesticides on human
health has been related by many different researchers.
In developing countries, such as Brazil, the use of
agrochemicals has increased considerably over the
years, and thus damage to human health resulting
from exposure has become greater, mainly due to a
lack of and/or incomplete use of appropriate clothing
as described in the product package insert, but also
to a lack of good practice in the use of pesticides,
especially in family farming. Objective: Considering
the above, the objective of this work was to evaluate
in vitro and in vivo dimethoate absorption through oral
and nasal mucous membranes. Methods: The in vitro
permeation test (IVPT) was performed in side-by-
side diffusion cells, 3.4 mL receptor solution, stirring
at 37 ± 1 °C, permeation area of 0,65cm2 by using oral
(cheek, sublingual, esophageal) and nasal mucosa.
7 collections at 0.25, 0.5, 1, 1.5, 2, 3 and 4h from
receptor solution were made. Dimethoate retention
was analysed in all mucosa. The in vivo study was
performed by collected saliva (spit in a test tube) and
collection of nasal fluid by using swabs, 30 minutes
before and four hours after dimethoate application on
a lemon plantation, in seven rural workers, 1 resident
and 1 researcher who monitored all applications. The
CEP/UFPE approval was n° 5.007.282. All dimethoate
analyses were performed by validated LC-MS/MS.
Results: The cumulative dimethoate permeation was
2.5 times greater through the esophageal compared
to the buccal mucosa. However, the thickness of the
buccal mucosa was about 3 times greater than that of
the esophagus (356.0 ± 48.79 and 962.0 ± 80.44mm),
justifying this result. The percentage of dimethoate
permeated was around 1, 2 and 8% of the amount
applied, from the buccal, esophageal and sublingual
mucosa respectively. The dimethoate permeated
through the nasal mucosa was 3,683.28 ng/cm2 after
the 4-hour experiment, which means 11,08 ± 2,04 %
of the amount applied. For the in vivo study, at the
end of 4 hours of application, the highest amount
of dimethoate in saliva was found in two workers, 3
and 6 (383.76 and 1,198.27 ng/mL) corroborating the
observations by the researcher (talking during the
application and stopping the application to smoke).
Dimethoate was found in the nasal fluid of three
workers at concentrations of 6.17, 3.16 and 4.36 ng/
mL. Discussion/Conclusion: According to Thredgold
(2019), in agricultural settings it is reported that
90% of total pesticide exposure occurs in ways
attributed to dermal absorption or ingestion, while
10% is attributed to respiratory absorption. Even so,
these less important routes are also responsible for
exposure of workers. The pesticide solubilized in saliva
and found in nasal mucosa can be directly absorbed
through the tissue. At the same time, nasal mucosa
has a large contact surface and high vascularization.
It is worth mentioning that a direct correlation cannot
be made between the amount of dimethoate found
in the participant’s saliva and nasal mucosa with the
IVPT results, since these fluids do not remain static
in the individual during the 4 hours of work. However,
these data are worrying, given that the application of
pesticides is a routine activity and can have a serious
impact on workers’ health. A great effort in the area of
education must be made in an attempt to reverse this
situation. Acknowledged: CNPq and FACEPE.

Photoprotective effect of Rapanea ferruginea
bark extract-loaded nanoemulgel
Cordenuzzi, Dorys Angela; Benvenutti, Larissa; Santin, José Roberto; Lucinda-Silva, Ruth Meri
Postgraduate Program in Pharmaceutical Sciences. University of Vale do Itajaí, Itajaí, SC, Brazil.
Background/Introduction: Rapanea ferruginea
is a brazilian plant popularly used to treatment of
itching, rashes, hives and eczema. The bark extract
has shown anti-inflammatory and anti-nociceptive
activities. Nanosystems, such as nanoemulsions
and nanoemulgel, are dermatological carriers that
improve drug release and dermal drug permeation.
Nanoemulsions with the R. ferruginea extract showed
improved anti-inflammatory capacity in in vitro and
in vivo models. Objective: To evaluate the effects
of nanemulgel containing Rapanea ferruginea bark
extract on cell viability, irritative potential and
photoprotection using in vitro models. Methods: The
glycolic extract of R. ferruginea bark was obtained by
the dissolution of the concentrated alcoholic extract
in propylene glycol. The nanoemulsion was prepared
by phase inversion method. The R. ferruginea bark
extract-loaded nanoemulgel was prepared using
nanoemulsion:carboxyvinyl gel proportion1:2. The
antioxidant activity of the extract was evaluated by
DPPH method. The extract cytotoxity (1, 10, 100 and
300 µg/mL) and the nanoemulsion on L929 cells was
evaluated by MTT method. The irritative potential
of the extract, nanoemulsion and nanoemulgel was
tested on L929 cells through an agarose-overlay
method. Then, the extract and the nanoemulsion were
tested for their photochemoprotective (UVA and UVB)
effect using L929 cells. A photoprotection study of the
R. ferruginea bark extract-loaded nanoemulgel was
performed in L929 cells using irradiation method and
quartz plates. A commercial sunscrenn FPS 30 and
the base nanoemulgel were utilized as controls. The
photoprotection factor was calculated by accessing
the cell viability of irradiated and non-irradiated
cells. Results: The extract presents antioxidante
activity in DPPH method with CE50 of 0.95 mg/mL
± 0.03. The extract and nanoemulsion preparation
57
did not exhibited cytotoxic effect in L929 cells. Pure
R. ferruginea extract and loaded formulations did
not present irritative potential using the agarose-
overlay method. The extract (100 and 300 µg/mL)
and the nanoemulsion show photoprotective effect
on both UVA and UVB radiations, protecting the L929
cells of the cytotoxicity irradiation effect. In the
photoprotection study, the nanoemulgel containing
R. ferruginea extract show a photoprotection
factor of 12.96 ± 2.29 and the base gel provided
an insignificant photoprotection factor of 1.49 ±
0.50. Discussion: The skin is highly exposed to the
deleterious effects of UV solar radiation, which
has turned into a major environmental carcinogen.
In vitro methods using fibroblasts showed that R.
ferruginea extract did not present cytotoxic effects
on L929 cells and the agarose-overlay method
demonstrated that the formulations are no irritative.
The extract and nanoemulgel showed capacity
of protecting L929 cell of UVA/UVB irradiations.
Nanoemulgel formulation containing R. ferruginea
extract showed photoprotective properties, similarly
as the one reported by commercial FPS15 sunscreen.
The biochemical, molecular and histological changes
induced by acute skin UVB exposure can lead to
oxidative stress, inflammation and photoaging. This
research showed that in addition to photoprotective
characteristics, the extract presents antioxidant
activity and literature data demonstrated that R.
ferruginea preparations decrease the release of the
TNF, IL-1β, and KC cytokines and MPO activity in skin
inflammation. Conclusion: The results show the
antioxidant and photoprotective potential of the R.
ferruginea bark extract and the respective loaded
nanostructured system, which can be considered a
promising active in the development of new products
for skin protection and care.
 58

Preliminary data on a newly developed

biomimetic Reconstituted Human
Ocular Epithelium Model using an
animal free defined medium
Bosquetti, Bruna1; Catarino, Carolina Motter1; Costa, Meg Cristina da Castilho1; Thá,
Emanoela Lundgren1; Silva, Artur Christian Garcia2; Schuck, Desiree Cigaran1; Canavez,
Andrezza Di Pietro Micali1; Brohem, Carla Abdo1; Valadares, Marize Campos2
1
Grupo Boticário; 2 Universidade Federal de Goiás.

Introduction: The physiological characteristic
of the human corneal epithelium makes it more
susceptible to tissue damage caused by substances
or environmental conditions. As such, eye corrosion
and irritation are important endpoints for the
toxicological evaluation of ingredients and cosmetics.
For a long time, these outcomes have been evaluated
using animal models (Draize test). However, social
pressure, ethical concerns and scientific motivations
lead to the development of alternative methods,
such as reconstituted human corneal epithelium
(RhCE) models. These models have been widely used
as assessment tools for scientific and regulatory
purposes. Currently, there are four commercially
available models validated for this purpose, which
integrate the OECD Test Guideline n. 492 (EpiOcular™
EIT, SkinEthic™ HCE, LabCyte CORNEA MODEL 24 EIT
and MCTT HCE™ EIT). In Brazil, due to bureaucratic
and customs issues, the acquisition of these
models is extremely challenging. In this context,
some laboratories developed their own models,
such as the Tox in Ocular model developed by Artur
and collaborators (Silva, 2019). All these models
are formed by a stratified epithelium nurtured
by media with different compositions, most of
which are based on commercial sources whose
exact content is not available. The dependence on
commercial media impacts the cost of tests and can
limit customization (e.g.: developing a free animal
content model). Furthermore, unavailability in the
market and discontinuation could impair the use of a
model or even require completely new development.
Objective: The goal of this project is to develop and
validate (interlaboratory) a new RhCE model based
on the Tox in Ocular using a chemically defined
culture medium. Methods: For the reconstructed
tissue, HaCaT keratinocytes were seeded in 24-well
inserts at a density of 5×105 cells/insert and cultured
for 4h under a submerged condition for adhesion.
Following that, the apical medium was removed,
and cells were maintained at an air-liquid interface
for 5 to 8 days until adequate stratification with the
specific media. Four media conditions were evaluated
(each condition had different composition of the
supplement blend), as well the presence or absence
of type I collagen matrix on the inserts. The choice of
the supplements for the culture medium was based
on the concentration scale provided in the commercial
kit. After the differentiation period, histological
analyzes of the models using hematoxylin-eosin were
performed. Results/Discussion: The four conditions
maintained at the air-liquid interface for 5 days
showed no significant histological differences. 4-5
layers of cells formed an epithelium with a thickness
comparable to Tox in Ocular model and the human
cornea. The morphology of the samples maintained
at the air-liquid interface for 5 days was comparable
to the Tox in Ocular model. The samples kept for
extra 3 days at the air-liquid interface, presented the
formation of many vacuoles, which resulted in a loss
of homogeneity of the tissue demonstrating cellular
stress. These results lead us to believe that the major
impact in the model is the stratification period time
and not the tested variations on media components
(such as the animal components elimination) or the
matrix absence. These remain to be confirmed with
further tests. Conclusion: With the results obtained
so far, we have demonstrated the possibility of
developing a biomimetic RhCE model using a known
media composition, bringing the advantage of a free
animal content model. The next steps of the study are
the characterization of the tissue through biomarkers
and the response using reference substances.
Acknowledgments: Grupo Boticário, Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Financiadora de
Estudos e Projetos (FINEP) e Fundação de Apoio à
pesquisa do Estado de Goiás.
 59

Safety evaluation of nanoemulsion and

emulgel containing pomegranate peel
extract using alternative in vitro methods.
Rudolf, Carline; Benvenutti, Larissa; Rocha, Anna Carolina Furaer; Cordenuzzi,
Dorys Angela; Santin, José Roberto; Lucinda-Silva, Ruth Meri

Postgraduate Program in Pharmaceutical Sciences. University of Vale do Itajaí, Itajaí, SC, Brazil.

Introduction: Plants rich in substances with antioxidant
and depigmenting potential can be useful to prevent
skin aging and the appearance of hyperchromias,
being used as actives in cosmetics. Several studies
indicate that the pomegranate peel extract has
strong antioxidant and depigmenting potential in in
vitro studies, but because they are phytocomplexes,
it is necessary to evaluate the safety of these
products before possible clinical tests on volunteers.
Objective: To evaluate the safety of nanoemulsion
and emulgel cosmetics containing pomegranate peel
extract using alternative in vitro methods. Methods:
The pomegranate peel extract was obtained by
dynamic maceration and characterized in terms of
total phenolic content, ellagic acid content by HPLC
and antioxidant activity (AA) by the DPPH method. The
extract was incorporated into a nanoemulsion (NE)
and emulgel (EM) cosmetic base at a concentration
of 0.1%. The cytotoxicity of pomegranate peel extract
(1, 10, 100 μg/mL) and products was determined
by cell viability assay using the MTT method on
L929 murine fibroblast cell line. The skin irritation
potential (Agarose overlay) of the products was
determined by the agar diffusion method, using L929
cells. Extract, nanoemulsion and emulgel samples
were tested at concentrations of 1, 10, 100 μg/mL.
The degree of irritation was evaluated by the lysis
zone, according to the classification described by the
American Pharmacopoeia. The irritating potential was
also analyzed by the HET-CAM model, applying the
nanoemulsion and the concentrated extract directly,
without dilution, and the emulgel with 1:10 previous
dilution. The samples were applied on the chorio-
allantoic membrane and the possible irritating effects
observed after 5 minutes. The results were compared
with the positive and negative controls, 0.1 M NaOH
and 0.9% NaCl, respectively. Results: The soft extract
showed AA with EC50 of 300.03 ± 0.52 μg/mL, total
phenolic content of 296.72 ± 6.92 mg EAG/g and
ellagic acid content of 2.73 ± 0.60%. Two phycosmetic
formulations containing 0.1% of the pomegranate
peels soft extract were obtained, NE and EM. In the
cell viability assay by the MTT method, the extract did
not promote cytotoxicity after 24 hours of incubation
at all concentrations evaluated (1, 10 or 100 µg/mL).
In the agarose ovelay assay, the extract samples at
concentrations 1, 10 or 100 µg/mL were not able to
cause the formation of a toxicity halo around the
sample, the nanoemulsion samples, with and without
extract, showed a toxicity halo with classification
3 – moderate, and the emulgel samples, with and
without extract, presented reactivity classification 2
– mild. The safety evaluation by the HET-CAM method
showed that the extract and tested formulations,
NE and EM, did not promote blood extravasation or
even the formation of blood clots in the microvessels
present. Discussion: The viability test by the MTT
method demonstrated that the extract does not
promote toxicity. In the agarose ovelay assay, the
extract did not show reactivity, the emulgel showed
mild reactivity, while the nanoemulsion showed
moderate reactivity, probably due to the presence
of the surfactants used with irritating potential.
The safety evaluation by the HET-CAM method
demonstrated that the soft extract and tested
formulations were classified as safe. Conclusion:
Conducting the research allowed obtaining scientific
knowledge about the safety of pomegranate peel
extract and phytocosmetic products containing
0.1% of the extract, as well as contributing to the
development of innovative cosmetics.
 60

Spectroscopic assessment of lung mucus

as a tool for toxicokinetic/toxicodynamic
assessment of chemical respiratory sensitizers
Mendonça, Izadora Caroline Furtado1; Silva, Artur Christian Garcia1;
Mendanha, Sebastião Antônio2; Valadares, Marize Campos1
1
Laboratory of Research and Education in In vitro Toxicology (Tox In), Faculty
of Pharmacy, Federal University of Goiás; 2 Center for Research, Technological
Development and Innovation in Pharmaceuticals, Medicines and Cosmetics
(FarmaTec), Faculty of Pharmacy, Federal University of Goiás.

Introduction: Low molecular weight (LMW) respiratory
sensitizers are chemical entities that trigger
hypersensitivity in the lungs and airways after being
inhaled, culminating in the development of allergic
asthma/rhinitis after long-term exposures mainly
regarding an occupational context. Contrastingly with
the dermal sensitizers, known by contact dermatitis
elicitation after cutaneous exposure, the mechanisms
that underly the toxic response to LMW respiratory
allergens are not entirely available, so up to now, there
are still no validated methods for addressing the lung
sensitization potential of chemicals. Besides, although
it is known that these toxicants must bind covalently to
proteins to be recognized by the immune system cells, it
is still not clear which are the molecular targets that are
involved in this haptenization process. Thus, even though
some attempts to apply skin sensitization assays to
evaluate respiratory sensitizers have been taken, there
are significant anatomical/structural discrepancies
regarding these two different biological compartments
(skin versus lungs), which can impact the toxicokinetics/
toxicodynamic and mode of action (MoA) of this class of
compounds. Bearing in mind that mucus is a considerable
physiological barrier overspread in the respiratory
tract and that it has a major role in the pharmaco/
toxicokinetic features of chemicals that enter this
system through inhalation, in this work, we investigated
the potential interactions of LMW respiratory sensitizers
with the mucin, the major protein mucus component,
using a spectroscopy-based method commonly used
for addressing mucus interaction with inhaled drugs.
Objectives: The purpose of this work was to perform
an in chemico spectroscopy analysis to evaluate the
potential interactions between LMW respiratory
sensitizers and mucus, which could drive the formation
of toxicant-mucin complexes. Methods: We evaluated
seven known respiratory sensitizers (Chloramine
T, Piperazine, Maleic Anhydride, Cyanuric Chloride,
Trimellitic Anhydride Chloride, Trimellitic Anhydride,
and Glutaraldehyde) and two non-sensitizers (Glycerol
and Lactic Acid). The test chemicals were solubilized in
Phosphate-buffered Saline (PBS) in four concentrations
ranging from 200 – 1,25µM and incubated with a
solution of isolated mucin at 0,12 mg/mL (pH 7.4) for 0
and 24 hours at 37°C. After incubation, absorbance was
measured in a spectrophotometer at the wavelengths
from 200 to 400 nm (10 nm steps) to evaluate changes
in the spectroscopy behavior of mucin resulting from a
toxicant-mucin complex formation. Results: The results
demonstrated that mucin had an absorption peak at 260
nm, as expected according to described in the literature.
Furthermore, we observed a slight absorbance increase
in the higher evaluated concentrations of glutaraldehyde
and cyanuric chloride (hyperchromism) and a decrease
in the chemical chloramine-T (hypochromism) after 24
hours of incubation. However, additional data evaluating
the chemical glutaraldehyde were obtained through the
Isothermal Titration Calorimetry (ITC) method, where no
potential covalent interactions were detected between
this chemical and isolated mucin. Nevertheless, the other
chemicals did not promote significant alterations in the
mucin spectroscopic profile. Discussion/Conclusion: In
this work, we demonstrated that, although some minor
alterations were detected, LMW respiratory sensitizers
do not potentially interact with mucin, the major protein
component of pulmonary mucus. These results clarified
two essential aspects of human response to these
toxicants: I) the lung mucus barrier does not seem to have
a mandatory role in the toxicokinetic of these chemicals
and II) the pulmonary mucin is not a worthwhile target
for the molecular initiation event (covalent binding
to proteins) of the respiratory sensitization, once no
possible interactions were detected between this protein
and the evaluated chemicals. Aknowledgements:
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) e Financiadora de Estudos
e Projetos (FINEP).
 61

Study of the toxicity of NPS amphetamines

and cathinones using in silico tools
Rodrigues, Caio Henrique Pinke; Castro, Jade Simões; Bruni, Aline Thais

Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.

New psychoactive substances (NPS) have emerged in
recent years to offer an alternative to classical drugs.
The idea behind NPS appearance is to provide recreative
effects to the users and circumvent the prohibition.
These substances can yield many threats to both law
enforcement and health. The most common NPS are
amphetamines and cathinones, which have similar
structures and stimulating effects. There is a lack in
both the structural and toxicological properties of
these drugs. Because of their complexity, experimental
approaches can be challenging to provide results.
Given these facts, in silico tools are an alternative to
obtaining data about NPS. The study’s primary goal
was to give an insight into the toxicological properties
using in silico evaluation. We studied 42 (forty-two)
pairs of homologous amphetamines and cathinones,
a total of 84 (eight-four) structures. For this purpose,
we calculated the toxicological properties of these
compounds from different in silico software: Protox
II, SwissADME, GUSAR, and TEST. The resulting data
matrix contained 84 samples split into two classes
(amphetamines and cathinones) and 24 (twenty-
four) collected variables containing calculated values
for LD50, solubility, and LogP using different models
according to each software. We evaluate the influence
of the variables on the samples by multivariate
tools. Principal Component Analysis – PCA was used
to explore the system, whereas Soft Independent
Modeling of Class Analogy – SIMCA and Partial Least
Square Discriminant Analysis – PLS-DA were used to
evaluate the classification. PCA showed a profile in
discriminating amphetamines and cathinones. This
behavior was further assessed through SIMCA and
PLS-DA. In both supervised techniques, we performed
a variable selection. According to their activity, SIMCA
results showed around 10% of error in the classification
of amphetamines and cathinones. Four principal
components and twelve variables were necessary to
model the classes. Solubility information was found as
those variables with the most discriminating power.
PLS-DA results showed that no misclassification was
found. Three principal components were necessary
to model the classes, containing around 93.5% of
the complete information. Both internal (Q2) and
external (R2) regression coefficients showed a good
fit (0.83 and 0.85, respectively). Calculated LogP
values were the most important variables in this case.
In conclusion, we can say that in silico tools raised
information about NPS amphetamines and cathinones.
Multivariate analysis showed that the software could
help give insight into the toxicological properties of
these drugs. Solubility and LogP variables were the
most important in discriminating amphetamines
and cathinones. It makes sense, given that the
only difference among homologous structures is a
carbonyl group. Finally, it is crucial to strengthen that
having values from different software and different
models for the properties is essential to establish the
classes. We thank the Brazilian Agencies Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq, grant 465450/2014-8) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES,
Finance Code 001) for financial support.
 62

Testing strategies for evaluation of eye

irritation potential of agrochemical formulations
as an alternative to animal testing
Choksi, Neepa1; Latorre, Andreia Oliveira2; Pais, Mariana Castello Novo3; Murata, Rosana
Zoriki Hosomi4; Catalano, Shadia M.I.5; Aguilera, Mariana6; Pires, Janaina Aparecida
Cardoso7; Ogasawara, Maryanne8; Habe, Priscila9; Perjessy, Gisele10; Allen, David1
1
Integrated Laboratory Systems, Research Triangle Park, NC, USA; 2 BASF SA, Sao Paulo, Brazil;
3
Syngenta Crop Protection, Sao Paulo, SP, Brazil; 4 Bayer SA, Sao Paulo, SP, Brazil; 5 Corteva
Agriscience, Barueri, SP, Brazil; 6 Ihara, Sorocaba, SP, Brazil; 7 Ourofino, Ribeirão Preto, SP, Brazil;
8
 FMC, Campinas, SP, Brazil; 9 Sumitomo, Sao Paulo, SP, Brazil; 10 Crop Life Brazil, Sao Paulo, SP, Brazil.

The in vivo rabbit eye test was developed over 75 years
ago to assess the irritation and corrosion potential
of substances that may come into contact with the
eye. Today, regulatory hazard classification and
labeling systems based on United Nations Globally
Harmonized System of Classification and Labeling
(GHS) are still based on this method. While multiple
in vitro methods have been developed as non-
animal alternatives, their applicability to evaluate
eye irritation potential of agrochemical formulations
still need confirmation. Due to the complex nature of
these products, they were not typically included as
reference chemicals in test methods validation. In an
attempt to identify one or more alternative methods
that are reliable to evaluate eye irritation potential
of end-use products, we conducted a retrospective
evaluation of 158 agrochemical formulations. These
agrochemical formulations had in vivo and in vitro
data available and the additivity calculation of GHS
was performed to determine if these non-animal
alternative methods could accurately differentiate
non-irritant and irritant formulations. This work is
sponsored by Crop Life Brazil Alternative Methods
Team (CLB_MAlt) to ILS and the data was provided
by associated companies. In vivo data were available
from the Draize test (OECD 405). In vitro results were
available from at least one of three test methods:
bovine corneal opacity and permeability (BCOP, OECD
437); isolated chicken eye (ICE, OECD 438); and the
Reconstructed Human Cornea-Like Epithelium (RhCE,
OECD 492). All in vitro methods were conducted
according to respective OECD test guidelines. The
results herein suggest that the individual methods
were highly predictive of end-use products that
do not require classification for eye irritation
hazard. However, even in vitro methods validated
for identifying chemicals GHS category 1 (BCOP and
ICE) did not reliably identify the agrochemicals that
induce serious eye damage. In fact, for category 1, the
major driver for classification of agrochemicals is the
persistence of effects, which is not evaluated by the
current in vitro methods. Nevertheless, the majority
of agrochemical formulations falls into category
that does not require classification (around 70%).
Thus, these data suggest that the four alternative
approaches can be used in different testing strategies
to identify agrochemical formulations that do not
require eye irritation hazard classification without
using animals, which could significantly reduce the
use of animals for this endpoint. Acknowledgments:
Kolle S, Stinchcombe S, Inforzato M, Masinja W, Grivel
A, Corvaro M and Baldassari J as representants of the
international working group supporting this project.
 63

The endothelial compartment can actively

participate in the adverse outcome pathway
(AOP) of chemical respiratory allergy
Silva, Artur Christian Garcia; Carvalho Filho, Sérgio de Morais; Valadares, Marize Campos

Laboratory of Research and Education in In vitro Toxicology (Tox In),

Faculty of Pharmacy, Federal University of Goiás.

Introduction: Occupational asthma is a workplace- Maleic Anhydride, Cyanuric Chloride, Trimellitic

related allergic condition characterized by airway
hyperresponsiveness and airflow limitation in
response to different agents exposure. Specifically,
the low molecular weight (LMW) respiratory
sensitizers are defined as chemical entities that can
cause airway hypersensitivity after inhaled, being
classified by the EU REACH as Substances of Very High
Concern (SVHC). For instance, this classification makes
the regulatory background behind these substances
as rigorous as those adopted for genotoxic/
mutagenic chemicals. Although the relevance in both
regulatory and human health contexts, there are
still no validated methods for the risk assessment of
LMW respiratory sensitizers, and one of the primary
reasons for this issue resides in the fact that the
Adverse Outcome Pathway (AOP), which precedes the
clinical manifestations of occupational asthma, is not
entirely elucidated. Bearing in mind the respiratory
tract multicompartmental structure and the massive
extension of the air-blood-barrier (ABB), composed
primarily by pneumocytes and endothelial cells, most
in vitro models for pulmonary toxicity are only based
on the assessment of the epithelial compartment
response to toxicant exposure. We hypothesized
that the endothelial cells also contribute actively to
the chemical respiratory allergy initiation/elongation
processes, responding directly to respiratory
sensitizers regardless of the cell crosstalk in the
asthma pathological microenvironment. Objectives:
This work proposed to evaluate in vitro the
alterations triggered by LMW respiratory sensitizers
in human endothelial cells to investigate this cell
type’s participation in the mechanistic framework
of chemical respiratory allergy, which clinically
leads to occupational asthma. Methods: The study
was conducted employing the endothelial cell line
EA.hy926, so that seven known chemical respiratory
allergens were evaluated (Chloramine T, Piperazine,
Anhydride Chloride, Trimellitic Anhydride, and
Glutaraldehyde). First, the cytotoxicity of the test
materials was determined using the MTT reduction
assay, following exposure for 24 hours. Thus, each
substance’s cell viability 80% concentration (CV80) was
defined. Posteriorly, endothelial cells were exposed to
this concentration for 24 hours, and the inflammatory
cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-α)
were measured afterward in both supernatant and cell
lysate samples using the cytometric bead array (CBA)
method. Moreover, the expression of the endothelial
activation biomarker ICAM-1 was performed by flow
cytometry analysis after exposing the EA.hy926 cells
for 24 and 48 hours to the toxicants CV80. Results: The
results have shown that endothelial cells exposed to
five of the seven evaluated respiratory sensitizers
displayed a significative increased production of IL-6
in both supernatant and lysate samples. Besides,
some chemicals also prompted an increase in IL-8
and IL-1β production. Considering the expression of
ICAM-1, five of the seven LMW sensitizers promoted
a significant increase in this biomarker expression
by endothelial cells not at 24 hours but 48 hours
after exposure. Discussion/Conclusion: The results
demonstrated that respiratory sensitizers can
directly activate the human endothelium by releasing
inflammatory mediators and increasing the adhesion
molecules expression. These findings support the
active participation of endothelial compartment in
the mechanistic framework of chemical respiratory
allergy, which might be encompassed considering
the development of New Approach Methodologies
(NAMs) for risk assessment of respiratory toxicants.
Aknowledgements: Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES), Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) e Financiadora de Estudos e Projetos (FINEP).
 64

The Implementation of Alternative Methods

to the Use of Animals for ocular toxicity:
difficulties and opportunities for innovation
Gimenes, Izabela1,2; Presgrave, Octavio1; Gonzalez, Marcelo2; Alves, Gutemberg2
1
Fundação Oswaldo Cruz-Fiocruz; 2 Universidade Federal Fluminense-UFF.

Brazil presents the fourth highest consumption of
personal hygiene products, perfumery and cosmetics,
on a market of around 2 billion dollars. Determining
the potential for eye irritation is a key safety estimate
for these products. The Normative Resolution (RN)
18/2014 brought a regulatory change that included
a mandatory replacement of animal testing by
alternative methods in Brazil. As a result, the interest
in validated alternative methods has increased.
The Bovine Cornea Opacity and Permeability Test
(BCOP) is among the alternative methods accepted
for regulatory purposes. Therefore, knowing the
challenges and limitations of its implementation
can contribute to a global diagnosis and increase
its benefits and applicability in Brazil. Objective: to
map the main challenges and technical limitations
faced by the performers of alternative methods, with
a focus on Eye Irritation and BCOP. Methods: This
project was approved by the local Research Ethics
Committee (CAAE: 17552519.1.0000.5243). Participants
were practitioners of alternative methods in Brazil,
including managers and professionals at academic
laboratories, research centers (public and/or
private). Volunteers were recruited through a list
of 51 participants from the National Network of
Alternative Methods-RENAMA/MCTI. Questionnaires
with open and closed questions and focal individual
interviews were performed with a discourse analysis
of records and transcriptions for a qualitative
synthesis. Results: The results show that 95% of the
participants perform and 5% have already worked
with alternative methods. Most respondents (71%)
stated that the greatest difficulty faced is related to
the high cost of validation/implementation, followed
by the limited access to training (61.9%) and high cost
of materials (61.9%), as well as purchasing reagents
and equipment (42.9%). Regarding of eye irritation
potential, 33% of respondents perform the BCOP
test and 66.7% perform the short-term in vitro test
(STE). The low availability of slaughterhouses to
obtain the bovine eye (50%) was its main limitation
followed by the fact that this method provides a less
assertive classification (42.9%) and a large waste
of the eyeballs due to scratches and other damage
(28.6%). Focal interviews (n=7) pointed out that the
low availability of the eyeballs sources is related to
the facilities of material disposal. Three participants
described a 2-3 hours travel to the slaughterhouses,
hindering the performance of the test on the same day.
The test is not validated for execution after 24 hours
of collection. Participants stated that preservation
or cryopreservation system could solve the problem
of material loss and reduce visits to collection sites.
All participants addressed difficulties with the OECD
Guide due to the lack of technical details, demanding
other support materials and contact with experienced
professionals. The need for access to more courses,
practical activities and training for implementation of
testes was presented, as RENAMÁ s actions through
PremaSul were not seen as sufficient to absorb all the
demand. Most participants from public laboratories
reinforced the need for investments and the high
cost of imported materials, indicating that the
relative costs for validation in Brazil are much higher
than in other countries. Discussion/Conclusion:
These findings indicate several challenges to the
implementation of BCOP and alternative methods,
related to infrastructure, access to information
and training, high costs, and technical limitations.
Overcoming such limitations may demand an open
innovation environment through dialogue between the
Triple Helix (Companies; Scientific and Technological
Institutions–ICTs and Government), with the
strengthening of initiatives such as the RENAMA and
the Brazilian Center for the Validation of Alternative
Methods. Keywords: alternative methods, qualitative
research, BCOP, eye irritation. Acknowledgements:
The authors thank the RENAMA/MCTI for the technical
assistance, and the financial support by Inova Fiocruz/
Fundação Oswaldo Cruz.
 65

The use of NAMs to evaluate the toxicity

of UV filters: emphasis on aquatic toxicity
and endocrine disrupting effects
Nonino, Elisa de Castro Wille; Leme, Daniela Morais; Pestana, Cynthia Bomfim

Department of Genetics, Federal University of Paraná, Curitiba, PR, Brazil.

Introduction: UV filters are widely used in the
composition of cosmetics (such as sunscreen lotions)
and a range of products to prevent from damage
caused by the ultra-violet (UV) component of sunlight.
Despite the benefits to human health, this group of
substances has been drawing attention due to their
possible environmental impacts when released into the
aquatic environment, including endocrine disrupting
effects. Objectives: Due to the restriction on the use
of animals in the cosmetic industry, endpoints should
be evaluated through alternative methods such as
in vitro and in silico tools. which are now referred to
as being New Approach Methodology (NAM) data.
In order to obtain information on the endocrine
disrupting effects as well as damage to aquatic
environments assessed through data on the potential
PBT (persistence/biodegradation, bioaccumulation
and toxicity), ten organic UV filters were investigated
using alternative methods. Methods: The following
organic UV filters were investigated: diethylamino
hydroxybenzoyl hexyl benzoate, octocrilene,
ethylhexyl triazone, tris-biphenyl triazine; bis-
ethylhexyloxyphenol methoxyphenyl triazine;
butyl methoxydibenzoylmethane, homosalate,
ethylhexyl salicylate, octyl methoxycinnamate and
phenylbenzimidazole sulfonic acid. In vitro assays
were retrieved from “ToxCast” (Toxicity Forecaster)”,
an USEPA database available at https://comptox.
epa.gov/dashboard. In silico data were retrieved
from VEGA (https://www.vegahub.eu), T.E.S.T.
(Toxicity Estimation Software Tool) and OECD Toolbox
(version 4.3). The following endocrine receptors
were investigated: androgen receptor (AR), estrogen
receptor (ER1 and 2) and thyroid receptor beta (TRb).
For environmental evaluation, biodegradation,
bioaccumulation and fish toxicity were searched.
Results: In vitro assays were available for 60% of the
UV filters: three of them were active as antagonists
(binding to the receptor blocking its action) to ER1
and ER2, AR and TRb receptors and inactive as
agonists for the same receptors. Two UV filters
were totally inactive (phenylbenzimidazole sulfonic
acid and ethylhexyl methoxycinnamate). One filter
(ethylexyl salicylate) was only active to estrogen
receptor antagonists. The in silico data confirmed
in vitro data for all compounds, except for butyl
methoxydibenzoylmethane, for which no activity
was predicted in silico and therefore was considered
inconclusive. From the 4 other UV filters investigated
using only in silico tools, no endocrine activity was
predicted for three of them. Discussion/Conclusion:
By analyzing all the collected data, consistency
in the results was verified for most (80%) of the
compounds, since 30% were classified as endocrine
disruptors (octocrilene, homosalate and ethylhexyl
salicylate) and 50% as non-endocrine disruptors
(bis-ethylhexyloxyphenol methoxyphenyl triazine,
ethylhexyl methoxycinnamate, ethylhexyl triazone,
tris-biphenyl triazine and phenylbenzimidazole
sulfonic acid). Only two UV filters (diethylamino
hydroxybenzoyl hexyl benzoate and butyl
methoxydibenzoylmethane) could not be classified. In
the environmental evaluation, although all UV filters
presented high LogPow values, low bioconcentration
fators were predicted in silico (categories 3 or 4). On
the other hand, all compounds presented high toxicity
to fishes and half of them were predicted as not
readily biodegradable and/or persistent. Our results
suggest that UV filters can be potentially harmful to
the aquatic environment due to their possible acute
toxicity and low biodegradability. The potential
endocrine disrupting activity of 3 out of 10 UV filters
also raises concerns, especially homosalate, which
is also not readily biodegradable and toxic for fishes.
Considering the environmental impact of substances
depends not only on their toxic potential but also on
exposure, further risk assessment analysis could
provide more relevant information on those health
care products. Acknowledgments: CNPq.
 66

Toxicity features of agrochemical formulations

related to eye and skin irritation potential that
are important to build weight of evidence for
integrated approach of testing strategies
Latorre, Andreia Oliveira; Pepato, Ana Claudia de Andrade; Faria, Patricia Miranda; Cazarin, Karen

BASF SA, Sao Paulo, Brazil;

The registration of agrochemical formulations
remains supported to a large extent based on
animal testing to characterize eye and skin irritation
potential, despite the multiple in vitro methods
already developed as non-animal alternatives.
Some advances toward regulatory acceptance
can be granted applying Integrated Approaches to
Testing and Assessment (IATA) developed by OECD.
IATAs are flexible frameworks that “integrates and
weights all relevant existing evidence and guides
the targeted generation of new data, where required,
to inform regulatory decision-making regarding
potential hazard and/or risk.” An essential step
of an IATA is to characterize the toxicity features
of the target chemicals. In fact, considering that
agrochemicals formulations are mixtures, the hazard
characterization related to eye and skin irritation
potential from large datasets can help building
weight of evidence (WoE) and improve the integrated
approach of testing strategies without animal testing
to predict these endpoints. Therefore, a public dataset
from the Brazilian Health and Regulatory Agency
(ANVISA) of registered agrochemicals formulations
until 2019 was retrospectively reviewed. This dataset
provides the hazard classification based on United
Nations Globally Harmonized System of Classification
and Labeling (GHS) adopted by ANVISA in that year.
The GHS hazard category for both endpoints, eye
and skin irritation, and the type of formulation were
available for a total of 1640 out of 1705 formulations.
The prevalence of the GHS hazard categories was
evaluated as well as the correlation between skin and
eye irritation, since IATA 263 for serious eye damage
and irritation considers data on skin corrosion (GHS
Cat 1) in the WoE analysis to predict the eye irritation
potential. The evaluation of this dataset revealed
around 70% (1128/1640) of the formulations did not
require classification for eye irritation, while 19%
(316/1640) and 12% (196/1640) were classified as
GHS cat 2 and cat 1, respectively. Once considered only
the liquid formulations water-based, the percentage
of formulations not classified for eye irritation was
even higher, 88.6% (444/501). For skin irritation, most
formulations did not require classification as well,
approximately 85% (1396/1640), while 9% (148/1640)
and 5% (85/1640) were classified as GHS cat 2 and cat
3, respectively and only 1% (11/1640) were classified
as skin corrosive GHS cat 1. The 11 formulations
classified as skin corrosive, 46% (5/11) did not require
classification for eye irritation, while 18% (2/11) and
36% (4/11) were classified as GHS cat 2 and cat 1,
respectively. Considering the formulations classified
as severe eye irritants (GHS cat 1), 127 out 196
formulations (65%) did not require classification for
skin irritation and only 2% (4/196) were classified as
skin corrosive. Overall, these data indicate that both,
skin corrosion and severe eye irritation potential,
should not be considered in the WoE analysis of
agrochemicals formulations to predict the hazard
potential of these endpoints. Nevertheless, it is likely
that the negative eye irritation potential can provide
evidence predicting the negative skin irritation. In
this dataset, 93% of formulations (1045/1128) not
classified for eye irritation were also not classified for
skin irritation. In summary, the higher prevalence of
formulations that did not require classification for eye
and skin irritation indicates that integrated testing
strategies with bottom-up approaches will be more
accurate predicting human outcome. Additionally,
data on skin corrosion or on eye damage should be
carefully considered in WoE analysis to predict eye and
skin irritation potential of agrochemical formulations.
Acknowledgments: Kolle S and Stinchcombe S

Toxicological evaluation of amphetamine-
likes employing in silico methodology
Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.
From the first decade of the 2000s onwards, a world order
phenomenon began to draw the attention of various
agencies. This phenomenon became known as New
Psychoactive Substances (NPS). They were spread as an
alternative to classic drugs, mimicking their effects in a
“legal” way. The main problem for individuals is the threat
to health due to the absence of toxicological information.
The most prominent NPS were amphetamines and
cathinones. As they appeared in the market quickly, the
experimental approaches classically employed faced
a challenge in providing results. Given these facts, we
found in silico tools a faster alternative for obtaining
data on NPS. For these reasons, the study aimed to study
and provide information on the toxicological properties
of amphetamines and cathinones using in silico
methodologies. In this work, we applied the MetadrugTM
software for analyzing 21 pairs of homologous
amphetamines and cathinones, a total of 42 structures.
This software is part of the MetaBaseTM API to access the
knowledge base behind all systems biology products
of Clarivate Analytics (https://www.cortellislabs.com/
page/?api=api-MB). Simulated variables are structural
properties, CYP450 QSAR Models, Protein binding QSAR
Models, ADME QSAR Models, predict the therapeutic
activity and toxic effects. The total number of variables
was 78. We used Soft Independent Modeling of Class
Analogy (SIMCA) to evaluate the answers. This method
considers the classification between amphetamines
and cathinones and indicates the most significant
parameters to define the groups. Results showed that
both classes needed three principal component analysis
to be modeled, and no misclassification was observed.
To amphetamines, three variables presented the highest
modeling power. They were i) affinity to human serum
albumin, log value of the retention time (ADME QSAR
Models); ii) potential to activate 5-hydroxytryptamine
(serotonin) receptor 2B (Protein binding QSAR Models);
and iii) Potential activity against arthritis (Prediction
of therapeutic activity). On the other hand, to classify
67
Rodrigues, Caio Henrique Pinke; Bruni, Aline Thais
the cathinones, the variables that result in the highest
modeling power were seven. They were: i) affinity to
human serum albumin, log value of the retention time
(ADME QSAR Models); ii) potential to be a substrate of
human P-glycoprotein transporter (Protein binding QSAR
Models); iii) potential to activate 5-hydroxytryptamine
(serotonin) receptor (Protein binding QSAR Models); iv)
potential for inducing liver lipid accumulation (Prediction
of toxic effects); v) potential to inhibit CYP2D6 (CYP450
QSAR Models); vi) potential activity against arthritis
(Prediction of therapeutic activity); and vii) Potential
activity against schizophrenia (Prediction of therapeutic
activity). These results indicated that amphetamines are
more stable in the transport protein. For cathinones, we
observed that the main variables were related to the liver
and correlated proteins. However, both groups showed
potential to activate the serotonin receptor and activity
against arthritis. These observations showed a lower
toxic potential for cathinones and their greater instability
when compared to amphetamines. In conclusion, we can
say that in silico methodologies can provide assertive
information about amphetamines and cathinones. The
SIMCA analysis showed that it is possible to obtain
toxicological properties that differentiate these drugs
and indicate parameters for educational and health
measures. The variables linked to the liver were the
most important in the discrimination of amphetamines
and cathinones, as cathinones are more water-soluble.
It makes sense, given that the only difference between
homologous structures is a carbonyl group. Because of
the responses of this work, it is essential to reinforce
the importance of in silico methods to assist in decision-
making in the face of the rapid emergence of different
available NPS. We thank the Brazilian Agencies Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq, grant 465450/2014-8) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES,
Finance Code 001) for financial support.
 68

Toxicological evaluation of effects

of bothropic venoms through the
chorioallantoic membrane assay HET-CAM
Braga, Jacqueline Ramos Machado1; Lima, Natália Cavalcante Barbosa2; Alves, Carolina Esmeraldo
Lima2; Rocha, Danilo Galvão2; Jorge, Roberta Jeane Bezerra2; Biondi, Ilka Borges3

Universidade Federal do Recôncavo da Bahia, Centro de Ciências Agrárias Ambientais

1

e Biológicas, Laboratório de Répteis e Anfíbios. Cruz das Almas, Bahia; 2 Universidade

Federal do Ceará, Núcleo de Pesquisa e Desenvolvimento de Medicamentos, Laboratório de
Farmacologia de Venenos e Toxinas. Fortaleza, Ceará; 3 Universidade Estadual de Feira de
Santana, Laboratório de Animais Peçonhentos e Herpetologia, Feira de Santana, Bahia.

Introduction: The Hen’s Egg Test – Chorioallantoic
Membrane (HET-CAM) is an alternative toxicological
method widely used to determine the irritation
potential of a substance, by monitoring the damage
caused to blood vessels, mainly in ocular toxicity
assessment tests. However, few studies have
investigated the usefulness of this method to evaluate
the properties of snake venoms, as an alternative for
hemorrhagic tests in animals. Objective: This study
aimed to evaluate the coagulant and hemorrhagic
effects of Bothrops leucurus (BlV) and Bothrops
erythromelas (BeV) snake venoms through the HET-
CAM assay. Methods: Five fertile fresh White Leghorn
hen eggs (n=80) were used for each group, and the
tests were performed in duplicate. In the HET-CAM
assay, the eggs were incubated horizontally and
rotated in a brooder for nine days (38 oC ± 5 oC, 70%).
On the 9th day of incubation, the CAM was exposed
and 200 µL of pool of bothropic venom diluted in
saline was added (50 µg/mL; 100 µg/mL; 200 µg/
mL; 1mg/ml), being the changes filmed (5 min) and
photo documented (0s; 30s; 3min; 5min). As negative
control, 200 µL of saline (0.9%) was used. The
irritation index (IS) was calculated using Luepke’s
classification system and the data analyzed by ANOVA
and Student’s t-test (p<0.05). Results: The results
revealed that the BeV has severe irritation properties
(IS 12.7 ± 0.5), even at low concentrations (50 µg/mL),
in a time-dependent manner, since the first signs of
severe effects (hemorrhage and coagulation) were
detected already after 30 seconds of exposure in the
CAM. However, BlV showed moderate irritation (IS
7.8 ± 0.3) from the concentration of 100 µg/mL, and
severe irritation properties (IS 11.8 ± 0.6) after 60s
of exposure. Conclusion: BeV was more irritating to
CAM than BlV. This can be explained by the already
known higher phospholipase activity of BeV, when
compared to BlV. Thus, the HET-CAM test proved to
be a good alternative model for toxicological analysis
of non-neurotoxic venoms. However, considering that
this was a pilot study used as an alternative method
for evaluating the effect of bothropic venoms, we
suggest that venom composition tested in the HET-
CAM assay and the ontogenetic and sexual variations
of the snakes must be considered when analyzing
the results. Keywords: Bothrops, embryo, chicken,
alternative methods.
 69

Use of a biomolecular decellularized bovine

cornea derived solution as a method of
evaluating opacity and ocular irritation
Santos, Jordana Andrade; Dias, Wanessa Amorim; Valadares, Marize Campos

Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, Goiás, Brasil.

Currently, there is a need to develop new
methodological approaches for the assessment of
ocular toxicity, which reproduce results more similar
to those found in humans and which aim to replace the
in vivo Draize eye test. Ocular opacity is one of the top
assessments for eye damage. This opacity is related
to the interaction of the biomolecules of the corneal
extracellular matrix with the substances to which it
was exposed. The extracellular matrix of the cornea
is composed of molecules such as proteins – mostly
collagen – and glycosaminoglycans, and they have an
important organizational characteristic that gives it
transparency. Once this organization is disturbed, by
different mechanisms such as ‘coagulation’ described
as the replication/denaturation of macromolecules
(particularly proteins) or ‘saponification’ described
as the breakdown of lipids, opacity is observed. One
of the main alternative tests for ocular assessment,
Opacity and Ocular Permeability of Bovine Cornea
(BCOP – OECD TG 437, uses opacity as one of
the classification parameters for substances as
categories at the extremes of the eye irritation
spectrum. These extreme categories are classified).
as ‘no categories’ and ‘category 1’ according to the
Globally Harmonized System (GHS) classification.
The test is already validated and well established,
however, some issues involving access to raw material
(bovine cornea), preservation of the same, and use
of specific equipment, end up making this test less
widespread. Since access to slaughterhouses may be
limited depending on the locations of the laboratories,
maintaining the viability and transparency of the
corneas is essential, something that can be obtained by
reagents that preserve the organ during transport but
have a high cost. Based on this, the present study aims
to propose accessibility and low-cost method, based
on the decellularized extracellular matrix derived by
bovine cornea for application as an in chemico test
of complexation of biomolecules with substance for
pre-screening and strategy for orientation of the
next steps for categorization of GHS Category 1 and
uncategorized chemicals. For this, bovine corneas
were collected and underwent decellularization
and digestion processes until obtaining a hydrogel,
which was previously characterized to confirm the
preservation of its compounds, mainly collagen and
glycosaminoglycans. The biomolecular solution was
prepared in different concentrations, 100%, 50%,
25%, 12,5%, 6% and 3% of Hydrogel in phosphate
buffer saline (PBS). The biomolecular solutions were
placed in 24-well plates to identify the most suitable
concentration for the method. For exposure to the
substances, a device was developed by 3D printing,
so that a cellulose membrane was suspended above
the biomolecular solution. PBS was used as a negative
control and 1-octanol (pure) and benzalkonium
chloride (5%) were used as test substances.
The biomolecular solutions were exposed to the
substances through the membrane for 24h at room
temperature, 25°. After that, the solutions were
evaluated for their turbidity, by spectrophotometry,
at 405nm, and compared with PBS control. Previous
results showed that there was a change in turbidity in
all concentrations of biomolecular solutions exposed
to the test substances when compared to PBS. Among
the concentrations of biomolecular solutions, those
that proved to be the most suitable for evaluation
in the spectrophotometer were 25% and 12.5%. This
indicates that the biomolecular solution, based on
natural extracellular matrix components, was able
to change its initial characteristics when exposed
to highly irritating chemicals with the potential to
generate ocular opacity, which indicates that the
test can be used pre-screening of Category 1 or
unclassified substances. A cheaper test, accessible
to researchers who do not have immediate access
to slaughterhouses and who do not have the specific
inputs to perform the BCOP test. Acknowledgments:
Capes and CNPq
 70

Use of CellFate®iN for a three-dimensional

in vitro model of human lung epithelium
Santos, Jeniffer Farias1; Reis, Emily Marques2; Berti, Fernanda Vieira2; Koepp, Janice2; Nunes, Viviane Abreu1
1
Laboratory of Skin Physiology and Tissue Bioengineering. University of Sao Paulo.
Sao Paulo, Brazil; 2 Biocelltis Biotecnologia S.A., Santa Catarina, Brazil;

In vitro assays have been a good tool to study the
pathophysiology of respiratory diseases and to identify
molecular targets for future clinical applications.
Bidimensional models of lung cell cultures are widely
used, but do not reflect the architecture of the lung
tissue. Recently, biotechnology has been used for
the establishment of three-dimensional models that
mimic the in vivo tissue microenvironment and to
accelerate the process of validation and efficacy of
drugs used in the clinical phase. The company Biocelltis
Biotecnologia S.A. recently launched CellFateiN®, the
extracellular matrix biocompatible functionalized
to mimic the lung tissue. To establish a functional in
vitro three-dimensional model of human pulmonary
epithelium cultivated on CellFate®iN, human
pulmonary fibroblasts and epithelial cells (CALU-3)
were cultured on the CellFate®iN for up to 21 days in an
air-liquid interface (IAL) and submitted to histological
analyzes. The activity of angiotensin converting
enzyme 2 (ACE2), was identified by hydrolysis of
fluorogenic substrate. The Solute Carrier Organic
Anion Transporter Family Member 3A1 (SLCO3A1)
and e-cadherin, which are proteins expressed by
pulmonary epithelial cells, was identified by qPCR.
SLCO3A1 was expressed on the first day of culture in
ALI, and e-cadherin at the end of the culture period (21
day). ACE2 has 4 times more activity than pulmonary
fibroblasts (control), with its highest activity identified
on the 14th day of IAL culture. CellFate®iN seems to be
a viable platform for in vitro cultivation of pulmonary
cells, and, after 21 days, these cells were organized
as a pseudostratified epithelium, reproducing
the pulmonary epithelium structure. Detection of
proteins expressed in pulmonary epithelium suggests
that a functional epithelium might be formed. These
data show that this three-dimensional model of
pulmonary epithelium might be used for in vitro 3D
assays for a better understanding of the physiology
of the respiratory epithelium. Keywords: human lung
epithelium, cellfatein, 3Dmodel
 02 
ANALYTICAL TOXICOLOGY
 72

Analysis of cannabinoids profile in

cannabis-based products by HPLC-DAD
Cardoso, Marilia Santoro1,3; Oliveira, Claudete C.4,5; Costa, José Luiz2,3
1
School of Medical Sciences, University of Campinas (UNICAMP), Campinas-SP, Brazil; 2 Faculty
of Pharmaceutical Sciences, University of Campinas (UNICAMP), Campinas-SP, Brazil; 3 Campinas
Poison Control Center, School of Medical Sciences, University of Campinas, Campinas-SP, Brazil;
4
Farmanguinhos- Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro – RJ, Brazil; 5 Associação
de Apoio à Pesquisa e Pacientes de Cannabis Medicinal (APEPI), Rio de Janeiro-RJ, Brazil.

Background: More than 500 constituents have been
identified in Cannabis sativa L., including cannabinoids,
terpenes, flavonoids, alkaloids and others. The interest
in Cannabis related studies grow even further with
endocannabinoid system’s discovery, which suggested
a pathophysiological modulation of this system useful
for treatment of several diseases. Due to high cost
of medicines and Cannabis derivatives (imported
and domestic) for patients who do not have financial
conditions for acquisition and / or medical prescription,
domestic cultivation of its plant-based extracts occur
to be the only alternative. Unfortunately, as these
products are not yet subject to the minimum quality
and safety tests and requirements in the production
chain, such as: quantitation of cannabinoids, terpenes
and contaminants (mycotoxins, residual solvents and
pesticides), there is probably an inadequate exposure
levels harmful to users. In this context, the main concerns
and considerations of this work are associated with
cultivation process and self-medication with Cannabis,
which can cause health risks. In addition, pharmacological
studies are also needed to evaluate plant profile
constituents’ variability and its therapeutic efficacy,
as they can be modified by geographic and climatic
factors. Objective: To develop and validate an analytical
method to determine 10 cannabinoids (cannabidivarin,
cannabidiolic acid, cannabigerolic acid, cannabigerol,
cannabidiol, cannabinol, Δ9-tetrahydrocannabinol,
Δ8-tetrahydrocannabinol, cannabichromene and
tetrahydrocannabinolic acid) in Cannabis-based
products (plant, oil and extracts) by high performance
liquid chromatography technique with diode array
detection (HPLC-DAD). And also analyze real samples
donated from Associação De Apoio À Pesquisa E Pacientes
De Cannabis Medicinal (APEPI), Brazil. Methods: For
quantification of cannabinoids, a representative aliquot
of the sample is diluted and analyzed by HPLC-DAD, using
a Nexera X2 chromatographic system (Shimadzu, Kyoto,
Japan), equipped with an C18 chromatographic column,
maintained at 35 °C. Mobile phase were composed of
ultra-pure water (A) containing formic acid (0.1%, v/v)
and acetonitrile (B) with flow rate of 2 mL/min and elution
in isocratic mode UV absorption spectra are acquired
from 200 to 400 nm, with quantification of cannabinoids
at 228 nm and a total analysis time of 22 min. Samples
were diluted factor range from 1:400 (for minority
cannabinoids) to 1:1000 (for major cannabinoids). Plants
samples (200 mg) were diluted in metanol (10 mL), and
then diluted (1:150 and 1:10 to majority and minority
cannabinoids, respectively). Results: The method
fulfilled all Brazilian’s Health Regulatory Agency (ANVISA)
recommended criteria (selectivity, accuracy, precision,
linearity, limit of quantitation, ruggedness and dilution
integrity), demonstrating to be adequate for all analytes.
Linearity was achieved between 2 to 50 µg/mL (r > 0.99,
1/x). Precision (% RSD) and bias (%) were investigated
for each analyte at three concentration levels (low,
medium, and high controls). The controls were evaluated
with one-way analysis of variance (ANOVA) performed
in each concentration to assess potentially significant
variability (p < 0.05). Precision results were below 1.8%
and bias results between 93.8% and 107.9%. No carryover
was observed. A total of 168 samples already were
analyzed and at least two cannabinoids were identified
in each sample, including: cannabidivarin (31.5% of the
samples), cannabidiol (75.6%), cannabidiolic acid (16.1%),
cannabigerol (58.9%), cannabinol (22.6%), cannabigerolic
acid (7.1%), Δ9-tetrahydrocannabinol (85.1%), Δ8-
tetrahydrocannabinol (0.0%), cannabichromene
(61.3%) and tetrahydrocannabinolic acid (10.7%). The
most identified substances were cannabidiol and Δ9-
tetrahydrocannabinol, with quantified concentration
between 0.8 – 48.2 mg/mL in Cannabis oil and 0.2
- 78.2% w/w in extract and between 0.8 – 88.6 mg/
mL in oil and 0.3 – 86.4% w/w in extract, respectively.
Discussion/Conclusion: The method was successfully
validated fulfilling all ANVISA’s recommended criteria
and will be applied for more quantification of Cannabis-
based products donated from APEPI. Acknowledgments:
This study was supported in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil
(CAPES) - Finance Code 001.
 73

Analysis of carbofuran and chlorpyrphos-methyl

in GC-MS under different conditions of the
injection port and column oven temperatures
Rosa, Victória Gomes1; Bairros, André Valle2; Ugalde, Gustavo Andrade2; Chimendes,
Nayomi de Andrade2; Santos, Lara Celestina2; Pacheco, André Lucas Bezerra2; Reginato,
Fernanda Ziegler2; Berlato, Dener Gomes2; Nascimento, Marcelo Henrique Santana2
1
Nucleus Applied to Toxicology (NAT); 2 NAT.

Introduction: Brazil is one of the biggest consumers
of chemical products such as carbamates and
organophosphates pesticides. According to the
Phytosanitary Pesticides System (Agrofit) from
Brazilian Ministry of Agriculture, in 2014 there
was the largest increase in the consumption or
sales of pesticides in the country. Carbamates and
organophosphates act as cholinesterase inhibitors,
as they prevent the inactivation of acetylcholine,
and they are responsible for most cases of poisoning
in rural workers. Objectives: Develop a method to
analyze carbofuran (carbamate) and chlorpyriphos-
methyl (organophosphorate), in a gas chromatography
system coupled with a single quadrupole mass
spectrometer (GC-MS). Methods: It was used a gas
chromatography coupled to a single quadrupole mass
spectrometry (GC-MS) with an automatic injector
(Shimadzu, Kyoto, Japan). The column used was a
Rtx®-5 MS (30m x 0.25mm, 0.25µm film thickness)
with 5% diphenyl and 95% dimethyl polysiloxane.
Helium gas was used as carrier gas at a flow rate of
1.20 mL/min and 1µL was injected in splitless mode.
Different temperatures at the injection port were
tested: 175, 200, 225, 250, 275 and 300°C. In addition,
different configurations in column oven temperature
settings were tested as follows: Method 1: 50ºC (held
for 1 min) → 50 °C (10 °C/min) → 300°C (held for 3
min.), totaling 29 minutes; Method 2: 50ºC (held for 4
min) → 50 °C (35°C/min) → 190°C (held for 7 min.) →
190ºC (40 °C/min) → 300ºC (held for 1 min.), totaling
15.75 minutes. Interface and ion source temperatures
were kept at 300 °C. Mass spectra were recorded over
50-700 atomic mass units (amu) in the range of 0.30
scan/second. The analytes were identified by a GC-
MS applying electron impact ionization (EI) at 70 eV
to form the ions of interest in scan acquisition mode.
For the tests, the analytical standards of carbofuran
and chlorpyriphos-methyl at a concentration of
5 µg/mL in methanol were used. Results: The
column oven configuration that presented the best
performance for tested molecules and its thermal
degradation products was the method 2, as it
reduced the total analysis time per 13.25 minutes.
Carbofuran was degraded at the injection port at all
tested temperatures. Due to the fact that most of
carbamates are thermally unstable, as is the case of
carbofuran, breakage occured at the injector port or
column to their phenolic and amino acid counterparts.
For the best column oven configuration (method 2),
carbofuran-phenol retention time was 5.134 minutes
while carbofuran retention time was 8.019 minutes.
Chlorpyrifos-methyl showed best chromatographic
behavior in method 2 applied in the column oven and
the temperature at the injection port that presented
the best chromatographic performance was 175 °C.
When the higher tested temperatures were applied at
the injection port, greater peak areas for the thermal
degradation product of chlorpyrifos-methyl (retention
time of 10.388 minutes) and formation of carbofuran-
phenol were obtained in a directly proportional way.
Conclusion: Concluded that the analytical method
used was efficient for chlropyrifos, and may be very
useful for toxicological analysis while carbofuran
required other strategies for its determination in GC-
MS. Acknowledgements: We wish to thank PROBIC/
FAPERGS-CNPq for providing financial support.
 74

Analysis of cholinesterase inhibitor pesticides

by GC-MS in larvae of Lucilia cuprina
(Calliphoridae) for entomotoxicological studies
Ugalde, Gustavo Andrade1; Chimendes, Nayomi de Andrade1; Pacheco, André Lucas Bezerra1;
Santos, Lara Celestina1; Reginato, Fernanda Ziegler1; Berlato, Dener Gomes1; Cardoso, Leonardo
Corrêa1; Nascimento, Marcelo Henrique Santana1; Rosa, Victória Gomes1; Santos, Rachel1;
Pereira, Alessandra de Oliveira2; Monteiro, Silvia Gonzalez2; Bairros, André Valle1
1
Nucleus Applied to Toxicology (NAT); 2 Parasitic Disease Laboratory (LAPAVET).

Introduction: When human body is in an advanced
state of decomposition where tissue toxicological
analysis is impracticable, insect larvae can be
considered an alternative option for researching
toxicants. Entomotoxicological studies with
cholinesterase inhibitors (carbamates and
organophosphates) are scarce using this alternative
matrix. Objective: The objective of this work was
the development of an analytical method able
to determine organophosphate and carbamates
pesticides through gas chromatography coupled to
mass spectrometry (GC-MS), followed by subsequent
application of recovery studies in L3 stage larval
matrix of Lucilia cuprina. Methods: Analyzes were
performed on a GC-MS with a Rtx®-5MS column (30
m x 0.25 mm, 0.25 µm film thickness). Helium was
the carrier gas at a constant flow rate of 1.20 mL/
min. The chromatographic parameters used were as
follows: injector temperature 175°C; 1 µL of injection
volume in splitless mode; column oven setting: 50°C
(held for 4 min) → 50°C (35°C/min) → 190°C (held for
7 min.) → 190°C (40°C/min) → 300°C (held for 1 min.),
totaling 15.75 minutes of chromatographic execution.
After obtaining the L3 stage larvae, the QuEChERS
method, proposed by Anastassiades et al. (2003),
with modifications and two variations in cleanup step
were employed in order to test analytes recovery. The
steps are summarized below: 1g of the homogenized
larvae was weighed into a 15 mL falcon tube,
then the analytical standards (Aldicarb, Carbaryl,
Carbofuran, Methomyl, Propoxur, Carbophenothione,
Chlorpyriphos Ethyl, Chlorpyriphos Methyl, Ethion,
Fenitrothion, Formotion, Malathion, Malaoxon,
Paraoxon Ethyl, Paraoxon Methyl, Pyrimphos Ethyl,
Pyrimiphos Methyl, Triazophos) and the internal
standard were spiked at a concentration of 50
ng/g were added in duplicate. Afterwards, 2 mL of
acetonitrile were added and the tubes were manually
shaked for 1 minute. Then 1 g of NaCl and 4 g of MgSO4
were added. Then, two methodologies were tested as
cleanup step. Method 1: After centrifugation (4,000
RPM, for 10 minutes), 1 mL of the supernatant was
transferred to another 15 mL falcon tube, with 150 mg
of NaCl and 25 mg of Primary Secondary Amine (PSA)
previously weighed. Method 2: After centrifugation
(4,000 RPM, for 10 minutes), 1 mL of the supernatant
was transferred to another falcon tube, with 150 mg
of NaCl, 25 mg of Secondary Primary Amine (PSA)
and 25 mg of C18 sorbent. Finally, the tubes were
manually shaked for another 1 minute, centrifuged
at 4,000 RPM and 500 µL of the supernatant was
transferred into a 2 mL chromatography vial and
dried in a sample concentrator, resuspended in 50
µL of ethyl acetate and 1 µL was injected into the
GC-MS. Results: Eight analytes (Ethion, Aldicarb,
Methyl and Ethyl Chlorpyriphos, Malaoxon, Methyl
and Ethyl Pyrimiphos and Triazophos) were
successfully recovered by both cleanup methods
tested, however a better recovery was obtained
with the method whose cleanup step was performed
with isolated PSA sorbent not associated with the
C18 sorbent with the following calculated recovery
values: 52.4% for Ethion, 33.1% for Aldicarb, 32.3%
for Methyl Chlorpyrifos, 20% for Malaoxon, 65.7%
for Methyl Pirimiphos, 32% for Ethyl Cholrpyrifos,
12% for Ethyl Pirimiphos and 9% for Triazofos.
Conclusion: The proposed methodology allowed
determinate eight from 19 analytes tested. This
work is part of doctoral thesis and will continue to
be optimized in order to involve the recovery of all
tested analytes. Acknowledgements: We wish to
thank PROBIC/FAPERGS-CNPq for providing financial
support.
 75

Case report of valproic acid intoxication:

the relevance of chromatographic methods
for elucidating cases in hospitals
Sampaio, Amanda Mello Kasper Vaz; Reginato, Fernanda Ziegler; Bairros, André
Valle; Roehrs, Miguel; Lovatel, Ivy Bauer; Ugalde, Gustavo Andrade

Núcleo Aplicado à Toxicologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria

Background/Introduction: A 15-year-old patient
was referred to the university hospital with high
sensory relegation, coma and under intubation.
The consumption of 60 quetiapine tablets (25 mg)
and the suspicion of other central nervous system
depressant drugs in the 12 hours prior to hospital
admission were reported. However, toxicological
analysis based in immunoassay did not detect any
drug. So, chromatographic techniques were employed
to elucidate this case. Objective: Emphasize the
importance of toxicological analysis, especially
chromatographic methods in cases of intoxication
arriving in hospitals. Methods: Rapid test for
qualitative detection of multiple drugs of abuse by
immunochromatography (Abon®) in NaF plasma and
urine samples was employed in the first moment.
Quetiapine analysis by liquid chromatography with
ultraviolet-visible detector (LC-UV/Vis) in 500 µL NaF
plasma sample in pH 10 extracted with hexane (2.5
mL). After homogenization (2 min) and centrifugation
(4000 rpm / 10 min), solvent was dried, resuspended
with 100 µL of mobile phase and 20 µL was injected.
Mobile phase was composed by phosphate buffer
pH 5.5/methanol/acetonitrile (30:30:40) in isocratic
mode (1.0 mL/min) using analytical column (C18 4.6
x 150 mm x 5 µm) at 225 nm. Medazepam was used
as an internal standard. Valproic acid determination
in serum sample (0.5mL) was based in liquid-liquid
extraction (LLE) by HCl (30 µL, 2M), after addition of
ethyl acetate (1.5 mL) and mixed on the vortex (1 min).
Centrifugation (4000 rpm for 5 min) and passage
of 1.25 mL of solvent into a tube containing sodium
sulfate. An aliquot of this extracted solvent (500
µL) is put into a vial and 1 µL is injected in the gas
chromatography coupled to mass spectrometry (GC-
MS) in split mode (1:10). Initial temperature was 70°C
(3 min), increasing 35°C/min to 180°C (3 – 6 min) and
50°C/min to 280°C (6 – 8 min). Final temperature fixed
for 4 min. Total chromatography time was 12 min. Flow
rate in the first 7 min was 1.5 mL/min. It was increased
to 2.5 mL/min over 6 min and held at that rate until
the chromatography was finished. Retention time of
valproic acid under these conditions was 5.50 min.
Mass spectrometer operated in electronic ionization
(EI) mode with 70 eV applying Selected Ion Monitoring
(SIM) mode [m/z = 73, 102 and 115]. Results:
Immunochromatography test was undetectable for the
tested drugs in urine and plasma samples. Quetiapine
was detectable (limit of detection = 150 ng/mL),
however it was not quantified (limit of quantification
= 500 ng/mL). Valproic acid concentration in serum
sample was 977.96 µg/mL, considered potentially
fatal. Discussion/Conclusion: The reports from this
case mentioned high self-administration of quetiapine
tablets. From an analytical investigation and new
information during the patient’s hospitalization,
we proved that quetiapine was not involved in this
case, while high concentrations of valproic acid were
determined in GC-MS, confirming the intoxication
of the patient. Toxicological analyzes are still rarely
used in the laboratory routine at a hospital in Brazil.
Chromatographic techniques, especially liquid
chromatography and gas chromatography, allow
the analysis of different class of drugs and other
substances that immunoassay tests did not evaluate.
The use of chromatographic methods in order to help
hospitals for intoxications cases are essential for the
diagnosis and prognosis of intoxications as well as
the choice of the best treatment. Acknowledgments:
UFSM
 76

Detection of cobalt in human urine by

liquid chromatography coupled with
high resolution mass spectrometry
for anti-doping control purposes
Santos, Vanessa Farelo1,2; Carneiro, Gabriel Reis Alves1; Coelho, Matheus Campelo da
Costa1; Machado, Sérgio de Paula2; Pereira, Henrique Marcelo Gualberto1
1
Laboratório Brasileiro de Controle de Dopagem, Chemistry Institute, Federal
University of Rio de Janeiro; 2 Laboratório de Química Inorgânica Computacional,
Chemistry Institute, Federal University of Rio de Janeiro.

Introduction: The World Anti-Doping Agency
(WADA) accredited laboratories must follow a List
of Prohibited Substances and Methods [1], updated
every year. Some analytical strategies are developed
to cover most of it. However, the analysis of cobalt,
which was incorporated in 2015 to the list, is yet a
challenge because of its incompatibility with the
classical strategies available in the anti-doping
laboratories (i.e. liquid chromatography and gas
chromatography coupled with mass spectrometry).
The supplementation with cobalt salts has effect
on stimulus of erythropoiesis via stabilization of the
hypoxia-inducible factors. In 2015, Thevis et al. firstly
reported products containing significant amounts
of undeclared cobalt and nickel species in products
advertised as erythropoiesis-stimulating agents
by inductively coupled plasma mass spectrometry
(ICP-MS) [2]. Objective: The project aims to develop
a cost-effective approach for the detection of cobalt
in athletes’ urine employing liquid chromatography
coupled with high resolution mass spectrometry (LC-
HRMS). Methods and results: To allow the detection
of metal ions in urine by LC-HRMS, it is necessary its
complexation from the matrix with a ligand. Based
on the reported formation of cobalt complex with the
diethyldithiocarbamate (DDC, (C2H5)2NCSS-) ligand [3],
it was the complexation strategy adopted to achieve
the results necessary to show the potentiality of
the proposed method. Due to the same coordination
number, oxidation state and geometry of the complex,
rhodium was proposed as one quality control for the
analysis. The method development was guided by
many stages of Design of Experiments (DoE) in both
instrumental analysis and sample preparation (e.g.
in the electrospray ionization, mass spectrometry
conditions, extraction with organic solvent,
complexation, composition of mobile phases and pH
of the synthesis). The final setup involves a liquid-
liquid extraction with methyl tert-butyl ether, in which
the organic layer is transferred to another tube and
evaporated to dryness in a thermostatic bath under a
nitrogen stream at 40°C. The samples are reconstituted
with 50 µL of the mixture water pH 4 (5mM ammonium
formate/formic acid) and acetonitrile (30:70).
A Thermo Dionex Ultimate 3000 UHPLC system
coupled to a QExactiveTM hybrid quadrupole-orbitrap
mass spectrometer equipped with an electrospray
ionization source is used. Separation is performed in
a reversed-phase column (Syncronis – Thermo, USA,
C18, 1.7 µm, 50 mm x 2.1 mm) at 40°C in a five-minutes
isocratic chromatographic run (30% water pH 4 and
70% acetonitrile), at a constant flow rate of 300 µL
min-1. Conclusion: As a conclusion, it was possible to
detect cobalt as its DDC complex in a cost-effective
and high throughput analysis by LC-HRMS. As a future
perspective of the project, the method will be validated
according to WADA guidelines for further application
in anti-doping purposes. Acknowledgments: The
authors thank Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) and Autoridade
Brasileira de Controle de Dopagem (ABCD).
REFERENCES
1. World Anti-Doping Agency. Prohibited List 2022, Montreal,
2022. Available at https://www.wada-ama.org/sites/default/
files/2022-01/2022list_final_en_0.pdf.
2. Thevis, M, et al. Solutions advertised as erythropoiesis-
stimulating products were found to contain undeclared cobalt
and nickel species. Int J Sports Med. 2015;36: 82-84.
3. Minakata, K, Suzuki, M, & Suzuki, O Application of electrospray
ionization tandem mass spectrometry for the rapid and
sensitive determination of cobalt in urine. Analytica Chimica
Acta. 2008;614:161-164.
 77

Determination of 5-fluorouracil in dried

blood spots (DBS) by UPLC/MS-MS
Silva, Laura Cé1,2; Grando, Ana Paula1; Hahn, Roberta Zilles1; Linden, Rafael1,2; Antunes, Marina Vezon1,2
1
Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate
Program on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil

Background: 5-Fluorouracil (5-FU) is an antimetabolite TQS-micro triple quadrupole mass spectrometer

drug that belongs to the class of fluoropyrimidines.
It is a compound analogous to endogenous uracil,
having a fluorine atom in the C-5 position. Since it was
synthesized in the 1950s, 5-FU remains one of the
most used chemotherapeutics to treat various types
of cancer, especially gastrointestinal tumors. Among
patients treated with 5-FU, only 20-30% reach the
therapeutic target and its use has been associated
with potentially severe toxicity, being a candidate for
therapeutic drug monitoring. Considering the ease
of collection and the intrinsic logistical advantages
of dried samples, the availability of a finger-prick
dried blood spots (DBS) assay for measuring 5-FU
concentrations has the potential to allow identification
of patients who needs dose adjustments. Up to date
there is no report of a DBS-based method for 5-FU
quantification. Objective: To develop and validate
a method for determination of 5-FU in dried blood
samples (DBS) by UPLC/MS-MS. Methods: 5-FU
were extracted from 8 mm DBS punches with 100 µL
of ultrapure water containing IS (5-clorouracil 0.3
ng mL-1) incubated for 10 minutes at 46 °C and 1000
rpm, followed by 10 minutes ultrasound incubation.
After, the aqueous extracted was precipitated with
200 µL of methanol cooled at -20 ºC for 15 min. After
centrifugation, 100 µL of supernatant was transferred
to a new tube and diluted with 100 µL of water with
0.5% acetic acid. After homogenization the extract
was transferred to vial and 1 µL injected onto UPLC/
MS-MS Acquity® I-Class UPLC coupled to a Xevo®
with electrospray ionization in positive mode.
Chromatographic separation occurred in an Acquity®
UPLC HSS C18 (1.8 µm 2.1 x 150 mm) at 25 °C. Mobile
phase was water with 0,5% acetic acid (eluent A) and
acetonitrile with 0.5% acetic acid (eluent B) eluted in
gradient mode from 95:5 to 10:90 (A:B, v/v) at flow rate
of 0.25 mL min -1. The MRM transition for quantification
were 5-FU m/z 131 → 58 and IS 5-CU m/z 147 → 74.
The method was validated according to FDA, to date
specificity linearity, precision/accuracy, sensitivity,
selectivity and matrix effect tests have been
completed. Results: Total analytical run time was 5
min, retention time was 1.94 min for 5-FU and 2.85 min
for IS. The method was selective, with no interference
peaks eluting at the same relation time as 5-FU. The
lowest limit of quantification 100 ng mL-1 presented
accuracy of 115% and 3.39 % inter assay and 2.50 %
intra assay CV%. The method was linear from 100 to
2000 ng mL-1 (r2 > 0.99), with accuracy of 91-102%,
precision with CV% intra-assay within 3.24-4.47%,
inter-assay within 5.0-14.51%, and mean extraction
yield 70%. The internal standard compensated
adequately the matrix effect from +4.96 to +14.49%.
Discussion/Conclusion: The method has been shown
to be adequate for determining the concentrations
of 5-FU in DBS samples. After completion of the
validation tests regarding the impact of hematocrit
on assay accuracy and extraction yield, the method
will be applied in a clinical study. Acknowledgments:
Financial support from Fapergs, CAPES and CNPq.
 78

Determination of cocaine and metabolites

applying dried spot oral fluid using
well plate by HPLC-MS/MS
Borges, Gabriela Ramos1,2; Santos, Bruno Pereira1,2,3; Gouveia, Giovanna Cristiano1,2; Dalanhol,
Carolina Silveira1,2,3; Scherer, Juliana2; Eller, Sarah1; Oliveira, Tiago Franco1
1
Pharmacosciences Department, Federal University of Health Sciences of Porto Alegre (UFCSPA), Brazil;
2
Center for Drug and Alcohol Research and Collaborating Center on Alcohol and Drugs, Hospital de
Clínicas de Porto Alegre (CPAD/HCPA), Brazil; 3 Federal University of Rio Grande do Sul (UFRGS), Brazil.

Background/Introduction: Cocaine is one of the most
illicit drugs used in Brazil. This substance is known
for the significant risks to users and leads up to
significant public health issues, compromising one’s
quality of living. In this context, the development of
new techniques for drug detection applied in a simple
way to unconventional biological matrices, such as
oral fluid, can be an important tool for toxicological
analysis. Objective: This study aims to develop and
validate a dried spot oral fluid using well plate sample
preparation technique by LC-MS/MS for analysis
of cocaine (COC), ecgonine methyl ester (EME),
benzoylecgonine (BE), cocaethylene (CE) e o-hydroxy
cocaine (COC-OH) in oral fluid. Methods: Oral fluid
samples, collected by expectoration, were obtained
from patients in different addiction treatment services
in, Porto Alegre, RS. Sample Procedure: For sample
processing, 24-well plates were used as support. In
each well were placed four uniform layers of filter
paper. Oral fluid samples were centrifuged during 5
minutes at 10000 rpm and diluted in ultrapure water in
the proportion 1:1 (v/v). An aliquot of 100μL of sample
was pipetted into each well and dried for 60 minutes
at room temperature. Then, 250μL of a mixture of
methanol and acetonitrile in the proportion 1:3 (v/v)
were added. The internal standards, BE-d3 and COC-d3,
were added to the solvent. The plate was shaken
for 5 min at 150 rpm, the supernatant was collected
and 2μL was injected in the analytical system.
The extraction solvent was determined through a
simplex-centroid design by the software Statistica
8.0. The drying time and the number of layers of filter
paper used were also evaluated. Instrumentation: A
Shimadzu liquid chromatograph mass spectrometer
LCMS-8045 was used with an electrospray source
operating in positive ion mode as follows: capillary
voltage, 4000 V; nebulizer gas, 3L/min; drying gas,
10L/min; drying temperature, 250°C; heating block
temperature, 400 °C. The chromatographic separation
was achieved on a Shim-pack GISS column (100 mm
× 2.1 mm, 1.9μm), eluted with a gradient of water
(solvent A) and acetonitrile (B) both fortified with
0.1% formic acid, at a 0.4mL/min flow rate and 40 °C.
Results: Following the recommendations of ANSI/
ASB Standard 036, the developed method has been
validated for lower limit of quantitation (LLOQ),
linearity, within-run and between-run precision, bias,
selectivity, ionization suppression/enhancement
and carryover. The LLOQ was 1 ng/mL for all of
the analytes. The concentration ranges used were
1-100ng/mL for EME, COC, BE, COC-OH, CE and all
analytes presented a coefficient of determination
(r2) ≥ 0,99. The occurrence of heteroscedasticity was
verified and the weighting factor of 1/x2 was applied.
The within-run and between-run precision range
between 2.2 - 12.04%, considering relative standard
deviation. The bias for all the analytes varied between
90.9 – 109.6%. No significant level of interfering
substances was observed. Suppression of ionization
was observed for all analytes ranging between -50.6
– 71.9%. No carryover effects were detected. After
the full validation, 110 samples were analyzed. Of
these, 90 samples (81.8%) were positive for BE, 81
samples (73.6%) for COC, 33 (30%) for EME, 4 (3.6%)
for CE and one (0.9%) for COC-OH. Discussion/
Conclusion: All parameters evaluated were within
the limits recommended by the guidelines. The
suppression in ionization was probably caused by the
phenomenon of the matrix effect, although the other
validation parameters were not negatively affected.
Furthermore, the sample processing has optimized,
up to 24 samples can be prepared, simultaneously.
The present study demonstrated that the method was
successfully developed and validated for determining
the presence of cocaine and its metabolites by LC-
MS/MS, proving to be an analytical alternative,
simplifying the process of analysis of fluid oral.
Acknowledgments: FAPERGS; CAPES; CPAD/HCPA.
 79

Determination of cortisol and cortisone in

urine samples from patients with Parkinson’s
disease by dispersive tip microextraction
solid phase and ultra-efficient liquid
chromatography tandem-mass spectrometry
Kakuda, Priscila1; Souza, Israel Donizeti1; Tumas, Vitor2; Queiroz, Maria Eugênia Costa1

Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto,

1

Universidade de São Paulo, Ribeirão Preto, Brazil; 2 Department of Neuroscience and

Behavior, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.

Parkinson’s disease (PD) is a progressive and
multifactorial neurodegenerative disease. Studies
in PD patients have evidenced augmented cortisol
levels, which can be related to anxiety and depression
disorders and may exacerbate motor symptoms
and compulsive behavior, besides contributing to
negative PD progression. We have developed and
validated new strategies to determine possible
endocrine biomarkers, like cortisol and cortisone,
in urine samples from PD patients by using a
dispersive tip microextraction solid phase and
ultra-high performance liquid chromatography
tandem mass spectrometry (UHPLC-MS/MS). This
efficient microextraction technique consumes less
organic solvent and requires small sample volume.
Turbulent air bubble mixing creates a suspension
of sorbent in the sample, ensuring optimal contact
and highly efficient extraction. Microextraction
was carried out by using modified pipette tips filled
with a HLB (hydrophilic lipophilic balanced) phase
loosely accommodated between two porous filters.
The variables washing solvent volume, washing
organic solvent concentration, sorption equilibrium
time, sample pH, sample volume, and elution solvent
were evaluated. The proposed method was linear at
concentrations ranging from 0.5 ng mL-1 (LLOQ) to 500
ng mL-1 (R = 0.9983) for cortisol and from 3 ng mL-1
(LLOQ) to 500 ng mL-1 (R = 0.9972) for cortisone. The
precision assays gave RSD values ranging from 0.9%
to 13.3% and accuracy ranging from 83.5% a 113.4%,
without significant matrix effect. We successfully
applied this innovative UHPLC-MS/MS method to
determine cortisol and cortisone in urine samples
from PD patients. Acknowledgments: CAPES -
Process numbers 88887.501207/2020-00
 80

Determination of Ethyl Glucuronide

and Ethyl Sulfate in urine samples
from brazilian truck drivers
Costa, C.D.D.; Oliveira Neto, J.R.; Oliveira, N.R.L.; Alcântara, K.C.; Cunha, L.C.

Universidade Federal de Goiás - Rua 240 - Setor Leste Universitário, 74605-170 Goiânia, GO, Brasil

Background: According to the National Registry of
Traffic Accidents and Statistics/RENAEST, in the last
four years Brazil recorded about 3,190,830 traffic
accidents on national highways, with the consumption
of licit and illicit substances by drivers, mainly the
alcohol, one of those responsible for these numbers.
Objective: The aim of this study was to identify the
presence of ethanol consumption markers, being
Ethyl Glucuronide (EtG) and Ethyl Sulfate (EtS) in urine
samples from truck drivers. Methods: A total of 321
truck drivers were interviewed at a gas station on
BR-153, in West Center, between February 2014 and
February 2015 and collected approximately 50 mL of
urine for toxicological evaluation for the presence
of alcohol metabolites. EtG and EtS measurements
were performed by liquid chromatography coupled to
mass spectrometry (LC-MSMS), using a reverse phase
column and a gradient of acetonitrile and ammonium
formiate as mobile phase. Deuterated analytes were
utilized as internal standards, EtG-D5 and EtS-D5.
Few volume of urine (100 µL) was used, with simple
dilution, followed by centrifugation, before injecting
into the chromatographic system. The lower limit
of quantification (LIQ) established for both markers
was 100 ng/mL. Basics descriptive statistics were
performed, including percentage frequency, median
and means were processed and analyzed using the
software Epi Info. To analyse the association between
the variables was used multiple correspondence
analysis and hierarchical cluster analysis. Results:
All of them were male with a median age of 42 years,
most of them were married, low scholarity. 47%
related consumption moderate or high risk of alcohol
and tobacco and 21% use of cocaine, cannabinoids,
amphetamine, hypnotics/sedatives, opioids,
inhalants. Measurement of urinary EtS and EtG was
performed by LC-MS/MS and showed 34.5% (n=111) of
positive results for both markers and 15% (n=48) for
just EtG. Discussion/Conclusion: Markers of recent
alcohol consumption were identified in the urine
samples, as well as it was found that truck drivers
consume tobacco, cocaine and other illicit drugs
during the working day and this behaviors increase the
risk of being involved in accidents on the highways.
from the country. Based on this, measures are needed
to improve the quality of life of drivers, as well as
to prevent and treat individuals who use alcohol in
traffic accidents. Acknowledgements: UNODC, UFG.
 81

Determination of pharmaceuticals
and personal care products (PPCPs) in
surface water in Southern Brazil
Bastiani, Marcos Frank; Hahn, Roberta Zilles; Lizot, Lilian de Lima Feltraco; Bondan, Amanda
Pacheco; Castilhos, Maria Amélia; Freitas, Mariana; Favreto, Camila; Linden, Rafael

Feevale University

Background: In recent decades, the consumption
of pharmaceuticals and personal care products
(PPCPs) grew up all over the world, both because
of the increased availability and the ease of access.
PPCPs are chemical products, synthetic or natural,
that can be found in pharmaceuticals, over-the-
counter products, deodorants, repellents, fragrances,
sunscreens, among others, all of which are widely
used throughout the world. PPCPs are known to be
released into aquatic environments through various
routes, including domestic wastewater, hospital
wastewater, improper disposal by the manufacturers,
wastewater treatment plants, and water treatment
plants. Objective: The objectives of this work were
to identify and endocrine disrupting chemicals
(bisphenol A, estradiol, and ethinylestradiol),
antimicrobials and other pharmaceuticals such
as antibiotics, anti-inflammatories, analgesics,
lipid regulators, β-blockers, antidepressant, and
antiepileptics in surface waters used as influents of
water treatment plants located in 5 cities of the Vale
do Rio dos Sinos region, the Rio Grande do Sul, Brazil.
Samples are being collected fortnightly. Methods:
Compounds of interest were extracted from 200 mL
surface water samples using solid-phase extraction
with an OASIS® HLB (3 ml/60 mg) cartridge. SPE was
automatically performed using an ASPEC GX-271
device. Estradiol, ethinylestradiol, and bisphenol A
were derivatized with dansyl chloride prior to mass
spectrometric detection. Derivatization conditions
were selected based on a statistically designed
experiment and response surface analysis. The
evaluation of the experimental data was performed
using Design Expert 13® (Stat-Ease, USA). The
optimized derivatization conditions were incubation
time of 5 minutes, incubation temperature of 60 °C,
and concentration of dansyl chloride solution of
2.5 mg/mL. The extracts were analyzed by liquid
chromatography-tandem mass spectrometry (LC-
MS/MS), with electrospray ionization both in positive
and negative mode. Results/Discussion: The curves
were linear with a correlation coefficient (r) greater
than 0.99. The compounds measured in surface
water and concentration ranges were the following:
amoxicillin - 300 to 765 ng L-1; atenolol - 5.64 to 89.5
ng L-1; azithromycin - 3.8 to 13.6 ng L-1; bezafibrate -
0.43 to 4.35 ng L-1; carbamazepine - 4.65 to 24.1 ng L-1;
caffeine - 207 to 4730 ng L-1; ciprofloxacin - 0.52 to
2.74 ng L-1; diclofenac - 9.8 to 58.2 ng L-1; fluoxetine
- 1.20 to 2.27 ng L-1; ibuprofen - 14.32 to 185.8 ng L-1;
propranolol - 13.95 to 77.24 ng L-1; salicylic acid - 7.55
to 20.5 ng L-1; sulfamethoxazole - 0.5 to 57.5 ng L-1;
triclosan - 6.48 to 39.5 ng L-1; bisphenol A - 12.15 to
86.67 ng L-1; β-estradiol - 44.2 to 697.8 pg L-1; 17-α-
ethinylestradiol - 132.15 to 819.6 pg L-1. Conclusion:
Significant concentrations of various compounds
were found in Sinos river and these concentrations
will be used to assess ecotoxicological risks. The
monitoring of PPCPs in surface waters is essential to
evaluate the presence of possible ecotoxicological
and human health risks. Acknowledgments: Feevale
University, CAPES.
 82

Determination of psychoactive substances

in sweat samples using disposable
pipette extraction (DPX) and GC-MS
Gomes, Nayna Cândida1; De Martinis, Bruno Spinosa2
1
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Ribeirão Preto, São Paulo, Brasil;
2
 Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Ribeirão Preto, São Paulo, Brasil.

Introduction: There is little scientific information
available on the actual composition of drugs sold
and consumed, which are often masked or unknown.
Objective: Develop and validate a method for
analyzing amphetamine, methamphetamine, MDMA,
MDA, MDEA, cocaine, cocaethylene, dibutylone,
n-ethylpentylone, 25E-NBOMe, 25C-NBOMe, 2C-E,
2C-C, fentanyl and carfentanyl in a sweat using the
DPX-CX1 and GC-MS. Methods: 650µL of artificial
sweat were applied on absorbent cellulose paper.
They were fortified with the analytical standards
and with the internal standards amphetamine-d11,
fentanyl-d5 and cocaine-d3. They were submitted
to the extraction process using the DPX-CX1 tip.
This filter paper was inserted into a glass tube. 2ml
of methanol was added, then vortexed and on the
shaker table. Subsequently, 1mL of this solution was
transferred to another glass tube and 100μL of 0.1 M
H3PO4 was added. The conditioning of the DPX-CX1
tip was done with 1mL of acetonitrile. Then, 1mL of
the solution containing 0.1MH3PO4 was aspirated.
Afterwards, it was washed with 500μL of methanol.
For the elution of the analytes, 1500μL of a solution of
dichloromethane:isopropanol:ammonium hydroxide
(78:20:2) was used.The eluate was evaporated
under nitrogen flow, reconstituted with 40μL of
ethyl acetate. It was then derivatized with 40μL of
MSTFA. The separation was carried out in a HP5 fused
silica capillary column (30mx0.25mmx0.25μm film
thickness). The injector temperature was 280°C in
splitless mode and the injection volume was 1µL. Helium
was used as a carrier gas. The initial oven temperature
was 90°C, remaining for 2 minutes, and later, it was
raised to 220°C at a heating rate of 10°C.min-1. Then,
the temperature was raised to 290°C at a heating rate
of 20°C.min-1, remaining for 4 minutes, totaling 22.5
minutes of analysis.The mass spectrometer with a
quadrupole mass analyzer was initially used in the full
scan operation mode (80 to 490m/z) and later it was
operated in the SIM mode.The ionization mode used
was electron ionization. The temperature used in the
MS interface, source and quadrupole was 280, 230
and 150°C, respectively. The parameters evaluated
during the validation of the method were those
recommended by resolution 27/2012 and 899/2003
of ANVISA. Results: The detection limit ranged from
1ng/patch to 15ng/patch for amphetamine, MDMA,
MDA, dibutylone and n-ethylpentylone analytes, 2C-E
and 2C-C, respectively. The analyzed interferents
did not interfere in the retention time, identification
and quantification of analytes. No residual effect
was observed either. The matrix effect ranged from
2.12% for fentanyl to 9.32% for MDEA. The method
was considered linear in the concentration range of
2 to 400ng/adhesive for amphetamine, MDMA, MDA,
dibutylone and n-ethylpentylone, from 3 to 400ng/
adhesive for methamphetamine, from 5 to 400ng/
adhesive for MDEA, 25C- NBOMe, fentanyl and
carfentanyl, from 10 to 600ng/adhesive for cocaine,
cocaethylene and 25E-NBOMe and from 30 to 1100ng/
adhesive for 2C-E and 2C-C. Analyte recovery values
range from 71.34% to 98.79% for the HQC of 2C-C and
dibutylone, respectively. The intraday precision ranged
from 1.13 to 14.79%, which correspond to the LQC and
MCQ values of the 2C-E and MDEA, respectively. And
the interday precision ranged from 1.23 to 18.50%,
which were the LQC values of 2C-E and LLOQ of MDMA,
respectively. The intraday accuracy values ranged
from -9.18% to 17.86% which correspond to the MCQ
and LLOQ values of amphetamine. And the intraday
accuracy ranged from -10.89% for the amphetamine
MQC to 19.11% for the MDMA LLOQ. The analytes were
stable in both the biological sample and the standard
solution. Discussion/Conclusion: The method
developed and validated met all the criteria required
by resolution 27/2012 and 899/2003 of ANVISA.
Acknowledgments: This research was supported
by CAPES – Brazil (Financing Code 001) and FAPESP
2017/18021-5.
 83

Determination of synthetic cannabinoid

receptor agonists (SCRAs) in infused papers and
herbal materials seized in Brazilian prisons
Martins, Aline Franco1,2; Barbosa, Ingrid Lopes3; Milanez, Guilherme Paier3; Costa, José Luiz2,4
1
Faculty of Medical Sciences, University of Campinas, Campinas, SP, Brazil; 2 Campinas Poison
Control Center, University of Campinas, Campinas, SP 13083-859, Brazil; 3 Superintendence
of the Technical-Scientifc Police, Institute of Criminalistics, São Paulo, SP 05507-060, Brazil;
4 
Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, SP 13083-871, Brazil .

Introduction: As reported by Rodrigues et al. (2021),
the number of synthetic cannabinoid receptor agonists
(SCRAs) seizures in Brazilian prison systems are
increasing, mostly as infused papers. A study realized
in infused papers seized in Scottish prisons from June
2018 to September 2019, observed an increase over
time in the number of samples in which multiple SCRAs
were detected and a variation of which SCRAs became
the most detected. 5F-MDMB-PINACA (5F-ADB),
5F-MDMB-PICA, 4F-MDMB-BINACA, and MDMB-
4en- PINACA were the most detected at different
times during the study, which reinforces the need
for constant monitoring of these substances in the
market. The present study described the identification
and quantitation of SCRAs in 23 infused paper and in 3
herbal material samples, seized in prisons located in
São Paulo state. Methods: The selected samples were
previously analyzed by the São Paulo State Police, in
a qualitative analysis by gas chromatography–mass
spectrometry (GC–MS) using full-scan and library
search for identification. Afterward, these samples
were analyzed by liquid chromatography-tandem
mass spectrometry in a triple quadrupole (LC-MS/MS)
system operating in MRM mode for determination of
the majority and the possible minority SCRAs present
in the samples. For the LC-MS/MS analysis the
samples were extracted with methanol and diluted
in a proportion of 1:1.000 and 1:10.000. Results and
discussion: All substances identified by GC-MS were
also identified by LC-MS/MS, but not the opposite,
showing GC-MS full-scan method employed were
unable to detect those SCRAs presented in lower
concentration levels. The presence of a single SCRA
in the samples was observed in 11 samples (27.5%),
while in 29 samples (72.5%) two or more SCRAs were
identified. The SCRAs most frequently detected in
the samples were MDMB-4en-PINACA and ADB-
BUTINACA, present in 72% and 70% of the samples,
respectively. The concentration of SCRAs shown great
variability, ranging from 0.2 µg/infused paper to 3,000
µg/infused paper. In cases with more than one SCRA
detected, the minority compounds did not exceed the
concentration of 50 µg/infused paper. Comparing
two different samples of a single case it is possible
to identify the same substances but in different
concentration levels, or different compositions.
The highest concentration of SCRA present in the
infused papers was for the cases involving MDMB-
4en-PINACA as the major compound, reaching until
3,000 µg/infused paper, but this SCRA was also found
as minority in other cases, with concentrations as
low as 0.9 µg/infused paper. Among all samples,
4F-MDMB-BUTICA was detected in only one case, at
a concentration of 125 µg/infused paper. For the three
samples of plant material, ADB-BUTINACA appears as
the major compound, with the presence of other minor
SCRAs ranging from 0.2 to 5 µg/mg. Conclusions:
These results shows that the studied samples did
not present homogeneity in the composition and
concentration levels, even when comparing a single
case, but it seems that some SCRAs are commonly
found together. The presence of different SCRAs in
the same sample may contribute to the increase in
the toxicity of these compounds, which reinforces the
need to obtain quantitative and qualitative data from
the seized samples. Acknowledgments: The authors
thank the Superintendence of the Technical Scientific
Police of the State of Sao Paulo and the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) (Projeto INSPEQT, Edital nº 16/2020, Processo
88887.516176/2020-00).
 84

Development and validation of a method

for the determination of meropenem
employing micro samples of dried blood
spots obtained from capillary punctures
Busato, Maria Amelia de Castilhos; Antunes, Marina Venzon; Linden, Rafael

Universidade Feevale - FEEVALE.

Background/Introduction: Meropenem is an analytical system. The analytes were quantified in

antimicrobial drug characterized as bactericidal, widely
used in intensive care units. The PK/PD parameter
best correlated with the efficacy of carbapenems is
the time in which the free fraction of the drug remains
above the minimum inhibitory concentration of the
bacteria. Based on preclinical models, this time should
be greater than 40%. Moreover, high concentrations
of meropenem are associated with nephrotoxic
effects. Despite having a known therapeutic range of
exposure, therapeutic monitoring of meropenem is
scarcely used in our Country due to the unavailability
of assays for quantification of the drug. An alternative
to increase access to these tests is the use of dried
blood spots (DBS), which allows facilitated collection
and transport of specimens for testing. Objectives:
To develop and validate an assay method for
quantification of meropenem in blood microsamples
obtained in the form of DBS, using LC-MS/MS.
Methods: DBS samples were obtained by pipetting 50
μL of whole blood on Whatman 903 paper. After drying,
6 mm diameter punches of the paper were transferred
to plastic microtubes and added with 300 μL of the
extraction solution (acetonitrile: water, 70:30 v/v,
containing ciprofloxacine-D8 3.3 μg/mL), followed by
vortex-mixing for 15 min and sonication for 5 min. An
aliquot of 50 μL of the supernatant was transferred
to another tube and added with 200 μL of water and
200 μL of dichloromethane. After homogenization and
centrifugation, the supernatant was injected into the
a liquid chromatography system associated with a
tandem quadrupole mass spectrometer (LC-MS/MS),
operating in positive electrospray ionization mode.
The separation was performed in an Acquity C8 column
(100 x 2.1 mm, 1.7 μm), with mobile phases composed
of water and acetonitrile, both containing 0.1% formic
acid and a flow rate of 0.3 mL/min. Calibrators were
prepared in blood at the concentrations of 0.5, 1, 2,5,
5, 10, 25, and 50 μg/mL. Accuracy and precision were
evaluated at the concentrations of 0.5, 0,75, 12.5,
and 35 μg/mL. The method was validated according
to international guidelines, including evaluation of
specificity, stability, matrix effect, extraction yield,
and hematocrit effect. Results: The method was
linear in the range of 0.5 to 50 μg/mL. The inter-assay
precision was between 5.2 and 7.5%, the intra-assays
precision was between 6.4 to 7.5%, and the accuracy
was between 101.2 and 101.7%. No interfering factors
were identified in blank samples. Hematocrit had
a limited effect on the accuracy and the extraction
efficiency. The matrix effect was adequately
compensated by the internal standard. Meropenem
was stable for up to 24 h in DBS maintained at high
temperature. Discussion/Conclusion: A method for
the determination of meropenem in DBS using LC-MS/
MS was developed and validated. The method presents
adequate analytical performance for use in a clinical
study, which is in progress. Acknowledgments:
UNIMED Vale dos Sinos, CNPq, Feevale University.
 85

Development and validation of analytical

method for the quantification of chemical
elements in whole blood with volumetric
absorptive microsampling by ICP-MS
Sousa Júnior, Wellington Tavares; Morais, Déborah Araújo; Oliveira, Silvana Ruella; Barbosa Jr, Fernando

Analytical and System Toxicology Laboratory, Department of Clinical, Toxicological and Bromatological
Analyses, Faculty of Pharmaceutical Sciences of Ribeirão Preto, Ribeirão Preto - SP, Brazil.

Introduction: The use of biological specimens for
biomonitoring is important for assessing the risk
of environmental and occupational exposures
to toxic elements and evaluate the deficiency of
essential elements in the human body. The most
common method of sampling blood is venipuncture.
Alternative blood sampling techniques have been
proposed, such dried blood spot (DBS). This technique
consists in a collection of small volumes of whole
blood on filter paper. However, a number of issues
and challenges have been associated with the use
of the DBS approach. Main issues are the hematocrit
effect and the homogeneity of the sample. Volumetric
absorptive microsampling (VAMS) is a novel sampling
blood method. This device consists an absorbent tip
attached to a plastic holder designed to absorb a
fixed sample volume. The VAMS technique provides
the same advantages of DBS: reduced blood sampling
volumes, simplification of the sample collection,
simplification of pre-treatment methods, reduced
costs of shipment and storage. Moreover, the
VAMS overcome the issues with hematocrit and
homogeneity. Objective: The aim of this study was to
develop and validate a method for the multielemental
quantification of Li, Be, Mg, Mn, Co, Ni, Zn, Cu, As, Se,
Rb, Sr, Cd, Cs, Ba, Hg, Tl, Pb e Bi in whole blood collected
with VAMS, by using inductively coupled plasma mass
spectrometry (ICP-MS). Methods: The analysis were
performed by dilute-and-shoot procedure. Briefly, the
VAMS was added in an extraction solution containing
0.2% v/v HNO3 and 0.05% (v/v) Triton X-100 in the ratio
1:50 (v/v). The resulting solution was directly injected
into the ICP-MS. The VAMS of 20 μL were obtained
from Neoteryx®. Reference materials of whole blood
were used for method optimization and validation
purposes. A pre-treatment with VAMS were evaluated:
contamination from blanks analysis, absorbed volume
in the device tip and extraction conditions (vortex for
5 minutes, shaking for 60 minutes, ultrasonic bath
for 30 and 60 minutes). The following validation
tests were performed: limit of quantification (LOQ),
limit of detection (LOD), linearity, accuracy, intra and
inter assay precision. Results: The linearity of the
calibration plots provided a correlation coefficient
higher than 0.99 for all chemical elements. Estimated
LODs and LOQs of the proposed method ranged from
0.10 to 29.2 ng L-1 and 0.35 to 88.6 ng L-1, respectively.
The blank of the VAMS showed values <1 µg L-1 for Li,
Mg, Ni, Zn, Cu, As, Sr and Ba while Be, Mn, Co, Se, Rb, Cd,
Cs, Hg, Tl, Pb and Bi were below the limit of detection.
The volume of blood absorbed in the device tip were
22,50 ± 0,80 µL with no statistical differences (p<0,05)
from the manufacturer’s certificate of conformance
of 22,80 µL. Shaking extraction for 60 minutes had
low performance with the lowest recoveries found
for Mn, Bi, Cu and Hg with values of 51.4, 54.3, 57.2
and 49.0%, respectively. Vortex and ultrasonic bath
extraction exhibited good recoveries for all analytes
(close to 100%). The average extraction recoveries
found ranged from 96 to 106% while RSD values were
<10%. Discussion/Conclusion: The proposed method
were satisfactory and met the acceptance criteria of
the validation guidelines. To avoid recovery issues,
the extraction must be optimized. The ultrasound
bath for 30 minutes is an effective method for
chemical elements extraction which allows the
preparation of more samples in a relatively short
time. Moreover, the VAMS technique procedure could
be an attractive method for human biomonitoring
studies when regular monitoring of a population’s
blood is required or special group with low adhesion,
such as childrens. Acknowledgments: We thank for
the financial support of CAPES (Finance Code 001) and
CNPq (168344/2017‑3).
 86

Development of a HPLC-DAD-RF method

for quantification of neurotransmitters
in heads of Drosophila melanogaster
Carriço, Murilo Ricardo Sigal1; Rodrigues, Marina Diaz2; Paz, Maria Elizabeth Gomes1; Molina, Higor
Severo2; Nogueira, Caroline Lacerda1; Denardin, Elton Luis Gasparotto3; Roehrs, Rafael3,4
1
 Student of the Postgraduate Program in Biochemistry, Federal University of Pampa
– Uruguayan campus. RS; 2 Undergraduate student in Pharmacy, Federal University of
Pampa Uruguayan campus. RS; 3 Professor at the Federal University of Pampa – Uruguayan
campus. RS; 4 Professor at the Federal University of Pampa – Bagé campus. RS.

Drosophila melanogaster is an important experimental
model due to its high evolutionary conservation
of many human-like genetic and physiological
processes. D. melanogaster is used for studies
involving cancer, metal toxicity, nanomaterials,
depression and human neurodegenerative diseases
such as Parkinson’s and Alzheimer’s. Clinical and
experimental evidence has shown that changes in
monoaminergic neurotransmitters in the nervous
system are indicators of these neurodegenerative
diseases. In this sense, techniques that can accurately
identify and quantify these compounds, such as high-
performance liquid chromatography (HPLC), can be
important tools for establishing these neurotoxicity
indicators in various studies. In this perspective, the
present study aims to develop a chromatographic
method by HPLC for the quantification of octopamine,
dopamine, serotonin and tryptophan in the head of D.
melanogaster. To quantify the compounds, we used
high-performance liquid chromatography equipment
coupled to a Young Lin (YL) 9100 diode array
detector (HPLC-DAD) equipped with an Inertsil ODS-
3 5 µm column (4.6 × 250 mm). The compounds were
separated using the mobile phases (FM) composed of
water acidified to pH 2.55 (H3PO4) (A) and methanol
(B). With a gradient that consisted of FM 98:2 (A:B -
flow 0.4 mL.min-1) up to 6 minutes, increasing the
organic phase to 50:50 (A:B) up to 11 min returning
to initial conditions in 13 min (flow 0.5 mL.min-1), and
I reestablish the initial flow in 20 min and keeping
it that way for up to 30 min. An injection volume of
20 µL was used, the compounds were identified by
comparing their retention time and DAD spectrum
with the reference standards. The analytical curve
was constructed with Octopamine (198 nm), Dopamine
(198 nm) and serotonin (204 nm) and consisted of 6
points at concentrations of 0.1; 0.5; 1.0; 2.0; 5.0 and
10.0 mg.L-1 for each compound. The validation criteria
evaluated were selectivity, linearity, repeatability,
detection limit (LD), quantification limit (LQ). As an
application of the previously validated methodology,
30 flies (age 0 - 4 days) were frozen (-80 °C) and their
heads were homogenized with 400 µL of saline (0.9%
NaCl and 0.5 mol.L-1 HCl - 96:4) (n=3). Then the samples
were centrifuged at 10000 RPM (4°C), the supernatant
was filtered on a 0.22 µm PTFE syringe filter and
analyzed on HPLC-DAD. The chromatographic method
proved to be selective, with the separation of the 4
compounds confirmed according to retention time
and DAD spectrum, as well as lineares with R2 of
0.9996 for octopamine, 0.9993 for dopamine, 0.9993
for serotonin and 0.9996 for tryptophan. In addition,
the method is repetitive and sensitive, with relative
standard deviations between 0.015% and 2.9% and LD
and LQ of 0.025 and 0.075 mg.L-1 for octopamine, 0.043
and 0.131 mg.L-1 for dopamine, 0.01 and 0, 03 mg.L-1 for
serotonin and 0.038 and 0.151 mg.L-1 for tryptophan.
For real head samples of D. melanogaster we were
able to quantify 3 of the 4 compounds present in the
calibration curve, octopamine with 1.01 ± 0.11 mg.L-1,
dopamine with 0.68 ± 0.01 mg.L-1, and serotonin with
0.64 0.08 mg.L-1. The chromatographic method met
all the validation steps within what is recommended
by national and international regulatory agencies,
proving to be a selective, linear, sensitive and repetitive
method for the quantification of neurotransmitters
present in the head of D. melanogaster. The
chromatographic method also uses a low amount of
organic solvents, in addition to not using derivatives
or detectors that require other consumables such
as the mass spectrometer (MS), which makes it an
inexpensive method when compared to others in the
literature. Furthermore, it can be an important tool
for investigations of diseases in D. melanogaster
that change the composition of these compounds.
Keywords: HPLC-DAD; neurotransmitters; method
validation.

Development of a method based on Green
Analytical Toxicology to determine the
active components of ayahuasca in hair
Santos, Fabiana Pereira; Fabris, Andre Luis; Bruno, Vitor; Yonamine, Mauricio
Introduction: Ayahuasca is a hallucinogenic beverage
originated in South America and obtained through
the fermentation of two plants, P. viridis, containing
N,N-dimethyltryptamine (DMT), and B. caapi,
containing the β-carbolines (harmine, harmaline,
and tetrahydro-harmine); the combined ingestion
of these substances allows DMT to be absorbed
through the gastrointestinal tract and exert its
psychoactive effects. This beverage is commonly
used recreationally, although recent research
shows therapeutical applications of ayahuasca
for the treatment of some psychiatric disorders.
Considering the growing of ayahuasca consumption,
analytical methods for detecting these analytes in
conventional matrices has become popular. However,
no methods so far have simultaneously detected DMT
and the main β-carbolines in hair samples, which is
a matrix that provides information on long-term
exposure. In addition, a modern trend is the Green
Analytical Toxicology (GAT) that aims at reducing the
environmental impact of such methods. Objective: To
develop an analytical methodology for the quantitation
of ayahuasca alkaloids in hair using GAT principles.
Methods: Twenty milligrams of hair samples were
chemically digested with NaOH 1M and the analytes
were extracted using the dispersive liquid-liquid
microextraction (DLLME). Briefly, a mixture of
tetrahydrofuran with chloroform (THF:CHCl3) was
added to the digested samples followed by 30s of
vortexing and centrifugation. Then, the organic layer
was transferred to another vial containing with water
and carbonate-bicarbonate buffer pH 10.8 0.1M. Next,
samples were again vortexed for 30s and centrifuged
so the organic layer could be transferred to another
vial, dried under nitrogen flow, and resuspended
with 50μL of mobile phase prior injection into the
UPLC-MS/MS. Results: First, different organic
solvents were tested to digest the hair samples and
87
Department of Clinical and Toxicological Analyses, School of
Pharmaceutical Sciences, University of São Paulo.
a solution of NaOH was the only that fully digested
hair fibers. Next, the Design of Experiment statistical
tool was used to optimize both sample digestion and
subsequent analyte extraction. The best condition of
sample digestion associated with less degradation of
the targeted analytes was 1 mL of NaOH 1M incubated
for 90min at 50°C. Furthermore, once the samples
were digested, different mixtures of extraction and
dispersive solvents were investigated and two main
candidates were obtained: THF:CHCl3 and hexane with
methyl acetate (HEX:MA) however, the first mixture
yielded the highest analyte recovery. Next, different
buffers were tested to maximize analyte recovery
and carbonate-bicarbonate pH 10.8 0.1M provided
higher recovery. Finally, the method was tested with
hair samples spiked at 10pg/mg and was able to
successfully recovery and detect all targeted analytes.
In addition, 2 authentic hair samples of ayahuasca
users were analyzed to test the applicability of the
method. Discussion/Conclusion: Although different
solvents were assessed, only a solution of NaOH
was able to fully digest hair fibers. In that context,
the common organic solvents employed for that
purpose (e.g. methanol) did not fully digest hair
fibers, thus NaOH was chosen. Furthermore, albeit
the analytes were exposed to harsh conditions during
alkaline digestion and suffered partial degradation,
satisfactory sensitivity was achieved. Moreover,
even though HEX:MA efficiently recovered all target
analytes, THF:CHCl3 was chosen due to the superior
recovery paramount to achieve the intended limits
of detection using only 20mg of hair samples (10pg/
mg). Despite this mixture is more hazardous than
HEX:MA, only 200µL was used. Finally, the method
accomplished not only to recover the analytes from
authentic hair samples, but also detected them at
the concentration of 10pg/mg. Acknowledgements:
CAPES-PROEX: 88887.499615/2020-00

Development of a method for analysis of
simvastatin and simvastatin hydroxyacid by
HPLC in human plasma using HF-LPME extraction
Vasconcelos, Mayrla Emilia Dantas1; Gaitani, Cristiane Masetto1; Moraes,
Simvastatin is a prodrug in the lactone form (SVL),
and must be hydrolyzed in a 1st pass metabolism by
CYP3A4 for its main active metabolite, simvastatin
hydroxyacid (SVA) to exert a pharmacological activity
consisting of the inhibition of hydroxymethylglutaryl
coenzyme A reductase, an enzyme that participates
in the metabolic pathway of cholesterol production,
thus being an important drug in the treatment of
dyslipidemias, and a potential marker for CYP3A4
activity. Associated with the fact that it is a drug
commonly used by obese patients or at risk of obesity,
SVL can be considered a marker of choice for monitoring
this enzyme in patients undergoing Roux-en-Y gastric
bypass (RYGB) bariatric surgery. Despite the benefits
of this surgery, there is a detriment to the patient
regarding the absorption and oral bioavailability of
nutrients and drugs, as well as an increase in hepatic
CYP3A4 expression to compensate for the reduction in
intestinal CYP3A4 activity, an overproduction that can
generate long-term liver damage. This study aims to
develop a method for the analysis of SVL and SVA by
high performance liquid chromatography in plasma,
using HF-LPME as the extraction method. Firstly, the
chromatographic conditions were defined having
as stationary phase a 5.0 µm reversed-phase C18
column (4.6 mm x 100 mm) from Waters coupled to
a guard column (2 µm, 2.1 mm x 5 mm) and as mobile
phase a mixture of methanol and ammonium acetate
buffer (pH 4) in gradient mode: 0.01-5.00 min→65
MeOH: 35 Buffer/ 5.01-10.00 min→80 MeOH:20 Buffer,
flow of 1.00 ml/min, temperature of 40º C, injection
volume of 20 ul and wavelength for UV detection of
88
Natália Valadares2; Demets, Mariana Barbieri Alvarez1
1
Universidade de São Paulo - USP; 2 University of Florida - , UFL.
238 nm. Using this method with the UV detector we
had a range of 100 ng/ml – 10 ug/ml for SVL and SVA
in standard solution. After defining the analytical
condition, the extraction of SVL and SVA in plasma
samples was performed using the two-phase HF-
LPME, which consists of a 15 cm MF-PP Q3/2 hollow
capillary membrane impregnated in organic solvent,
coupled to two tips and then immersed in a test tube
with the donor solution that contains 500 ul of plasma
previously centrifuged, 50 ul of standard solution (SVL
and SVA) and 3.5 ml of 0.25 mol/l acetic acid solution
(pH 2.0). For the chromatographic analysis under
these conditions, the SVA had a retention time of 6.7
minutes and the SVL of 8.0 minutes having satisfactory
resolution value between peaks (0.893). For the
extraction, the solvent that had the best performance
was octanol, being used in the two-phase HF-LPME,
the extraction was selective because there were no
interferences from the plasma sample in the analysis
and the recovery of the method was 19,7% for SVL and
21,5% for SVA, it is worth mentioning that according
to the literature this is a method that usually offers
low recovery values, however, as long as they remain
constant between analyzes and provide detection
of the necessary concentration range of the analyte,
there is no compromise of the method. Further studies
will evaluate other solvents, improve parameters
such as resolution and detection range and finally
validate the analytical method making it suitable
for its application in plasma samples from patients
undergoing bariatric surgery.
 89

Development of a miniaturized sample

preparation method for determination of
ketamine, its metabolites and analogs
in oral fluid samples using Dispersive
Liquid-Liquid Microextraction (DLLME)
Kahl, Júlia M.M.1,2; Berlinck, Débora Zorrón2; Chinaglia, Kauê de Oliveira1,2;
Rodrigues, Leonardo Costalonga1,2; Costa, Jose Luiz1,2
1
Campinas Poison Control Center, University of Campinas (UNICAMP), Brazil; 2 Faculty
of Pharmaceutical Sciences, University of Campinas (UNICAMP), Brazil.

Background/Introduction: Ketamine is a dissociative
anesthetic, which present heterogeneous
pharmacology, as it interacts with several receptors.
This molecule is metabolized into three active
metabolites, such as 6-hydroxynorketamine.
Nowadays, ketamine has been found at parties
(mainly electronic music parties, “raves”), being
used as drug of abuse, and representing 87% of the
quantity of hallucinogens seized in the last 5 years.
Objective: the aim of this work was the development
and validation of an analytical methodology based on
dispersive liquid-liquid microextraction (DLLME) and
liquid chromatography–mass spectrometry (LC-MS/
MS) for simultaneous determination of ketamine, its
metabolites norketamine and 6-hydroxynorketamine,
and its analogs dechloroketamine and
2-fluorodeschloronorketamine (sold as New
Psychoactive Substances) in oral fluid samples.
Methods: The DLLME procedure was optimized
during the method development. Parameters such as
the extraction and dispersive solvents, and solvents
volume were studied and optimized. In this stage
were used drug-free blank samples, fortified with
solutions of analytes at the calibrators and controls
concentrations. For extraction, a 50μL aliquot of oral
fluid (collected with Quantisal) was extracted using
100μL methanol and 50μL chloroform, as the disperser
and extractor solvents, respectively. Subsequently
to this stage the samples are centrifuged and the
extractive solvent, sedimented, containing the analyte
of interest. The organic phase was transferred to
another polypropylene tube and evaporated under a
nitrogen stream (30°C). The residue was resuspended
with 100μL of methanol and 2μL were injected into
the LC-MS/MS. Results: The instrumental conditions
were optimized, and the analyses were performed in a
Nexera-UHPLC chromatographic system, coupled with
a LCMS8060 mass spectrometer. The chromatographic
separation was performed with a Raptor™ Biphenyl
column (100×2.1mm, 2.7μm), maintained at 40°C. The
mobile phase consisted of ultra-pure water (A) and
methanol (B), both containing formic acid (0.1 %) and
2 mmol/L ammonium formate. The flow rate was set
to 0.4mL/min, and the elution gradient was isocratic,
with 35%B. The total run time was 4.5min. The method
presents good linearity from 10 to 1000ng/mL (r>
0.995, 1/x2 weighted linear regression). Discussion/
Conclusion: A reliable and accurate method was
developed for all the compounds. In the next step,
the method will be applied to the analysis of oral fluid
samples collected from volunteers at parties and
electronic music festivals. Acknowledgments: the
authors thanks to Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) and Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq).
 90

Development of a simple and fast method for

iodine determination in hair samples by ICP-MS
after alkaline solubilization at room temperature
Morais, Déborah Araújo; Sousa Junior, Wellington Tavares;
Oliveira, Silvana Ruella; Barbosa Junior, Fernando

Analytical and System Toxicology Laboratory, Department of Clinical, Toxicological and Bromatological
Analyses, Faculty of Pharmaceutical Sciences of Ribeirão Preto, Ribeirão Preto - SP, Brazil.

Introduction: Iodine is an essential chemical element,
which participates in the biosynthesis of thyroid
hormones (tyrosine derived), thyroxine (T4) (4 iodine
atoms) and triiodothyronine (T3) (3 iodine atoms).
Iodine deficiency and excess consumption can lead to
health problems. Thus, iodine human biomonitoring
is very important, and its determination in urine
is the most commonly used procedure. However,
information about the consumption of iodine in a
long-term period of time can be most relevant than a
single point determination in urine. Hair is a promising
matrix, as it accumulates the analyte excreted along
the strand, registering variations in consumption
over months or years, depending on the length of the
sample. However, studies aiming at the determination
of iodine in this matrix are still scarce, probably
due to the limited number of analytical methods
available in literature. Objective: The present study
aims to develop and validate a fast, simple, and,
low-cost sample preparation procedure for iodine
determination in hair samples by ICP-MS employing
alkaline solubilization with tetramethylammonium
hydroxide (TMAH) at room temperature. Methods:
Two different Human Hair Reference Materials
(QM-H-Q1616 and QM-H-Q1725) from the Institut
Nacional de Santé Publique du Quebec (INSPQ -
Centre de toxicologie, Canada, Quebec) were used
for method optimization and validation purposes.
The concentration of TMAH (10, 25, 50, 75 and
100% (v/v)), the solubilization time (7, 10, 12 h and
overnight) and the sample mass (between 15 and
20 mg and between 70 and 100 mg) were optimized.
Then, high purity de-ionized water was added to the
samples until maximum concentration of 1% (v/v)
TMAH. Finally, samples were directly analyzed by
ICP-MS (PerkinElmer, NexION® 2000 B, Waltham,
EUA). Results: No statistical differences were found
between the results for solubilization times of 10, 12
h, and overnight, TMAH concentrations of 50, 70, and
100% (v/v) and, sample masses (between 15 and 20
mg and between 70 and 100 mg) at 95% confidence
level on applying the t-test. Good analytical iodine
recoveries (p > 0.05) were obtained after solubilizing
the samples at least for 10h using 50% (v/v) of TMAH
(Human Hair QM-H-Q1725 reference material) and
mass between 15 and 20 mg (Human Hair QM-H-Q1725
and QM-H-Q1616 reference materials), attesting the
accuracy of the method. The intra (n = 4) and inter
(n = 4) precision were 6.2% and 6.3%, respectively,
showing good analytical repeatability (Human Hair
QM-H-Q1725 and QM-H-Q1616 reference materials).
Discussion/Conclusion: A new and promising
analytical method for iodine determination in hair
samples by ICP-MS was developed employing 50%
(v/v) TMAH, overnight solubilization, and sample
mass between 15 and 20 mg. This method is useful
to analyze small hair segments (≤ 1 cm) since it
requires low hair masses (< 20 mg). The lower TMAH
concentration evaluated (50% (v/v)) provided good
analytical results with the advantage of avoiding
damages on the ICP-MS cones (sample, skimmer,
and hyper skimmer). With the proposed procedure,
at least 400 samples can be weighed, left overnight
in the alkaline medium for complete solubilization
at room temperature, diluted, and analyzed the next
day, considerably increasing the analytical capacity
in large-scale routine compared to methods that
require microwave digestion procedures. Therefore,
the sample preparation procedure using TMAH to hair
samples solubilization, is a quick, simple, and a low-
cost alternative to be applied in studies of body iodine
evaluation. Acknowledgments: We thank for the
financial support of Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES, Finance Code
001).
 91

Development of an analytical method based

on Green Analytical Toxicology employing
umbilical cord tissue for the evaluation
of maternal fetal exposure to cocaine
Meirelles, Gabriela de Paula; Pereira e Silva, Jefferson; Yonamine, Mauricio

Department of Clinical and Toxicological Analyses, School of

Pharmaceutical Sciences, University of Sao Paulo.

Introduction: The use of cocaine (COC) during
pregnancy has deleterious effects on both the
pregnant woman and the child, in prenatal and
postnatal periods, as well as obstetric complications.
However, this subject is still relatively unexplored, and
since the toxicological evaluation of in utero exposure
is challenging, the development of new analytical
methodologies for better toxicological management
urges. The umbilical cord has been regarded as an
alternative sample for such analyzes because of its
advantages, such as its presence throughout the
whole pregnancy and the fact that it is the biggest
channel for fetus exposure to drugs. Therefore, this
work intends to develop an ecologically friendly,
simple and low cost analytical method based on Green
Analytical Toxicology. Objective: This project aims to
develop and validate a method for the determination
of COC and its metabolites in umbilical cord through
green extraction and gas chromatography coupled
to mass spectrometry. Methods: For sample
preparation, 500 mg of umbilical cord tissue (UCT)
was placed in an eppendorf, 500 µL of 0.1 M phosphate
buffer (pH 6) was added and homogenized using
Ultra-Turrax for 1-2 min, followed by addiction of
1,5 mL of 0.1 M phosphate buffer. The homogenized
tissue was centrifuged and the supernatant (500
µL) was transferred to another eppendorf. Then,
1,5 ml of dichloromethane: isopropanol: ammonium
hydroxide (12:3:0.3) were added for the purpose of
extraction. The mixture was stirred and centrifuged.
The extract was collected and transferred to a vial,
being dried at 40°C/10 min under nitrogen flow.
Finally, it was derivatized with 30 µL of PFP and PFPA
at 70°C/30 min, after being dried under nitrogen flow.
It was resuspended with 50 µL of ethyl acetate and
injected into the GC-MS. Results: This method was
applied in a possibly positive UTC from a mother
with detection of 2478,03 ng/mL of benzoylecgonine
in urine. The results have shown the determination
of COC, benzoylecgonine (BZE), ecgonina methyl
esther (EME), ecgonine (EC) and anydroecgonine
methyl esther (AEME). Although the method worked,
an optimization was performed in order to improve
the extraction of the analytes. To optimize sample
preparation, an experimental design was carried
out by factor analysis, using the statistical tool DOE
(Design of Experiment), and Response Optimizer, in
Minitab® Statistical Software. An experimental model
was built to evaluate 3 factors, at 2 levels, with critical
influence on the extraction yield of all analytes. The
model evaluated the following effects:
• Volume of solvent mixture: 1,5 and 2 mL
• Agitation time: 2 and 10 min;
• PSA: 0 and 25 mg.
Only for EC, the factors of volume of solvent mixture,
PSA and the interaction between them presented
significant difference, so the extraction was improved
with 2 mL of solvent mixture and without PSA.
In addition, an optimization of the response was
performed with the prediction showed that volume of
solvent mixture of 1,7828 mL, agitation for 10 min, and
the absence of PSA were the best conditions for the
extraction of the analytes (D=07508). Conclusions:
This extraction was able to detect COC and its
metabolites from the positive sample. According
to the results, DLLME (dispersive liquid-liquid
microextraction), without PSA, proved to be a more
promising procedure than QuEChERS, with PSA. Due
to the sample complexity and despite not having an
especially micro volume, it can still be considered a
green method.
 92

Development of an extraction method for

the pesticide 2,4-dichlorophenoxyacetic
acid and its metabolite 2,4-dichlorophenol
from Apis mellifera
Paz, Maria Elizabeth Gomes1; Carriço, Murilo Ricardo Sigal1; Rodrigues, Marina Diaz2;
Ramborger, Bruna Piaia1; Denardin, Elton Luis Gasparotto3; Roehrs, Rafael3
1
 Student of the Postgraduate Program in Biochemistry, Federal University of
Pampa, Campus Uruguaiana; 2 Pharmacy Student, Federal University of Pampa,
Campus Uruguaiana; 3 Professor, Federal University of Pampa.

2,4-dichlorophenoxyacetic acid, known as 2,4-D, octadecylsilane followed by centrifugation (7000

which is an organochlorine herbicide used in post-
emergence against broadleaf weeds. This herbicide
is made from the compound 2,4-dichlorophenol (2,4-
DCP), which is also its main metabolites. Both 2,4-D
and 2,4-DCP are considered highly toxic, with known
genotoxic and hepatotoxic potential. The intoxication
of unwanted places occurs through drift, this process
occurs when the product is applied in inappropriate
weather conditions, being carried by the wind to
other places, contaminated by non-target native
organisms. Apis mellifera is an important bioindicator
for this contamination, as they travel large distances
daily in search of resources for the hive, they may
come into contact with pesticides. Thus, the aim of
this study was to develop a QuEChERS method for
the extraction of the 2,4-D herbicide and its 2,4-DCP
metabolite in Apis mellifera. The initial parameters
of the extraction method were performed with
Drosophila melanogaster (matrix), due to its greater
availability. Initially, 0.5 g of frozen D. melanogaster
were homogenized with 5 mL of water in Falcon tubes
(15 mL) and fortified with the standard solution of
2,4-D and 2,4-DCP to reach the final concentration
of 5 mg.L-1, and vortexed immediately for 1 minute.
Then, we evaluated the recovery of compounds
from a partition step with acetonitrile (MeCN) also
evaluating the influence of different proportions of
H3PO4 in MeCN (1, 2, 3, 5 and 10%) on the extraction
of 2,4-D and 2,4-DCP. The tubes were again shaken
and taken to an ultrasound bath (1 minute) for the
subsequent Salting-out step. This step consisted of
adding 2 g of MgSO4 and 0.5 g of NaCl followed by
vortexing (1 minute) and centrifugation for 5 minutes
at 7000 rpm. Then 1 mL of the organic phase was
collected and its cleaning was done by vortexing
the aliquot with 150 mg of MgSO4 and 50 mg of
rpm) for 5 minutes. The supernatant was filtered on
a hydrophobic PTFE syringe filter (0.22 µm). After
defining the extraction phase, we applied the method
to A. mellifera samples. All samples were analyzed by
Young Lin high performance liquid chromatography
(YL 9100) with diode array detector (HPLC-DAD).
Chromatographic separation was performed
isocratically with an Inertsil ODS-3 column (GL
Sciences) and mobile phases acetonitrile/ultrapure
water acidified to pH 3 with H3PO4 (87.5:12.5 v/v) with
an analysis time of 16 minutes, flow of 0.8 mL.min-1
and 20 µL injection volume. The detection of 2,4-D
(230 nm) and 2,4-DCP (220 nm) was confirmed by
comparing the retention time and DAD spectra of the
analytical standards obtained by the calibration curve
with 6 points between 0.5 and 12 mg.L-1. The results
were expressed as mean ± relative standard deviation
(%RSD), considering as ideal recoveries between 70%
and 120% with RSD < 20%. The extraction phase that
recovered the greatest amount of compounds and
met these criteria was MeCN with 5% H3PO4, which
recovered 103.6% ± 8.85% of 2,4-DCP and 104.8%
± 9.9% of 2,4-D. The application of the extraction
method in A. mellifera showed a recovery of 75.4% ±
3.3% for 2,4-DCP and 73.9% ± 13% for 2,4-D. Another
important aspect for choosing the extraction phase
was the lower extraction of interferents from the
sample, as we observed that by increasing the
proportion of acid, the extraction of interferents was
reduced. The results presented demonstrate that the
extraction method allowed the extraction of the 2,4-
D herbicide and its metabolite 2,4-DCP in samples of
A. mellifera reliably and can be an important tool for
environmental monitoring of non-target organisms
intoxicated by 2,4-D and its metabolite.
 93

Development of dispersive solid phase

microextraction (d-SPE) and HPLC-DAD
for emergency toxicological analysis
of toxic drugs in urine samples
Luz, Heloisa Peres1; Silva, Bruna Espíndola2; Santos, Claudia Regina3; Marchioni, Camila3

 Pharmacist Student, Federal University of Santa Catarina, Florianópolis, Brazil; 2 Resident

1

pharmaceuticals, University Hospital, Federal University of Santa Catarina, Florianópolis, Brazil;

3
 Department of Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

Introduction: Assistance and Toxicological
Information Center of Santa Catarina (CIATox/SC)
registered, in 2020, 5824 cases of intoxication involving
toxic agents. Among the 10 drugs with the highest
frequency of registration in humans are amitriptyline
and diazepam, totaling 343 and 303 registered cases,
respectively. Cocaine is the second drug of abuse most
related in intoxication cases registered in CIATox/
SC, with 221 cases. In this scenario, the emergency
toxicological analysis has become relevant due to
the constant and growing number of cases of acute
intoxication, since it helps in the diagnosis, prognosis
or support treatment. Objective: Develop a dispersive
solid phase microextraction (d-SPE) and analyze by
high-performance liquid chromatography coupled to
a diode-array detector (HPLC-DAD) that will be used
for the identification of amitriptyline, diazepam and
cocaine/ benzoylecgonine in urine samples. Methods:
Analytical standards of amitriptyline, cocaine and
diazepam were used at concentrations of 1μg/mL,
1μg/mL and 0.5μg/mL, respectively. The first step
consisted of the evaluation of the chromatographic
conditions. For the tests, the isolated standards
prepared in acetonitrile or the mixture of all drug
standards were dried and resuspended in 100µL
of initial mobile phase (MP). Thermo Scientific®
HPLC-DAD (Massachusetts, USA) and C18 column
(250 mm x 4.6 mm x 5 μm) was used. The following
parameters were evaluated: wavelength, composition
and proportion of MP, isocratic or gradient elution,
MP flow and run time. d-SPE was tested using three
different sortive phases (DSCMCAX, Strata-X e Strata-
X-Aw, 20 mg). 500μL de enrichment urine sample (pH
5) and 1000μL of ammonium acetate (pH 5) was mixed
with sortive phase. The system was shaken and
centrifuged. Supernatant was discarded and 100μL
de acetonitrile with 2% of ammonium hydroxide was
added. The sample was shaken for 10 minutes and the
supernatant was dried. The residue was resuspended
in 100μL of MP. Results: DAD detector conditions
were evaluated by scanning (190–800 nm), where
the best wavelengths were: 200 nm for amitriptyline,
225 nm for cocaine, and 198 nm for diazepam. In the
development of the chromatographic method, the
isocratic elution presented the best results, with a MP
composed of acetic acid buffer/ammonium acetate
0.25 M pH 5.5: acetonitrile (80:20, v/v) with a flow rate
of 0.7mL/min, resulting in a better chromatographic
resolution, where amitriptyline presented a retention
time of 10.4 min, cocaine 6 min and diazepam 5.3 min.
In the first d-SPE test the best phase was DSCMCAX
for amitriptyline (recovery = 26.1%). For cocaine
(recovery = 16.6%) and diazepam (recovery = 25,1%)
Strata-X-Aw was better. Conclusion: Until now, all
aspects of the chromatographic run and detection
system were evaluated obtaining a fast and simple
run, that minimally affected in the chromatographic
system. At this stage, it was possible to perform the
chromatographic analysis of 3 different substances
using a run of only 14 minutes and with an adequate
chromatographic resolution. The next step will be the
optimization of d-SPE to obtain better recovery. With
the development and validation of this method, it will
be possible to apply it in the routine of emergency
analysis. Keywords: Amitriptyline, Diazepam,
Cocaine, HPLC-DAD, d-SPE, Clinical Toxicology.
Acknowledgement: CNPq for the financial support
and CIATox/SC for the partnership.
 94

Ethanol quantification in ethanol-

based hand sanitizers: alert in the
COVID-19 pandemic scenario
El Haddad, Lohanna Pereira1; Costa, Bruno Ruiz Brandão2; Bigão,
Vitor Luiz Caleffo Piva2; De Martinis, Bruno Spinosa1
1
 Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de
São Paulo, Ribeirão Preto, Brazil; 2 Faculdade de Ciências Farmacêuticas de
Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil.

Background: The COVID-19 pandemic sharply
increased the demand for ethanol-based hand
sanitizers (EBHS). To meet this great demand,
regulatory health agencies worldwide have altered
their regulatory guidelines, raising concern about
the products quality. Objective: This study aimed to
quantify ethanol content and to qualitatively assess
common impurities in EBHS products marketed in
Brazil. Methods: For the quantitative evaluation, 0.10
g of the EBHS were weighed in a 20 mL headspace (HS)
vial, to which 5 mL of deionized water and 20 μL of
isobutyl alcohol (internal standard for quantitative
analysis) were added. The samples were vortexed for
10 s and incubated at 90°C for 6 min, and then 400 µL
of the vapor phase was injected (on split mode 50:1)
into a Gas Chromatography with Flame Ionization
Detector (GC/FID) system. This quantitative method
was validated according to the Anvisa Guideline on
Analytical Method Validation. For the qualitative
evaluation, 0.25 g of the EBHS were weighed in a 20
mL HS vial, to which 4 mL of deionized water and 5
µL of acetonitrile (internal standard for qualitative
analysis) were added. The samples were vortexed for
10 s and incubated at 90 °C for 10 min, and then 400
µL of the vapor phase was injected (on split mode 10:1)
into the same GC/FID system mentioned previously.
Forty-eight commercial samples marketed in Brazil
were analyzed. Results: The Anvisa establishes
that the ethanol content in EBHS commercialized in
Brazil must be at least 70% (w/w) and that the actual
quantity should not vary more than 10% in relation
to the declared amount. Moreover, products with
ethanol content less than 52.1 % (w/w) may not be
effective against virus and bacteria. In this context,
27.1% of the analyzed products (n=13) presented
unless one nonconformity regarding the amount
of ethanol: seven products declared an ethanol
content less than 70%, and in eight other products
the actual ethanol content presented a difference
superior to 10% than what was labeled (two samples
presented both nonconformities). Furthermore,
two samples contained ethanol levels less than the
amount considered effective against microorganisms.
According to the qualitative results, 12 samples (25%)
presented acetaldehyde or ethyl acetate. However, the
estimated levels for these substances were not toxic
considering the common use of EBHS. Discussion/
Conclusion: A simple and fast HS-GC/FID method to
quantify ethanol in EBHS was developed, validated,
and applied to commercial samples in Brazil. The
huge demand for EBHS may have impacted their
quality. Thus, the regulatory authorities must be more
vigilant to ensure that the commercially available
products meet the recommended specifications.
Because concern with proper hand hygiene tends to
remain an issue for a long period, more studies about
quality control of hand sanitizers will be needed.
Acknowledgments: This work was supported by
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES).

Evaluation of cork as a natural extraction
phase for the determination of new
psychoactive substances in blood samples
Background: New psychoactive substances (NPS)
represent a worldwide concern since distribution
and synthesis of new compounds continues to
increase. In this scenario, there is a need in developing
methodologies that can efficiently extract these
analytes from biological samples and provide a
straightforward analysis. In addition, new analytical
methods should be concerned with the use of
environmentally friendly alternatives following the
principles of “green analytical chemistry”. Therefore,
the use of natural materials as biosorbents in solid
phase-based techniques can be explored. Objective:
This study aimed to investigate and optimize the
variables of a solid phase-based extraction coupled
to a 96-deepwell plate employing cork for LC-MS/MS
determination of NPS in blood. Methods: An aliquot
of 200 µL of blood was priorly submitted to protein
precipitation with 250 µL of water, 250 µL of zinc
sulfate and 50 µL of NaOH, followed by agitation and
centrifugation. Afterwards, 500 µL of the supernatant
was submitted to the proposed procedure, which
consisted in its addition to a 96-deepwell plate
containing cork. The extraction occurred in an
orbital shaker at 300 rpm, followed by the removal
of the aqueous phase and addition of the desorption
solvent with further agitation. The organic layers
were recovered and dried under nitrogen stream,
reconstituted in 30 µL of methanol and analyzed. A
Nexera UFLC system coupled to a LCMS-8045 triple
quadrupole mass spectrometer (Shimadzu, Japan) was
used for the analysis. Regarding optimization studies,
blank blood samples were spiked with selected
analytes (phenethylamines, cathinones and classic
synthetic drugs), in a concentration of 50 ng/mL. First
evaluation was the selection between cork sheet or
cork powder for the procedure. Selection of extraction
significant variables was performed through
fractional factorial design (25-1) evaluating cork
particle size, cork mass, extraction time, desorption
time and desorption solvent volume. Variables
95
Birk, Letícia; Eller, Sarah; Oliveira, Tiago Franco
Graduate Program in Health Sciences, Federal University of
Health Sciences of Porto Alegre (UFCSPA), Brazil.
confirmed as significant were optimized by response
surface methodologies and desorption solvent choice
by a simplex-centroid design. Resulting data from the
optimization studies was analyzed by the Statistica®
software. Results: The use of cork powder was
selected for the procedure since it presented higher
analytical responses based on chromatographic peak
areas. Significant variables for the process were cork
mass, extraction time and desorption solvent volume.
These were evaluated through a central composite
design (CCD) for the achievement of optimum
values. CCD analysis of cork mass vs. extraction time
resulted in two optimum areas: (1) less cork mass
and greater extraction time, (2) greater cork mass
and less extraction time. These two conditions were
evaluated in triplicate and shown no statistically
significant difference (p > 0.05). Therefore, the use of
a minor extraction time and greater mass of cork was
chosen. Simplex-centroid results pointed to the use
of 100% of acetonitrile as desorption solvent. Final
procedure stands as follows: addition of 500 µL of
the supernatant to 30 mg of cork and extraction for
15 minutes at 300 rpm, followed by the removal of the
aqueous phase and addition of 1.15 mL of acetonitrile
with desorption for 2 minutes at 300 rpm. LLOQs
for the analytes ranged between 0.5 and 5 ng/mL.
Discussion/Conclusion: Optimal responses achieved
for cork powder over cork sheet can be explained by the
higher contact surface between sorbent and sample. A
higher mass of cork was chosen because it is a natural
material with low-cost, while reduced extraction time
improves the method high-throughput. In conclusion,
this study demonstrates the applicability of cork
as extraction phase for the determination of NPS in
blood and optimizes the parameters that influence
extraction efficiency. Nevertheless, the use of the
96-deepwell plate allows simultaneous extraction of
multiple samples in the same batch, with possibility
of automation. Acknowledgments: CAPES.
 96

Evaluation of the spontaneous

hydrolysis of cocaine under different
pH and temperature conditions
Gomes, Geovana Maria de Lima1; Santos, Vanessa Farelo1; Carneiro, Gabriel Reis
Alves1; Trajano, Christian Farias2; Pereira, Henrique Marcelo Gualberto1
1
 Laboratório Brasileiro de Controle de Dopagem, Chemistry Institute,
Federal University of Rio de Janeiro; 2 Comitê Olímpico Brasileiro.

Introduction: Cocaine (COC) is a stimulant of the
central nervous system, and it is included World Anti-
Doping Agency (WADA) Prohibited List1 to athletes.
The metabolism of COC is well described and its main
metabolites are ecgonine methyl ester (EME) and
benzoylecgonine (BZE), the last being generated by
the liver carboxylesterase or through spontaneous
hydrolysis2. In the light of the new WADA regulation
regarding COC cases, only the concentrations of COC
and BZE are considered during analysis and results
management. The project aims to evaluate the stability
of COC in different pH and temperature conditions.
Methods: Stability of COC was evaluated using water
and urine for comparison reasons. For water matrix,
COC reference material was spiked at a concentration of
100 ng/mL buffered at ranges from pH 4 to 9 and kept in
ambient temperature (25 ± 5°C). Another batch, with the
same composition was kept at refrigerator (4 ± 1°C) for
seven days. One aliquot of 70 µL per day was collected
and a “dilute-and-shoot” approach was performed with
30 µL of methanol containing the internal standard
7-propyltheophylline. For the stability study in human
urine, the pH considered was intrinsic to the sample. No
buffer was added. Real urine human samples from pH 5
to 8 range were used. Three samples per interval of pH
was chosen according to the division: interval 1 (pH 5.0
to 5.9), interval 2 (pH 6.0 to 6.9), interval 3 (pH 7.0 to 7.9)
and interval 4 (pH 8.0 to 8.9). COC reference material was
spiked at 100 ng/mL in all samples, which were exposed
to the same conditions of the study in water, for the same
period. One aliquot of 1.00 mL per was daily collected
and stored in freezer (-28 ± 2°C) until preparation
by solid-phase extraction and analysis using liquid
chromatography coupled with high resolution mass
spectrometry (LC-HRMS)3. Results and Discussion:
In both matrices evaluated, the instability of COC was
more pronounced in the pH 8 and 9, in which only small
fractions of initial COC were detected after seven days
of experiment in ambient temperature. In human urine
samples, COC in pH 8 decreased 78% in relation to the
initial condition under ambient temperature and 34%
under refrigeration. COC were still detected in all samples,
even in alkaline pH. However, it could be attributed to
the high sensitivity of the analytical approach adopted
(LOD < 1 ng/mL). Higher instability was observed in urine
samples and compared with water matrix, what suggest
that other factors, endogenous to urine samples,
developed a hole in the COC – BZE conversion. Water and
human urine samples were considered to evaluate the
difference in the spontaneous hydrolysis of COC to BZE.
Conclusion: As described in the literature, samples with
acid pH have shown the inhibition of the spontaneous
hydrolysis. In contrast, as much alkaline the samples,
more significant is the instability of COC. Hence, the
influence of temperature and natural excretion pHs
on stability of COC shows the possible impact when
considering the sample transport conditions and mid-
term storage until analysis. During results management
related to antidoping cases, pharmacokinetics illations
is not advisable, considering the lack of information of
the original concentrations of COC and BZE at the sample
collection time and the impact of temperature and
pH in the spontaneous reaction observed. Excretions
studies of COC administration becomes necessary to
better understand the impact of the spontaneous COC
- BZE conversion in antidoping cases interpretation.
Acknowledgments: The authors thank Autoridade
Brasileira de Controle de Dopagem (ABCD) for financial
support.
REFERENCES
1. World Anti-Doping Agency. Prohibited List 2022, Montreal,
2022. Available at https://www.wada-ama.org/sites/default/
files/2022-01/2022list_final_en_0.pdf.
2. Hoffman, R, et al. Experimental treatments for cocaine toxicity.
The Journal of pharmacology and experimental therapeutics,
2013, 347.
3. Sardela VF, et al. Comprehensive analysis boy liquid
chromatography Q-Orbitrap Mass Spectrometry: Fast Screening
of peptides and organic molecules. J. Mass Spectom., 2018, 1-28.
 97

First proof of concept of a new passive

sampler for marine biotoxins
Fitarelli, Bruna1; Fabichak, Ana2; Deolindo, Carolina Turnes Pasini3; Kleemann, Cristian3; Hoff, Rodrigo3
1
Departamento de Farmacociências, Universidade Federal de Ciências da Saúde de Porto
Alegre, Porto Alegre, RS, Brazil; 2 Departmento de Agricultura, Biodiversidade e Florestas,
Universidade Federal de Santa Catarina, Curitibanos, SC, Brazil; 3 Ministério da Agricultura,
Pecuária e Abastecimento, Laboratório Federal de Defesa Agropecuária, São José, SC, Brazil

Introduction: Marine biotioxins (MBs) are toxins
produced by algae. These compounds can be absorbed
by edible bivalve mollusks such as oysters and
mussels. The ingestion of such organism containing
MBs can lead to several health damages such as
diarrhea, paralysis, and even death. To assure the
seafood safety to the consumers, the production of
oyster and mussels in Santa Catarina state is strictly
regulated, with weekly laboratorial monitoring of
MBs. However, these compounds can be precociously
detected in the water near to the sea farms. Thus, the
use of direct detection or the use of passive samplers
can be very useful to detect MBs before the seafood
contamination. Objective: To perform a proof of
concept of an innovative model of passive sampler,
using a small scale prototype and okadaic acid and
domoic acid as MBs models. Methods: The passive
sampler is composed by a floating basis constructed
with high density expanded polystyrene containing
six wells with small holes. At the bottom of each
well, a pellet with adsorbent was inserted. A test
solution composed of ultrapure water was fortified
with analytical standards of domoic acid (DA, 44.3
µg mL-1) and okadaic acid (OA, 16 µg mL-1), producing
a solution with 17.72 ng mL-1 (DA) and 6.4 ng mL-1 (OA).
This test solution (TS) was placed into a beaker. After
that, the floating prototype was placed in the TS with
the following adsorbent pellets: i) three pellets of
bamboo activated carbon (BAC) with approximately
4-5 mm of diameter; ii) two pellets of cork with the
same dimensions used for carbon; iii) one pellet of
silicon rubber coated with cork powder. The beaker
was covered and sealed. A halogen lamp was placed
near to simulate solar heating. The experiment was
carried out during 68 hours. Three aliquots of TS was
taken at zero time and at the end of the experiment.
After the experiment period, the pellets were removed
and transferred to a polypropylene tube of 15-mL. To
each tube, 5 mL of methanol was added. Then, the
tubes were mixed submitted to ultrasound-assisted
extraction during 20 minutes. After that, samples
were filtered and the solution was evaporated in a
water bath (40 °C). The dry residue was reconstituted
and submitted to LC-MS/MS analysis according to the
official method (Molognoni et al, 2018). Results: The
TS analysis showed a decreased in the MB levels from
the 0 h until 68 h later of 27.7% and 3.3%, respectively.
Pellet analysis showed that BAC is effective to adsorb
both MBs. For OA, the initial calculated concentration
was 6.1 ng mL-1. At the end of the experiment, the OA
level was calculated as 4.8 6.1 ng mL-1. The OA mass
estimated as adsorbed by the activated carbon pellet
was 1.82 ng. The cork pellet was not able to adsorb
detectable amounts of OA. However, a relevant peak of
DA was identified. For DA, results closer to the OA was
obtained using BAC. However, calibration curves were
performed just for OA, not allowing the estimation
of DA in terms of mass. A signal comparison shows
an intense peak corresponding to DA and confirmed
using three m/z transitions. Discussion/Conclusion:
A proof of concept was performed for a new passive
sampler to MBs detection in sea water. DA and OA
was effectively adsorbed using BAC, showing that
the concept of the floating passive sampler is valid.
Further studies will be carried out to optimize the
main parameters of the model in order to achieve
a prototype able to be evaluated under real filed
condition in large term studies.
REFERENCES
Molognoni L, Dos Santos JN, Kleemann CR, Costa ACO, Hoff
RB, Daguer H. Cost-Effective and High-Reliability Analytical
Approach for Multitoxin Screening in Bivalve Mollusks by Liquid
Chromatography Coupled to Tandem Mass Spectrometry. J
Agric Food Chem. 2019 Mar 6;67(9):2691-2699. doi: 10.1021/acs.
jafc.8b06600.
 98

In vitro metabolism studies of ADB-4en-

PINACA, ADB-FUBIATA and BZO-CHMOXIZID
using human liver microsomes
Cunha, Kelly F.1,2; Krotulski, Alex J.2; Walton, Sara E.2; Papsun,
Donna M.3; Costa, Jose Luiz4; Logan, Barry K.2,3
1
 Faculty of Medical Sciences, University of Campinas, Campinas, SP, Brazil; 2 Center for
Forensic Science Research and Education, Fredric Rieders Family Foundation, 2300 Stratford
Ave, Willow Grove, PA 19090, USA; 3 NMS Labs, 3701 Welsh Rd, Willow Grove, PA 19090, USA;
4
 Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, SP, Brazil

Background/Introduction: The synthetic cannabinoids (SC)
was the largest group (209) of new psychoactive substances
monitored by the EU Early Warning System in 2020, in which 11
had being reported for the first time that year (1). Occasionally,
the SC consumption has led to fatal intoxications worldwide.
Considering their prevalence in the USA, ADB-4en-PINACA
and ADB-FUBIATA have been strongly recommended to be
incorporated into the SC scope analysis performed by toxicology
laboratories; while BZO-CHMOXIZID is also recommended to be
included after a slightly increasing trend (2). Objective: The aim
of this work was to identify the main phase I metabolites of 3 SC
(ADB-4en-PINACA, ADB-FUBIATA and BZO-CHMOXIZID) obtained
by in vitro metabolism assay using human liver microsomes
(HLMs) and analyzing by liquid chromatography coupled to
high-resolution mass spectrometry (UHPLC-HRMS). Methods:
HLMs incubation was performed following previously published
protocol (3). To 520 µL phosphate buffer 100 mM (pH = 7.4) was
added 5 µL phosphate buffer/acetonitrile mixture solution
(1:1, v/v) containing the analyte at 1,000 µg/mL, 50 µL NADPH
aqueous solution (10 mM) and 25 µL pooled HLMs. The tubes
were maintained at water bath (37 °C) under shaking during 2h,
and the reaction was stopped with 500 µL acetonitrile. After
centrifuged, samples were dried down to reduce volume and
centrifuged (10,000 rpm/5 min) again using a tube filter. The
extracts were transferred to vials and injected into an UHPLC
Nexera XR (Shimadzu, Japan) coupled to a HRMS TripleTOF® 5600+
(Sciex, Canada), using electrospray in positive mode. Acquisition
was performed using Information Dependent Acquisition (IDA).
Diazepam was used as positive reaction control. Results:
During the ADB-4en-PINACA ([M+H]+ at m/z 343.2129) in vitro
assay was produced and identified 11 phase I metabolites. Major
metabolites were obtained through hydroxylation combined
with internal hydrolysis (m/z 377.2183), hydroxylation (m/z
359.2078) and amide hydrolysis combined with hydroxylation
(m/z 360.1915). For ADB-FUBIATA ([M+H]+ at m/z 396.2082),
6 metabolites were identified. The most intense metabolites
were formed by hydroxylation (m/z 412.2038), followed by
N-dealkylation combined with loss of NH and dehydrogenation
(m/z 271.1444) and amide hydrolysis combined with
dehydrogenation (m/z 395.1774). Four metabolites had been
identified related to BZO-CHMOXIZID ([M+H]+ at m/z 362.1863).
The most prevalent identified metabolic pathways were
hydroxylation (m/z 378.1812), dihydroxylation (m/z 394.1763)
and ketone formation (m/z 376.1657), respectively. Discussion/
Conclusion: Our work provides the identification of several
metabolites related to 3 SC of interest currently. There are no
other published works related to in vitro metabolism studies
of the emerging “OXIZID” generation of SC, as well regarding
ADB-FUBIATA. Due to structural similarities, part of ADB-4en-
PINACA metabolic pathway is compared with those reported
for MDMB-4en-PINACA during in vitro metabolism study and
confirmed in in vivo authentic samples (4), with metabolites
formed via hydroxylation, dihydroxylation, N-dealkylation
and double bond oxidation. Nine metabolites for ADB-4en-
PINACA were formed when incubation was performed using
human hepatocyte (5), in which 7 were also identified on this
present work. Analysis of one authentic femoral blood and urine
samples from a postmortem case kindly provided by the NMS
Labs (Willow Grove, PA, USA) corroborated with 7 of 11 ADB-
4en-PINACA metabolites identified during our in vitro assay.
Metabolites formed by hydroxylation, hydroxylation combined
with internal hydrolysis, dihydroxylation and/or double bond
oxidation combined with hydroxylation are suggested to be
used as biological marker. Although authentic ADB-FUBIATA
and BZO-CHMOXIZID positive-biological samples have not been
identified yet, the detection of these analytes in drug material
recently suggests that they should be investigated and the
potential metabolites identification during our in vitro studies
could help in the development of future analysis methodology.
Acknowledgments: This study was financed in part by the
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -
Brasil (CAPES) - Finance Code 001.
REFERENCES
1. EMCDDA. European Drug Report 2021: Trends and Developments.
Publications Office of the European Union. 2021. p. 60. https://
www.emcdda.europa.eu/system/files/publications/13838/
TDAT21001ENN.pdf. Accessed Feb 2022.
2. Discovery N. Recommended Scope for NPS Testing in the United
States - Q4 2021. 2022. https://www.npsdiscovery.org/wp-content/
uploads/2022/02/Q4-2021-NPS-Scope-Recommendations_NPS-
Discovery_021722.pdf. Accessed Feb 2022.
3. Krotulski AJ, Mohr ALA, Papsun DM, Logan BK. Metabolism of
novel opioid agonists U-47700 and U-49900 using human liver
microsomes with confirmation in authentic urine specimens from
drug users. Drug Test Anal. 2018;10(1):127–36.
4. Erol Ozturk Y, Yeter O. In Vitro Phase I Metabolism of the Recently
Emerged Synthetic MDMB-4en-PINACA and Its Detection in Human
Urine Samples. J Anal Toxicol. 2021;44(9):976–84.
5. Kronstrand R, Norman C, Vikingsson S, Biemans A, Crespo BV,
Edwards D, et al. The metabolism of the synthetic cannabinoids
ADB‐BUTINACA and ADB‐4en‐PINACA and their detection in
forensic toxicology casework and infused papers seized in prisons.
Drug Test Anal. 2021;
 99

Liquid chromatography–tandem
mass spectrometry method for
simultaneous quantification of
neurotransmitters in rat brain tissue
Viana, Roberta Rodrigues1; Pego, Ana Miguel Fonseca2; Dallegrave,
Eliane1; Oliveira, Tiago Franco1; Eller, Sarah1
1
 Pharmacosciences Department, Federal University of Health Sciences of Porto
Alegre, Porto Alegre, RS 90050-170, Brazil; 2 John Jay College of Criminal Justice,
City University of New York, 524 W 59th St, New York, NY 10019, USA.

Background: The combination of advanced analytical
techniques makes it possible to determine the
concentrations of neurotransmitters (NTs) in
different biological matrices, providing a complex and
comprehensive study of the effects of psychoactive
substances. Therefore, methods must be specific,
sensitive, and robust to simultaneously monitor
different NTs, which can then be used to evaluate
the neuronal modulation caused by the effects of
xenobiotics. Objective: The present study aimed to
develop and validate a fast and simple analytical method
for the determination of NTs acetylcholine, serotonin,
γ-aminobutyric acid (GABA), glutamate, dopamine,
and metabolites 3-4-dyhydroxyphenilacetic acid
(DOPAC) and homovanillic acid (HVA) in rats brain
tissue by liquid chromatography coupled to tandem
mass spectrometry. Methods: An aliquot of rats brain
tissue was homogenized in type 1 water containing
2% formic acid to a final concentration of 100 mg/
mL. The brain homogenate (50 μL) and 20 μL of
dopamine-d4 (internal standard, 250 ng/mL) were
added to plastic tubes, followed by the addition of
930 μL of acetone prior to agitation. The samples
were then centrifuged, and the supernatant was
collected and dried under nitrogen stream. After
cooling down, the residue was reconstituted in 50 μL
of type 1 water, transferred to an auto‐sampler vial
and 20 μL were injected into the analytical system. A
Nexera-i LC-2040C Plus system coupled to a LCMS-
8045 triple quadrupole mass spectrometer was used
for analysis. The chromatographic separation was
conducted using a Shim-pack CLC ODS 4.6 mm x 25 cm
column eluted with water (solvent A) and methanol
(solvent B) both fortified with 0.1% formic acid with
a flow rate of 0.5 mL/min. Results: The optimization
of the best extraction solvent was performed through
a simplex-centroid design by testing the solvents
methanol, acetonitrile, and acetone. The best results
were obtained using acetone as an extraction
solvent (100%) as it yielded the region with the
best response. Moreover, the mathematical model
exhibited satisfactory results with a coefficient of
determination (R2) of 0.9059, which indicates that
the experimental results can be well explained with
this modeling strategy. The proposed method was
validated providing determination coefficients higher
than 0.99 for all analytes; the LLOQ values were 0.001
ng/mg for dopamine, serotonin, DOPAC and HVA and
0.1 ng/mg for acetylcholine, GABA, and glutamate;
intra-run precision ranged from 0.47 up to 11.52%;
inter-run precision from 0.68 to 17.54% and bias from
89.10 to 109.60%. Discussion/Conclusion: The NTs
are considered external and independent markers,
capable of quantitatively demonstrating the effects of
neuropsychiatric conditions and the consumption of
psychoactive substances on the signal transmission
of neuronal synapses, having wide applicability in
metabolomics studies. However, current techniques
described in the literature show the need for multiple
sample preparation steps, as well as considerable
volumes of sample and organic solvents, which results
in time-consuming analysis, the need for high sample
volume, in addition to having a greater negative
impact on the environment. The analytical strategy
developed in this study was not only able to overcame
the afore-mentioned issues but it has also proven to
be efficient for the simultaneous determination of
NTs at basal levels in rats brain tissue homogenate. In
conclusion, this analytical tool can be used to support
the study of changes in the neurochemical profile for
the characterization of the mechanism of action of
psychoactive substances, and both neurologic and
psychiatric diseases.
 100

Metabolism study of coca leaf tea in urine

by liquid chromatography coupled with
high resolution mass spectrometry
Gomes, Geovana Maria de Lima1; Santos, Vanessa Farelo1; Carneiro, Gabriel Reis
Alves1; Trajano, Christian Farias2; Pereira, Henrique Marcelo Gualberto1
1
Laboratório Brasileiro de Controle de Dopagem, Chemistry Institute,
Federal University of Rio de Janeiro; 2 Comitê Olímpico Brasileiro

Introduction: Cocaine (COC) is the main alkaloid
present in Coca leaf (Erythroxylum coca), and it is
included in the Prohibited List of World Anti-Doping
Agency (WADA). However, in the antidoping context,
the use of cocaine is prohibited only in-competition.
Currently, in-competition period shall in principle
be the period commencing just before midnight (at
11:59 p.m.) on the day before a competition in which
the athlete is scheduled to participate until the end
of the competition and the sample collection process.
WADA accredited laboratories findings for COC must
be reported as an Adverse Analytical Finding (AAF) if,
and only if, either (or both) of follow conditions is met:
i) The estimated concentration of the COC is above
10 ng/mL, and/or the estimated concentration of the
metabolite benzoylecgonine (BZE) is above 50 ng/mL.
The aim of the project was to evaluate the COC and
BZE level in urine from an excretion study from coca
tea administration and evaluated these levels under
the light of WADA rules to trigger AAFs. In addition,
the impact of the pH in the spontaneous hydrolysis
of COC in BZE was also evaluated. Methods: Coca
tea was administrated to 12 volunteers distributed
in two groups, one in the morning and other in the
afternoon. Volunteers were 6 females and 6 males
of different ages between 22 and 50. A commercial
tea bag of coca leaf was prepared by infusion for 5
minutes in 100mL of hot water. An aliquot of 5mL was
collected for analysis. A single administration of tea
was made to each volunteer and aliquots of all urine
of the firsts 48h and first urine in the morning after
48h until the fifth day after administration. Also, a
fraction of each urine aliquoted was transferred to
a Falcon tube containing a buffer solution of formic
acid/ammonium formate 2.0M in pH4 to control the
sample pH. Samples were kept under refrigeration
(-305°C) until preparation by solid-phase extraction
and analysis using liquid chromatography coupled
to high-resolution mass spectrometry (LC-HRMS).
Results and Discussion: Preliminary results showed
COC and BZE concentration values much higher than
necessary for the configuration of an AAF during the
firsts 24h after administration. COC concentrations
decreased fast, reaching the LOD of the method (1ng/
mL). The interval of Cmax of COC excreted among the
volunteers was of 2.4-87.1ng/mL and BZE was off
630.9-2432.1ng/mL, showing a large inter-individual
variation. Buffered samples showed the inhibition of
spontaneous hydrolyses of COC in volunteers’ urine.
The impact of pH and the inter-variability of the
excretion profiles observed among the volunteers
hamper the pharmacokinetics approach in results
interpretation. Continuously analysis will be done to
verify if the spontaneous reaction o hydrolysis can
influence the result management of COC and BZE in
doping samples. Conclusion: Excretion of coca tea
has shown high concentrations of COC and BZE in
urine samples, exceeding the established values for
doping control several hours after the administration.
An in-competition interval discussion is interesting to
evaluate if a consumption outside of this range could
be seen within the criteria currently adopted for an
adverse analytical result. The inter-variability of the
excretion profiles and the wide range observed in pH
values in real urine samples make the interpretation
of results very challenge related to the estimative of
the consumption time. Samples with buffer addition
will be compared with the samples without pH control
to further evaluation of the impact of spontaneous
hydrolysis of COC. Athletes should be aware about the
potential to an AAF from the consumption of coca tea
even out of completion period. Acknowledgments:
The authors thank Autoridade Brasileira de Controle
de Dopagem (ABCD) for financial support.
 101

New tools in analytical toxicology: analysis

of Paraquat in urine with a mobile phone
Chinaglia, Kauê de Oliveira1,2; Lanaro, Rafael2; Arantes, Ana Carolina Furiozo1,2; Costa, José Luiz1,2
1
 Faculty of Medical Sciences, University of Campinas, Campinas, SP,
13083887, Brazil; 2 Campinas Poison Control Center, Faculty of Medical
Sciences, University of Campinas, Campinas, SP, 13083859, Brazil.

Background/Introduction: Paraquat is a widely used
herbicide which use is prohibited by Brazilian’s Health
Regulatory Agency (ANVISA). Its toxic effects relate to
respiratory oxidative stress, which affects liver, heart,
and mainly lungs, causing hemorrhages, fibrosis,
edema, severe lungs failure and death. Paraquat’s
high toxicity and the absence of a specific antidote
leads to treatment difficulties, intensifying the need
for a fast intoxication diagnostic. Considering that, a
rapid identification for paraquat’s acute intoxication
can be realized through a colorimetric assay using
sodium dithionite, which changes the urine color to
blue in the pesticide’s presence. The color change can
be measured using PhotoMetrix, a free application
for smartphones developed for in situ chemical
analysis that decompose, and process digital images
obtained by the phone camera. It can be used for
univariate (calibration) and multivariate analysis
(principal component analysis [PCA]). Considering
that the application works with signal intensity, it
can be employed to toxicological colorimetric assays,
as in paraquat in urine, measuring the concentration
through the difference in color and signal intensity,
emerging as solution for identification and
quantification of paraquat in acute intoxication.
Objective: Development of an analytical method for
the quantification of paraquat in urine using the mobile
phone application PhotoMetrix and colorimetric
reaction with sodium dithionite. Methods: For sample
preparation, 2 mL of basic buffer solution (pH 9.0)
were transferred to a cell culture plate containing 6
flat-bottomed wells, followed by 1 mL of urine with
paraquat and 1 mL of solution of sodium dithionite at
0.1 g/mL. PhotoMetrix application was installed on a
mobile phone Xiaomi Redmi Note 7 with a camera of
48 Mpx and it was calibrated with 6 points from 10 to
150 µg/mL on univariate analysis, Vector RGB mode.
The region of interest chosen was 96x96, Flash mode
On, Exposure 0, Focus Mode on infinity, auto White-
Balance and resolution of 640x480. A ring light was
also used to improve illumination. Then, samples
were analyzed on a spectrophotometer DU-8200
Drawell® set at 600 nm to compare the correlation
of absorbance and concentration values. Results:
The developed method was able to quantify paraquat
in urine with linearity from 10 to 150 (r = 0.98). The
application generated the calibration curve equation y
= 1.893x + 10.439 while the plotted absorbance values
from the spectrophotometer generated y = 0.0126x +
0.0546 (r = 0.99). Urine sample from an intoxicated
patient was analyzed and a concentration of 152.23
ng/mL was estimated with the PhotoMetrix while on
the spectrophotometer the calculated concentration
for the same sample was 169.62 ng/mL. Discussion/
Conclusion: PhotoMetrix presented satisfactory
results for the analysis of paraquat in urine showing
good correlation with the spectrophotometry
technique. The application emerges as good
alternative due to its low cost, practicality and
efficiency, being an accessible technique to provide
reliable information about estimated concentration
after paraquat’s intoxication. Acknowledgments:
Materials and reagents used for this research were
financed by Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP).
 102

Preliminary results of method validation

for dihydropyrimidine dehydrogenase (DPD)
deficiency screening and 5-fluorouracil
determination in plasma by UPLC-MS/MS
Grando, Ana Paula1; Silva, Laura Cé1,2; Hahn, Roberta Zilles1; Linden, Rafael1,2;Antunes, Marina Vezon1,2
1
 Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate
Program on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil.

Background: 5-Fluorouracil (5-FU) is an antimetabolic
drug widely used for the treatment of gastrointestinal
tumors, analogous to the endogenous uracil.
Dihydropyrimidine dehydrogenase (DPD) deficiency
is responsible for approximately 80% of severe
toxicities related to fluoropyrimidines. DPD also
converts endogenous uracil (U) to dihydrouracil
(UH2), with U levels > 16 ng/ml and the ratio U/
UH2 <4.0 predictors of DPD deficiency, allowing
the identification of patients at risk for 5-FU severe
toxicity even before the first chemotherapy cycle.
During chemotherapy, plasma area under the curve
concentrations of 5-FU in the range of 20 to 30 mg.h/L
have been associated with better outcomes. A method
for determination of 5-FU and endogenous U and UH2
in plasma would help to individualize and improve
cancer treatment. Objective: To develop and validate
a method for the simultaneous determination of 5-FU,
U and UH2 in plasma by UPLC/MS-MS. Methods: As
U and UH2 are endogenous compound, the calibration
and control samples were prepared with the isotopes
uracil (U 2-13C15N2) and dihydrouracil (UH2-13C15N2)
standards. The analytes were extracted from 200 µL
of plasma after addition of 150 µL ammonium sulfate
1 M, 100 µL of internal standard (UH2-D4 200 ng/ml
and clorouracil 5-CU 4 µg/ml) and 2.1 mL of solvent
ethyl acetate:isopropanol (85:15, v/v). Samples
were homogeneized by rotation for 10 minutes and
centrifuged at 3000 rpm for 10 minutes. The organic
layer was transferred to a clean tube and concentrated
in an Eppendorf vacuum concentrator at 60 °C for 1 hour.
The extract was recovered with 200 µL of acetic acid
0.5% in water and after the lipid content was cleaned
out with the addition of 50 µL of dichloromethane.
After homogeneization and centrifugation, the
aqueous phase was transferred to a vial and 1 µL
injected onto UPLC/MS-MS Acquity® I-Class UPLC
coupled to a Xevo® TQS-micro triple quadrupole mass
spectrometer with electrospray ionization in positive
mode. Chromatographic separation occurred in an
Acquity® UPLC HSS C18 (1.8 µm 2.1 x 150 mm) at 25 °C.
Mobile phase was water with 0,5% acetic acid (eluent
A) and acetonitrile with 0.5% acetic acid (eluent B)
eluted in gradient mode from 100:0 to linear gradient
of 10:90 (A:B, v/v) at 4.7 mi, maintained for 1 min,
with flow rate of 0.25 mL min -1. The MRM transition
for quantification were 5-FU m/z 131 → 58 and it’s IS
5-CU m/z 147 → 74, and UH2-13C15N2 m/z 118.1→ 70,
U 2-13C15N2 m/z 116.1→ 71 and their IS UH2-D4 m/z
119→ 70. The method was validated according to
FDA, to date specificity linearity, precision/accuracy,
sensitivity and selectivity were performed. Results:
Total analytical run time was 8 min, retention time
was 2.68 for U-13C15N2, UH2-D4 and UH2-13C15N2, 2.97
min for 5-FU and 3.8 min for 5-CU. The method was
selective, with no interference peaks eluting at the
same relation time as the analytes with areas above
20% of those from the lower limit of quantification.
The method was linear from 2.5 to 250 ng mL-1 for
U-13C15N2, 5 to 500 ng mL-1 for UH2-13C15N2 and 50 to
2000 for 5-FU (r2 > 0.99 for 1/x fit for all analytes),
with accuracy of 89-105%. Precision with CV% intra-
assay within 1.90-9.87%, inter-assay within 4.9-13.5%
for all analytes. Discussion/Conclusion: The method
has been shown to be adequate for determining the
concentrations of 5-FU and U in plasma samples. After
conclusion of the validation tests for matrix effect
and extraction yield, the method will be applied in a
clinical study. Acknowledgments: Financial support
from Fapergs, CAPES and CNPq.
 103

Safety studies of a potential cathepsin

K inhibitor: 4-methoxy chalcone
and its degradation products
Benvenutti, Danyela Francine1; Buzzi, Fatima de Campos1; Corrêa, Rogerio1; Couto, Angélica Garcia1;
Wagner, Theodoro1; Paula, Favero R.2; Giovagnoli, Stefano3; Vivani, Riccardo3; Ricci, Maurizio3;
Santos, Carlos Eduardo Matos4; Santin, José Roberto1; Bresolin, Tania Mari Bellé1
1
Pharmaceutical Sciences Graduate Program, Universidade do Vale do Itajaí (UNIVALI),
Itajaí, SC, Brazil; 2 Pharmaceutical Sciences Graduate Program, Universidade Federal
do Pampa, Uruguaiana, RS, Brazil; 3 Department of Pharmaceutical Sciences Università
degli Studi di Perugia, Perugia, Italy; 4 Altox Lab, São Paulo, SP, Brazil.

Background: The chalcone (2E)-1-phenyl-3-(4- of seven major degradation products, in agreement

methoxy-phenyl)-2-propen-1-one, called 4-MC, has
demonstrated important anti-inflammatory and bone
regeneration activities, being a potential inhibitor of
cathepsin K. However, little is known about its toxicity.
Studies with cathepsin K inhibitor drugs have shown
important toxic effects, increasing the risk of stroke,
adverse dermatological effects, and others. Objective:
This work aims to provide a toxicological analysis
through in silico, in vitro techniques, in addition to
the in silico toxicology of major degradation products.
Methods: The molecule was synthesized, purified and
characterized as to its solubility, crystalline habit, and
purity. Stability was addressed by in silico and in vitro
models (photostability), monitored by HPLC-UV and
UPLC-MS/MS. Results: The 4-MC has a crystalline
form, without polymorphism, being practically
insoluble in water. The in silico analysis suggested the
hydrogens attached to the carbon located at the C6
position of both rings of 4-MC as the most probable
positions for auto-oxidation. The 4-MC showed photo
instability especially against visible light (loss of 22.1
and 56.4% at 1.2 and 2.4 million lux/h, respectively).
The UPLC-MS/MS analysis revealed the appearance
with in silico prediction. The probable degradation
products are isomers and dimers of 4-MC, formed
by loss of double bond and rotation of the molecule
followed by the addition process. In silico toxicological
analysis suggest that chalcone 4-MC, and its major
degradation products, showed potential mutagenicity
or skin sensibility being moderately toxic for liver
tissue. However, 4-MC did not show inflammatory
angiogenic or hemorrhagic activity, suggestive of
biocompatibility with mucous membranes in the
HET-CAM test. Discussion: Thus, these results
enabled the understanding of the intrinsic lability
and pointed out the potential toxicity profile of 4-MC,
which must be proven by in vitro and in vivo studies,
aiming to compare with other drug candidates,
previously reproved in clinical trials. Keywords: in
silico toxicology; in silico stability; HET-CAM test;
photostability. Acknowledgments: CAPES (PVE, grant
88887.116106/2016- 00), CNPq (J.R.S. -process #
429505/2018–3;310326/2020–6 and T.M.B.B. -process
# 305484/2018–4 are researcher professor granted
by CNPq with the respective processes and also to
Edital Universal (grant 88887.122964/2016‑00).
 104

Use of design of experiment tools to

enhance the recovery of synthetic
cannabinoids in dried blood spots (DBS)
Berlinck, Débora Zorrón2; Cunha, Kelly Francisco1; Costa, José Luiz1,2
1
 Toxicology Assistance and Information Center (CIATox), University of Campinas, Campinas,
Brazil; 2 Faculty of Pharmaceutical Sciences, University of Campinas (UNICAMP), Brazil.

Background: The concept of statistical analysis
during the planning stages of research was
introduced by Sir Ronald Fisher in the beginning of
the 20th century. This approach presents among
its benefits the rationalization of experiments and
process optimization. The pharmaceutical industry
has traditionally used the one-factor-at-a-time
(OFAT) model to study process optimization. Despite
the recent introduction of new and potentially better
statistical models, the pharmaceutical industry
has continued to use the previous system. This is
potentially due to lack of studies regarding new
models. Conversely, others such as chemical and
food industries have already introduced new models
such as Design of Experiments (DoE) and Principal
Component Analysis (PCA) in their routine. Among
such recent mathematical modeling models, DoE
has been shown to better understand the effects of
multidimensional and interactions of input factors
on the output responses of pharmaceutical products
and analytical methods. Objective: The aim of this
study was to access the effect of the implementation
of DoE on the extraction efficiency (recovery) of
synthetic cannabinoids in dried blood spots (DBS)
samples. Further objectives were to further analyze
17 of synthetic cannabinoids’ analytes by liquid
chromatography–mass spectrometry (LC-MS/MS)
method. Methods: The Plackett-Burman method
was be used for the screening variables, followed
by central composite designs (CCD) to determine
the best conditions for the extraction of synthetic
cannabinoids. Other statistical parameters used were:
analysis of variance (ANOVA), regression significance,
analysis of residuals, determination coefficients (R2
and R2-adj) and lack-of-fit of regression model. Six
variables were used as input factors: resuspension
volume, material used to resuspend extract, rotation
type, rotation time, vortex time and material for
extraction. The STATISTICA 10.0® software package
was used for analyses. Results: Of the six input
factors studied, two achieved statistical significance
(p<0.05) and hence taken to the next step of the
process (i.e. resuspension volume and rotation
time). From the four remaining factors, one was
not significant for any responses and three were
qualitative factors. For the next step, four different
levels and respective centre points were used for
each factor. Resuspension volumes were set as 40 µL
and 110 µL for the “star” points and 50 µL and 100 µL
for the factorial design; and rotation time as 6 and 34
minutes and 10 and 30 minutes. The optimal condition
found after the experiments was a resuspension with
50 µL mobile phase and rotation time of 34 minutes.
In these conditions, the results obtained varied
between 70 and 185% for the increment of analyte
recovery, when compared to the other conditions.
Discussion/Conclusion: The determination of the
optimal conditions for the extraction of synthetic
cannabinoids in DBS samples is essential bearing
in mind the limited amount of sample available for
analysis. With this study, the interaction of two
factors was taken into account in order to determine
the optimal conditions, what cannot be achieved with
the OFAT model. Our results show that the use of DoE
in the optimization process is crucial for obtaining the
best possible results. This is of essence especially
for large scale experiments used in industry, as it
contributes to lower costs and greater efficiency. In
addition, it optimizes the use of limited samples, a
common scenario in forensic toxicology.
 105

Validation and pre-clinical evaluation of

pharmacokinetic profile of antineoplasic
prototype LQMF030 in rats by LC-MS/MS
Pereira, I.B.1; Zoghaib, I.V.J.1; Gomes, S.A.1; Menegatti, R.2; Cunha, L.C.1
1
 Núcleo de Estudos e Pesquisas Toxico-Farmacológicas (NEPET), Faculty of Pharmacy, Federal
University of Goiás (UFG), Goiânia, Brazil; 2 Laboratório de Química Farmacêutica Medicinal
(LQFM) - ), Faculty of Pharmacy, Federal University of Goiás (UFG), Goiânia, Brazil.

Introduction: LQFM030 was obtained by molecular
simplification of nutlins and having demonstrated
excellent in vitro antineoplastic activity. Therefore,
a preclinical pharmacokinetic study was planned
and a rapid, sensitive, and selective LC/MS-MS
bioanalytical method was developed and validated to
quantify LQFM030 plasmatic concentrations in rats.
Methods: Chromatographic analysis was performed
using a 3200 QTRAP LC-MS/MS System and ACE®
C18 analytical column (5 µm 100 x 4.6 mm) and 4
minutes total run time. An (50:50) methanol and 2
mM ammonium acetate with 0.025% formic acid was
used as mobile phase. A multiple reaction monitoring
(MRM) method transitions were used m/z = 319.162
/ 191.20 and m/z = 426.032 / 175.20 for LQFM030
and internal standard respectively. Post validation
was performed after administration of LQFM030 100
mg/kg by gavage and 1.0 mL blood samples were
collected at intervals 0-8 h. The kinetic parameters
were calculated by WinNonlin 5.0 software
(Pharsight™). Results: Calibration curve was linear
over the concentration range examined 10-15000 ng/
mL with r> 0.99 and 10 ng/mL limit of quantitation.
Intra-assay precision was 0.6 to 5.5% and accuracy
from 95.5 to 111.3%. Inter-assay precision was 1.8 to
6.7%, and accuracy was 99.0 to 107.0%. The average
recovery was 74.1% and the matrix effect from -7.9 to
1.5%. The prototypes were considered stable in the
biological matrix and the solution proposed tests.
Pharmacokinetic results (mean ± SD): half-life (t1/2)
3.61 ± 0.68 h, total clearance (CLT) 36.49 ± 2.23 mL/
min/kg, volume of distribution (Vd) 11,40 ± 1.58 L/kg.
Conclusion: The validated analytical method proved
to be adequate to quantify low plasma concentrations
of LQFM030, allowing to establish the relationship
between dose and its pharmacological effects. As
proof of concept, pharmacokinetic parameters of
LQFM030 were calculated in experimental animals.
Keywords: LQFM030. LC-MS/MS. Preclinical
pharmacokinetics. Acknowledgments: Instituto de
Ciências Farmacêuticas - ICF; FAPEG, CNPQ, CAPES.
 106

Validation of a DPX-GC-MS method

for simultaneous quantification of
psychoactive substances and its
metabolites in human breast milk
Santos Junior, Wilson José Ramos1; Gomes, Nayna Cândida1; Costa, Bruno Ruiz
Brandão1; Bigão, Vitor Luiz Caleffo Piva1; De Martinis, Bruno Spinosa2
1
 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São
Paulo, Ribeirão Preto, Brazil; 2 Faculdade de Filosofia, Ciências e Letras de
Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil.

Introduction: Due to physicochemical properties,
several psychoactive licit and illicit compounds
consumed by a lactating woman can be transferred from
her bloodstream into human breast milk (HMB), which
can cause damage in newborn’s short and long term
development. Previous research show the presence of
drugs of abuse in breast milk samples from surveys’
volunteers or milk bank donations, such as marijuana
(THC) and cocaine. In this context, a method based on
Disposable Pipette Extraction (DPX) with analysis by
gas-chromatography coupled to mass spectrometry
(GC-MS) was developed and validated for the
quantification of several of psychoactive substances
in HBM. Objectives: To validate a DPX-GC-MS method
for simultaneous quantification of psychoactive
substances and its metabolites in human breast milk.
Methods: An HBM pool was spiked with the analytes
at 100ng.mL-1 (amphetamine, methamphetamine,
methylenedioxymethamphetamine, nicotine, cotinine,
methadone, morphine, cocaine, cocaethylene and
benzoylecgonine) and deuterated internal standards
at 200ng.mL-1. The sample preparation was previously
optimized by design of experiments and consisted of
adding 2.5 mL of acetonitrile and 100 µL of phosphoric
acid to 1 mL of HBM. Then, the samples were agitated
using a horizontal shaker (200 rpm for 10 min) and
centrifuged (2000 rpm for 10 min). After the protein
precipitation, DPX tips of cationic exchange phase
were used for extraction and pre-concentration: 0,5
mL of acetonitrile for solid phase conditioning (30
seconds); 0,5 mL of methanol/water 70% (v/v) for
washing (10 seconds); 875 µL of extraction solution
(dichloromethane: isopropanol: ammonium hydroxide
78:20:2 v/v/v, repeated three times for 30 seconds
each). The extraction solution was vaporized at 30 °C
using N2 and then reconstituted with 35 µL of ethyl
acetate. Finally, 2 µL of the sample were injected
in splitless mode in the GC-MS. The method was
validated based on ANVISA and SWGTOX Guidelines
on Bioanalytical Method Validation. Results and
Discussion: The limits of detection (LODs) reached
from 2,0 to 5,0 ng.mL-1 and the lower limit of
quantification (LLOQ) was 5,0 ng.mL-1 for all analytes.
Calibration curves were linear between 5.0 and 500
ng.mL-1 with correlation coefficient values higher
than 0.99 using weighted linear regression (1/x2).
Recovery values ranged from 15.0 to 86.0%, intra-
day precision 2,0-19,0% (CV%), inter-day precision
4.0-19.0% (CV%) and accuracy of 5.0-13.0% (RSE%).
Quality controls (QCs) samples were stable in long-
term stability, post-processing and thawing cycle
tests. No carryover effect was observed. Conclusion:
The method validation indicated accurate sensibility
and reproducibility on evaluated parameters. A
large volume of acetonitrile had to be used during
protein precipitation, although in DPX procedure
was possible to reduce solvent consumption after
statistical optimization. DPX-GC-MS demonstrated to
be a suitable technique for complex matrices analysis
for forensic toxicology purposes, such as HBM, with
quick and less laborious steps and also meeting Green
Analytical Chemistry principles. The validated method
will be applied for analysis of HBM collected at milk
banks in the city of Ribeirão Preto, São Paulo, Brazil.
Acknowledgements: This work was supported by
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES Pro-Forenses 25/2014) and Fundação
de Amparo à Pesquisa de Estado do São Paulo (FAPESP
– 2017/18021-5).
 107

Validation of a method for plasma vancomycin

therapeutic monitoring by High Performance
Liquid Chromatography with UV detector
Paula, Eliza Bianchini1; Santos, Claudia Regina2; Marchioni, Camila2
1
Federal University of Santa Catarina (UFSC), Brazil; 2 Department of Pathology,
Federal University of Santa Catarina, Florianópolis, Brazil.

Introduction: Vancomycin is a potent hospital
antibiotic used in the treatment of gram positive
bacterial infections including methicillin-resistant
Staphylococcus aureus (MRSA). The recommended
plasma concentration is 15 to 20 µg/mL. Lower
values cause development of resistant strains and
can lead to therapeutic failure. However, plasma
values above 20 ug/mL can cause nephrotoxicity and
ototoxicity. Therefore, a narrow therapeutic window
is observed for the treatment, besides, it’s known
that to be a drug with high intra individual kinetic
variation, hepatic and renal insufficiency patients
have different plasma concentrations with the same
dose applied. Consequently, patients’ therapeutic
monitoring becomes vitally important to prevent the
adverse effects caused by the high concentration
and also avoid the bacterial resistance development.
Objective: Validate an analytical method capable
of determining plasma vancomycin, using High
Performance Liquid Chromatography with UV detector
(HPLC-UV). Methodology: For plasma vancomycin
quantification, a method was developed using
Shimadzu HPLC-UV system, reverse C18 column (4.6
mm x 15 cm x 5 µm) adopting as mobile phase a two-
phase system constituted by acetic buffer pH 4.0 (A)
and methanol: acetonitrile in proportion to 70:30 (v/v)
(B). The gradient starts 90A:10B (v/v), at 4 minutes the
race reaches a concentration of 40A:60B (v/v), at 9
minutes 70A:30B (v/v) and back to 90A:10B (v/v) up to
12 minutes, maintaining this ratio up to 22 minutes. The
vancomycin retention time obtained was 11.5 minutes.
As an Internal Standard, it was used imidacloprid (50
µg/mL), which obtained a 14.2 minutes retention time.
The method analytical validation was performed based
on the standards of the National Health Surveillance
Agency of Brazil (ANVISA). Results: The method
presented a Limit of Detection (LOD) of 2 µg/mL and
a Limit of Quantification (LOQ) of 5 µg/mL. Linearity
was determined in the range of 5 to 100 µg/mL. The
intraday and interday precision were approved with
a Coefficient of Variation (CV) at 9.27%. The intra and
interday accuracy was also adequate with a Relative
Standard Deviation (RSD) at 10.52%. No residual
effect was observed. As for selectivity, there were no
significant peaks in the same retention time of interest
analytes. There was also no matrix effect (p > 0.05).
For stability, the samples were stable for 24h at room
temperature, with up to 3 freeze and thaw cycles,
post-processed for 24h and long term (3 and a half
months to date). Conclusion: The chromatographic
method for vancomycin quantification proved to be
sensitive, precise, accurate, with no residual or matrix
effect. It has been proven yet vancomycin stability
in biological matrix in freeze/thaw cycles, post-
processing and short/long duration. In conclusion,
the validated method complies with the requirements
to be used in hospital routine for necessary dose
adjustments in patients on antibiotic treatment.
Keywords: Vancomycin, Therapeutic monitoring,
Plasma, Validation, HPLC-UV.
 108

Validation of an analytical method for the

determination of psychoactive substances
in an oral fluid sample using the DPX-
SCX disposable tips and the GC-MS
Gomes, Nayna Cândida1; De Martinis, Bruno Spinosa2
1
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Ribeirão Preto, São Paulo, Brasil.
2
 Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Ribeirão Preto, São Paulo, Brasil.

Introduction: The emergence and dissemination
of new psychoactive substances around the world
represent a worrying public health problem. Objective:
Develop and validate a method for the analysis of
amphetamine, methamphetamine, MDMA, MDA, MDEA,
cocaine, cocaethylene, anhydroecgonine methyl
ester, dibutylone, n-ethylpentylone, 25E-NBOMe,
25C-NBOMe, 2C-E, 2C-C, fentanyl and carfentanyl
in oral fluid sample using the GC-MS. Method: In
a glass tube were pipetted: 500 µL of oral fluid, the
analytical standards and the internal standards
amphetamine-d11, fentanyl-d5 and cocaine-d3. 2 mL
of methanol was added, vortexed and shaker table,
followed by centrifugation. 2 mL of this solution was
transferred to another glass tube and 100 μL of 0.1 M
H3PO4 was added. The conditioning of the DPX-SCX tip
was done with 1mL of acetonitrile. 2mL of solution
containing 0.1 M H3PO4 were aspirated. Washing was
performed using 500 μL of methanol and, for elution,
1500 μL of dichloromethane: isopropanol: ammonium
hydroxide (78:20:2) solution was used. The eluate
was evaporated, reconstituted with 40 μL of ethyl
acetate and derivatized with 40 μL of MSTFA. HP 5
fused silica capillary column (30 m x 0.25 mm x 0.25
μm film thickness) was used. The injector temperature
was 280 °C (splitless mode), the injection volume was
1µL and the carrier gas used was helium. The initial
oven temperature was 90 °C for 2 minutes. This was
raised to 220 °C at a heating rate of 10 °C.min-1. Then,
elevation to 290 °C with a heating rate of 20 °C.min-1
for 4 minutes. The temperature used in the MS
interface, source and quadrupole was 280, 230 and
150 °C, respectively. The parameters evaluated in the
validation of the method were those recommended by
ANVISA resolution 27/2012 and 899/2003. Results:
The detection limit ranged from 1 to 15 ng/mL. and no
residual effect was observed. The matrix effect ranged
from 1.62% for dibutylone to 10.45% for 2C-E. The
method was considered linear in the concentration
range from 2 to 400 ng/mL for amphetamine, MDMA,
MDA, dibutylone and n-ethylpentylone, from 3 to
400 ng/mL for methamphetamine, from 5 to 400 ng/
mL for MDEA, 25C-NBOMe, fentanyl and carfentanyl,
from 10 to 600 ng/mL for cocaine, cocaethylene,
anhydroecgonine methyl ester and 25E-NBOMe and
from 30 to 1100 ng/mL for 2C-E and 2C-C. The analyte
recovery values ​​range from 74.45% to 97.38% for the
LLQ of 2C-E and n-ethylpentylone, respectively. The
intraday precision ranged from 0.32 to 14.03% for the
LQC and LLQ values ​​of dibutylone and 25C-NBOMe,
respectively. The interday precision varied from 1.15
to 13.93%, which were the LLQ values ​​of 25E-NBOMe
and HQC of 2C-C, respectively. The intraday accuracy
values ranged
​​ from -12.98% to 15.79% for the LLQ and
MQC values ​​of amphetamine and 2C-C, respectively.
The intraday accuracy ranged from -15.60% for the
amphetamine LLQ to 12.86% for the carfentanyl
LLQ. Analytes were stable in oral fluid and standard
solutions. Discussion/Conclusion: The method
developed and validated met all the criteria required
by resolution 27/2012 and 899/2003 of ANVISA.
Acknowledgments: This research was supported
by CAPES – Brazil (Financing Code 001) and FAPESP
2017/18021-5.
 109

Validation of disposable pipette

extraction method with quantification
by GC-MS to determine ethylenethiourea
in human urine samples
Romoli, Jéssica Cristina Zoratto1; Scanferla, Deborah Thais Palma2; Aguera, Raul Gomes2; Lini,
Renata Sano2; Castro, Juliana Cristina3; Bando, Érika4; Alves, Gessé de Souza4; Nerilo, Samuel
Botião4,5; Mossini, Simone Aparecida Galerani2,4; Marchioni, Camila6; Machinski Junior, Miguel1,4
1
 Postgraduate Programin Health Sciences, State University of Maringá, Paraná, Brazil;
2
 Postgraduate Program in Bioscience and Physiopathology, State University of Maringá,
Paraná, Brazil; 3 Postgraduate Program in Pharmaceutical Sciences, State University of
Maringá, Paraná, Brazil; 4 Laboratoty of Toxicology, Departament f Basic Health Sciences,
State University of Maringá, Paraná, Brazil; 5 University Center Ingá, Maringá, Paraná, Brazil;
6
 Department of Pathology, Federal University of Santa Catarina, Florianopolis, Brazil.

Introduction: Ethylenethiourea (ETU),
biotransformation product of dithiocarbamates
(DTCs) fungicides, has been suggested as a biomarker
of occupational exposure, requiring the development
and validation of methods for its quantification.
Some researchers have been used analysis by Gas
Chromatography-Mass Spectrometry (GC-MS) and
sample preparation by classic methods, like liquid-
liquid extraction. The Disposable Pipette Extraction
(DPX) technique is a miniaturization of solid phase
extraction that allows rapid and simple extraction
of analytes due to sorption equilibrium. Objective:
Validate a methodology for quantification of ETU in
urine samples by DPX-GC-MS. Methods: A 1 mL tip
containing 40 mg of silica gel freely accommodated
between two porous polymer filters was developed
for use in the DPX procedure. With the aid of a syringe
device, HCl 6N (100 µL) was aspirated as a conditioning
solvent from the extractor phase. Afterwards, the
urine sample (200 µL) was inserted into the pipette
followed by aspiration of air (turbulent air bubbles)
for 1 min, to create a sorbent suspension in the
sample, which was subsequently discharged. Then,
the sorbent was washed (cleaning step) with 100 µL
of hexane:dichloromethane (95:5, v/v) with a draw/
eject cycle. Finally, the ETU was eluted with 200 µL
of hexane:acetone (50:50, v/v) by aspirating air for
1 min and the eluate was dried in a water bath at 40
°C. The dried extract was reconstituted with 50 µL of
derivatizing solution (ethyl acetate: BSTFA: t-BuMe2Si-
Cl (5:4:1, v/v/v)) and incubated at 60°C/30 minutes,
modified from Fustinoni et al. (2005). The extract
was analyzed by GC-MS, quadratic high-quadratic
ISQ TRACE 1300, coupled to a mass spectrometer
(ThermoScientific®). Capillary column of Rxi®-5ms
capillary column (30m x 0.25 mm x 0.25 µm) was used
and helium was a carrier gas at 1.0 mL/min. Injections
were made in the pulsed splitless mode. The injector
liner (250 °C) injected 1 µL of the sample into the
chromatographic column. The oven temperature was
kept at 150 °C for 1 min, then the temperature was
increased to 240 °C at the rate of 20 °C min-1. The oven
was kept at 240 ºC for 5 min. The total run time was 11.5
min. Thus, the method was validated, evaluated linear
range, limit of quantification, precision, accuracy,
matrix effect, carryover, selectivity and stability.
Validation followed current guidelines published
by the European Medicines Agency (EMA), Food
and Drug Administration (FDA) and National Health
Surveillance Agency (ANVISA) Results: Two ions were
selected form ETU monitoring and confirmation, m/z
159 and m/z 231. The linear range was 100 to 1000 ng/
mL (r=0.999). Values of precision had ranged from 0.6
to 10.8% and accuracy was in the range -13.2 to 13.4%.
The limit of quantification was 100 ng/mL. Carryover
was not observed according to acceptance criterion. It
was verified that there was a matrix effect (r<0.001),
being of the rotational type. Therefore, analysis
by standard addition will be used to circumvent
this effect, since tests in sample preparation were
performed and satisfactory results were not obtained.
Stability was not obtained for the freezing/thawing
(two cycles at -20°C) and post-process (-20°C/96h)
tests, however, long-term stability studies are being
carried out. Discussion/Conclusion: Even though
the method DPX-GC-MS should a matrix effect, the
validation was effective, once it is possible for the
analysis of samples to be performed by standard
addition. The method developed is innovative,
cheap, simple, fast and requires a small amount
of biological sample and organic solvent, making
it a good option for application in rural workers in
order to assess occupational exposure. Keywords:
Ethylenethiourea, gas chromatography, DPX,
validation. Acknowledgements: CNPq, CAPES and
COMCAP-UEM for their support.
 03 
CLINICAL AND
LABORATORIAL TOXICOLOGY
 111

A simple procedure for the determination

of mercury in urine by Inductively Coupled
Plasma Mass Spectrometry (ICP-MS)
Sugawara, Eduardo Kinio; Silva, Graciele Machado; Paulucci, Leticia Trevisan;
Ramadan, Debora R.; Tufik, Sergio; Soares, Marcela de Oliveira

Associação Fundo de Incentivo à Pesquisa - AFIP.

Background: The material (Hg) is distributed in
different media, circulating in different states, being
found in different states: gaseous, liquid and solid.
Different degrees of forms, exhibiting varying degrees
of toxicity. H can cause kidney damage, central
nervous system disorders, intellectual disorders and
even death. Therefore, an efficient assessment of
suspected intoxication through the determination of
Hg levels in various biological matrices, mainly blood
and urine, is of vital importance. The aim of the study
was to validate an easy and suitable for analysis of
both organic and inorganic Hg in urine. Methods:
Validation was performed using a Inductively Coupled
Plasma Mass Spectrometry, 7850 ICP-MS. Analytical
analysis is ensured using the most abundant isotope
200
Hg as monitoring. The procedure involves a small
amount of 500 µL of urine, followed by dilution with
500 µL of internal standard at concentration of 1.0
µg/L more 4000 µL of hydrochloric acid and 2%
nitric acid. The internal standard was purchased
from Agilent at a concentration of 10.0 µg/mL. After
preparation the samples are followed by aspiration
into the SPS 4 model. A cleaning solution used was
hydrochloric acid and 2% nitric acid at a rate of 0.1
rps. Quantitation is achieved by the comparison of the
responses from a given sample with the responses
of calibrators with known concentrations prepared
through Multi Element Cabibration at concentration
of 10.0 µg/mL of Hg. Linearity was observed in the
expected concentration range from 0.1 to 1.6 µg/L
and urine samples were evaluated at six different
concentrations and six times each at the same time of
the study. For validation, the same extraction diluent
was used and to evaluate the matrix effect, tests
were performed with a white urine sample. Standards
acquired by Agilent. Results: The linearity coefficient
of determination (R) was 0.9931. The method showed
100% selectivity and no residual effect interference.
In order to determine the average inter-assay CV%,
three different concentrations were analyzed over
three days and the results for each low, medium, and
high concentration level were 7.64, 8.08 and 5.82%,
respectively. The accuracy of the method was cheeked
by analyzing samples of known concentration and
expressed as a percentage. A time total analysis
time was 3.5 min. Conclusion: The method was fast
and efficient for the determination of urine Hg. The
efficiency and selectivity combined with the technical
robustness can be used in the diagnosis of intoxication
and monitoring also in cases of toxicity.
 112

Analysis of plasmatic levels of

antipsychotics through an absorptive
paper-based extraction followed by fast
gas chromatography-mass spectrometry
Gouveia, Giovanna Cristiano1; Borges, Gabriela Ramos1; Santos, Bruno Pereira1,3; Sebben,
Viviane Cristina2; Arbo, Marcelo Dutra3; Eller, Sarah1; Oliveira, Tiago Franco1
1
 Graduate Program in Health Sciences, Federal University of Health Sciences of
Porto Alegre (UFCSPA), Brazil; 2 Toxicological Information Center of Rio Grande do
Sul (CIT/RS), Brazil; 3 Federal University of Rio Grande do Sul (UFRGS), Brazil.

Background/Introduction: Antipsychotics are
commonly used in the treatment of patients with
psychic disorders symptoms. In 2021, the cases of
antipsychotic exposure represented almost 20%
of all medicine intoxication cases attended by the
Toxicological Information Center of Rio Grande do
Sul (CIT/RS). Therefore, the development of quick,
easy and efficient methodologies for the diagnosis of
these intoxications is essential for clinical practice.
Objective: The aim of this study was to develop and
validate an absorptive paper-based extraction for
fast gas chromatography-mass spectrometry (fast-
GC/MS) analysis of chlorpromazine, haloperidol,
levomepromazine, and promethazine in human
plasma samples. Methods: The samples were
obtained from emergency cases attended by the
Toxicological Analysis Center at the CIT/RS. Sample
procedure: To perform this technique 24-well plates
were used with two discs (16 mm) of Whatman®
qualitative filter paper Grade in each well. An aliquot
of 50 μL of plasma was transferred into the plate
and dried for 10 min at room temperature. Then, 500
μL of a mixture of methanol and acetonitrile in the
proportion 1:1 (v/v), and 15 μL of internal standard
(chlorpromazine-d4) was added to the well. The
plate is placed on the orbital shaker for 5 min at 200
rpm. After that, aliquots of 450 μL of the solvent of
extraction were collected and dried under a nitrogen
stream. The residue was reconstituted with 20 μL of
methanol followed of 2 μL injection with a split ratio
of 1:5 into the analytical system. Optimization: The
solvents were optimized using the simplex-centroid
design and the volumes of sample and extraction
solvent were determined using the Doehlert design,
both by software Statistica 8.0. Instrumentation:
The chromatographic analysis was performed in a
GC-MS-QP 2010 Plus gas chromatography coupled
to mass spectrometry and autosampler model AOC-
20i (Shimadzu, Kyoto, Japan). Chromatographic
separation was carried out in a Rxi fused silica
capillary column model RH-Rix-5MS (20 m × 0.18 mm ×
0.18 μm). The ultrapure helium was used at a constant
flow of 1.42 mL.min−1. The injector temperature was
set at 250 °C and the initial oven temperature was
set at 200 °C, increasing to 310 °C, and held for 0.6
min, then increased to 330 °C and holding for 1.78
min, totalizing 4.0 min of chromatographic run. The
pressure was set at 297 kPa. The quantification of the
analytes was performed in selected ion monitoring
(SIM) mode using the highest intensity m/z ratio for
each substance. Results: The method validation was
conducted according to European Medicines Agency
guidelines on bioanalytical method validation. The
developed method has been validated for the lower
limit of quantitation (LLOQ), linearity, selectivity, and
carryover. The LLOQ was 50 ng/mL for all the analytes.
The concentration ranges used were 50-800 ng/mL for
chlorpromazine, haloperidol, levomepromazine, and
promethazine. All analytes presented a coefficient of
determination (r2) ≥ 0,99. The heteroscedasticity was
calculated, and the correction factor was applied. No
carryover effects were observed. Total of 28 samples
were analyzed. Six samples were found to be positive
for promethazine (21%), including one case reaching
a toxic concentration and three with concentrations
higher than the therapeutic recommendation.
Fourteen samples were positive for chlorpromazine
(50%) and nine were above the therapeutic range.
Discussion/Conclusion: The technique proposed
in this work presents a quick analysis considering
the extraction and chromatography procedure.
Therefore, the implementation of this methodology
in toxicological centers can optimize the analytical
procedure supporting the decision-making and,
consequently, more effective management, and
treatment, for the intoxicated patient. The method will
still be completed with all the parameters required by
the guideline. Acknowledgments: CAPES; FAPERGS.
 113

Assessment of mercury exposure and

health of riverside populations affected
by the Brumadinho disaster
Moreira, Camila Francisco; Nolasco, Daniela; Souza, Tainá Brumate; Mendes,
Michele Polyana Rocha; André, Leiliane Coelho; Paiva, Maria José Nunes

Toxicological Analysis Laboratory, Department of Clinical and Toxicologic Analysis, College

of Pharmacy - Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.

Introduction: On January 25, 2019, dam I at the
Córrego do Feijão mine in Brumadinho, Minas Gerais,
collapsed releasing more than 10 million cubic meters
of tailings into the environmental and water body of
Paraopeba river. This occurrence had several social,
economic, political, environmental and health impacts
on people living in the municipality and also in nearby
communities. However, the magnitude of these
effects on the population needs further investigation,
especially regarding environmental expousure to
contaminants. The research addressed here portays
the assessment of the health of comunities close to
the Paraopeba river throught the toxicology of metal
and metalloids, being the first work of this nature
in these locations. Objective: The purposes were to
evaluate the environmental exposure to mercury, as
well as the health of the population residing in the
communities near the Paraopeba River. Methods: 121
volunteers from the municipalities of Mário Campos,
Juatuba e São Joaquim de Bicas participated in this
cross-sectional study. Data were obtained through
self-repot in a pre-collection interview, blood and
urine samples Blood were collected for analysis of:
blood count, renal and hepatic function biomarkers
by biochemical measurements and metals and
metalloids by DMA (Direct Mercury Analysis). Results:
Volunteers reported hypertension (47), dermatites,
allergies and skin irritation (26), diabetes (17) and
mental health conditions (11). The blood count
demonstrated probable incidences of erythrocytosis
(23,3%), erythropenia (8,6%), hemoglobin deficiency
(6.9%), reduction in RDW-CV (68.1%), leukocytosis
(12.1%) and eosinophilia (9.5%). The measurement
of liver and kidney function biomarkers showed
an increase in: alkaline phosphatase (23.5%), AST/
TGO (47.3%) and serum urea (75%). In addition, 13
volunteers had an AST/ ALT ratio greater than 2 and
4 albumin results were above 30 mg g -1 creatinine.
The validated methodology for mercury analysis was
satisfactory, there were 15 levels above the detection
limit of the method (0,1212 ng; 0,606 µg L-1), 2 of them
above the quantification limit (0,2108 ng; 1,054 µg L-1),
in whole blood. The multivariate analysis indicates
similarity between the populations evaluated in terms
of global health parameters accessed Discussion and
Conclusion: The data presented in this work show
important hematological and biochemical alterations
in individuals living in communities along the
Paraopeba River after the Brumadinho disaster. These
events may be associated with exposure to metals
and metalloids in the region, however, mercury levels
were similar to those found in other populations
not occupationally exposed. Other potentially toxic
elements have been measured by ICP-MS to help
understand changes in tests performed and the
magnitude of impacts generated in these locations.
Acknowledgment: We thank the Ezequiel Dias
Foundation partners for their collaboration and
enabling important stages of this work.
 114

Case Report: follow-up care of a cocaine

intoxication case at a Toxicological Information
and Assistance Center in Fortaleza - Ceará
Salles, Gabriela Pereira1; Ferreira e Silva, Hendyelle Rodrigues4; Silva, Victória da
Costa2; Batista, José Márcio Machado3; Albuquerque, Polianna Lemos Moura Moreira3;
Moreira, Rhubens Levy Rodrigues4; Ferreira, Maria Augusta Drago1
1
 University of São Paulo, Ribeirão Preto – SP, Brazil; 2 Federal University of Ceará, Fortaleza – CE,
Brazil; 3 Dr. José Frota Institute, Toxicological Information and Assistance Center, Fortaleza-
CE, Brazil; 4 Toxicology Study Center of Federal University of Ceará, Fortaleza-CE, Brazil.

Introduction: Cocaine is a tropane alkaloid derived
from the leaves of the plant species Erytroxylum coca
and used worldwide as a drug of abuse. According to
the Single Convention on Narcotic Drugs of 1961 and
to the United States Convention against Illicit Traffic
in Narcotic Drugs and Psychotropic Substances of
1988, the distribution, import, export, manufacture
and production of this drug or derivative compounds
are illegal in most countries. To evade detection
and apprehension by law enforcement agencies,
traffickers employ a vast and diverse array techniques,
notably the swallowing of drug packets containing
cocaine. Objective: To describe patient care, trying
to assess the therapeutic procedures adopted by the
health team in handling a case of cocaine poisoning
related to the ingestion of drug packets. Methods: All
data pertinent to this case report were elicited from
the health team of the Toxicological Information and
Assistance Center located in the city of Fortaleza,
state of Ceará. A thorough review of the specialized
literature on the subject was also carried out. Results:
On the day of 03/10/2019, a 34-year-old male was
forwarded to the Toxicological Information and
Assistance Center about twelve hours after having–
according to the patient himself–ingested twenty
cocaine packets. By the time the Toxicological Center
was called upon to assist in treatment, the patient,
having already excreted nine intact packets and
manifesting hematemesis, had a blood pressure of
123/69 mmHg (120/80 mmHg) and a cardiac frequency
of 103 beats per minute (60-64 beats/min). Initial
management consisted of intravenous hydrations
to avoid hypotension, and of the administration of
mineral oil to facilitate the excretion of the remaining
drug packets; also, an echocardiogram (ECHO) and
other biochemical tests were requested. The latter’s
results, which were issued only in the morning of the
next day (04/10/2019), revealed high levels of AST: 41
U/L (0 - 35 U/L); CPK:1291 U/L (32 - 294U/L); and of
CKMB: 32 ng/mL (< 5 ng/mL). Hemoglobin (Hb) level
was below normal: 12,9 g/dL (14 - 17 g/dL). Tests
performed in the afternoon of 04/10/2019 detected
a higher-than-normal lactate dehydrogenase (LDH)
level–that is, 485 IU/L (115 - 225 IU/L)–and a further
elevation of the CPK level–to 4.371 U/L (32 - 294 U/L).
In view of such results, the medical staff decided
to keep the patient intravenously hydrated and to
request an abdominal x-ray to learn if the “packets”
had been eliminated. On 05/10/19, the patient fled the
hospital. Discussion/Conclusion: Depending on its
clinical manifestations, cocaine poisoning treatment
consists initially of procedures aiming ventilatory and
hemodynamic stabilization; additionally, vital signs
and body temperature should be carefully monitored.
Given that cocaine may cause an increase of
catecholamines release, cardiac frequency elevation
was to be expected. Considering that the use of
laxatives, by stimulating peristalsis, may cause the
drug packets to rupture, the administration of mineral
oil, in general, preferable in facilitating excretion.
The patient’s low Hb count may be related to the
episodes of hematemesis he reported. According to
the specialized literature, the concomitant rise of CPF,
CKMB and LDH levels within 24 hours after exposure
is indicative of heart damage and/or distress
consistent with cocaine poisoning. As this Center does
not perform toxicological analyses, the adoption of
therapeutic procedures was based solely on the signs
and symptoms manifested, laboratory test results
and on information provided by the patient himself.
This case-study reveals that the transportation of
drugs inside the gastrointestinal tract represents an
enormous health hazard. In addition, it was observed
that the therapeutic procedures adopted by the
health team were in accordance with what up-to-
date scientific literature recommends. Keywords:
Intoxication, Drug trafficking, Cocaine, Toxicological
Information and Assistance Center
 115

Case Report: follow-up care of a kerosene

intoxication case at a Toxicological Information
and Assistance Center in Fortaleza - Ceará
Salles, Gabriela Pereira1; Ferreira e Silva, Hendyelle Rodrigues4; Silva, Victória da Costa2;
Farias, Beatriz Valentim2; Magalhães, Karla do Nascimento3; Batista, José Márcio Machado3;
Albuquerque, Polianna Lemos Moura Moreira3; Ferreira, Maria Augusta Drago2
1
 University of São Paulo, Ribeirão Preto – SP, Brazil; 2 Federal University of Ceará, Fortaleza - CE,
Brazil; 3 Dr. José Frota Institute, Toxicological Information and Assistance Center, Fortaleza-
CE, Brazil; 4 Toxicology Study Center of Federal University of Ceará, Fortaleza-CE, Brazil.

Introduction: a mixture of hydrocarbons distilled
from petroleum, kerosene was very much present
in the domestic environment as it fueled oil lamps,
heaters and stoves before the development of
electricity as a source of power. In our times, the
substance is used mainly as a solvent, degreaser, or
lubricant in transport vehicles. Although nowadays
less frequent, intoxications caused by this substance
still occur and generally due to improper handling or
storage. Objective: To describe patient care, trying
to assess the therapeutic procedures adopted by the
health team in handling a case of kerosene poisoning.
Methods: All data pertinent to this case report was
elicited from the health team of the Toxicological
Information and Assistance Center located in the city
of Fortaleza, state of Ceará. A thorough review of the
specialized literature on the subject was also carried
out. Results: On the day of 11/09/2019, a 1 year and
3 months old male patient was forwarded to the
Toxicological Information and Assistance Center by
an Emergency Care Unit just four hours after having
accidently ingested kerosene while in his family
home. Medicated with a corticosteroid, a gastric
protector and dipyrone by the emergency healthcare
team, by the time the Toxicological Center was called
upon to assist in treatment, the patient, tachypneic
and tachycardic, presented a cardiac frequency of
149 beats per minute (80-130 beats per min.) and a
respiratory rate of 80 breaths per minute (24-40
breaths per min.). Thoracic x-ray, complete blood
count and a set of biochemicals tests (Prothrombin
Time, Activated Partial Thromboplastin Time, Urea,
Creatinine, Potassium, Chlorine, Magnesium, Sodium,
Amylase, Lipase) were requested and performed,
revealing, however, no significant abnormalities.
The initial management, using corticosteroids and a
gastric protector, was maintained. On the very next
day (11/10/2019), the diagnose of pneumonitis was
confirmed by a new thoracic x-ray. In view of the result,
the medical staff recommended the administration
of the intravenous antibiotic Cefepime (2g), and the
patient was subsequently discharged. Discussion/
Conclusion: Given its low rate of gastrointestinal
absorption, kerosene, when ingested, usually does
not to produce significant systemic toxic effects.
However, the substance, an irritant, may cause
inflammation of GI tract tissues and, consequently,
vomiting, which, in turn, if kerosene is aspired along
with other gastric contents, may cause pneumonitis–
that is precisely why the induction of emesis is not
recommended as a method of decontamination in
such cases. Regarding the therapeutic procedures
adopted by the health team in this specific case,
the use of gastric protectors, as to avoid possible
damage to the stomach mucosa, and of dipyrone,
for relieving pain, were in accordance with the most
up-to-date scientific literature recommendations,
as was antibiotic prophylaxis–to prevent secondary
infections or other respiratory complications. On
the other hand, the use corticosteroids in the face
of possible kerosene pneumonitis is no longer
advised, studies showing it has uncertain results
in improving patient outcome. Being physically and
mentally immature, given to curious and exploratory
behavior, children are particularly vulnerable to
accidental poisoning by household products–which
is a fairly common toxicological pediatric emergency.
Additionally, factors such as the inadequate storage
of these products, inadequate parental supervision,
economic and educational problems within the family,
contribute to the occurrence of these intoxications.
Accidents such as the one pertaining to this case
study, though deemed unforeseen, might be avoided
by promoting public awareness on the toxicity and
possible health hazard posed by household available
chemicals. Keywords: Intoxication, hydrocarbons,
kerosene, children, Toxicological Information and
Assistance Center
 116

Case Report: Phencyclidine cross-reaction

investigation in immunochromatographic
tests for rapid drugs detection
Cardoso, Leonardo Corrêa1; Rosa, Victória Gomes2; Pacheco, André Lucas Bezerra2; Santos, Rachel2;
Ugalde, Gustavo Andrade1; Berlato, Dener Gomes1; Reginato, Fernanda Ziegler1; Chimendes, Nayomi
Andrade2; Santos, Lara Celestina2; Nascimento, Marcelo Henrique Santana2; Oliveira, Sarah
Carobini Werner de Souza Eller Franco3; Oliveira, Tiago Franco3; Bairros, André Valle1
1
 Nucleus Applied to Toxicology, Postgraduate Program in Pharmaceutical Sciences,
UFSM; 2 Nucleus Applied to Toxicology, Course of Pharmacy, UFSM; 3 Federal
University of Health Sciences Foundation of Porto Alegre, UFCSPA.

Introduction: According to recent data from the
National System of Toxic-Pharmacological Information
(SINITOX), Brazil recorded more than 27.000 cases
of poisoning during 2017. This includes poisonings
caused by drugs, precursors, household products,
industrial agents, cosmetics, metals, drugs of abuse,
plants, food, venomous animals and unknown agents
whose diagnosis indicates the action of a xenobiotic
(SINITOX, 2017). Drugs and drugs of abuse account for
more than 33% of intoxication cases in the Rio Grande
do Sul (SINITOX, 2017; CIT, 2019), however the report
of cases of intoxications by drugs and medicines
that are not common to be found in the state are
important, especially considering the analytical
screening methodologies such as immunoassays. The
case report relates a 50-year-old male patient who
attempted suicide in his residence through massive
ingestion of prescribed drugs. Toxicological analysis
was performed with an immunochromatography
test and detected phencyclidine (an uncommon
drug in Brazil). Considering the patient’s history, the
greatest suspicion was a cross-reaction not studied
by the scientific literature. Objective: To evaluate
whether the suspected substances the patient had
contact with are likely to cause cross-reactions with
the immunochromatographic kit. Determine possible
drugs and medicines in the patient’s urine and blood
samples through confirmatory equipment such as
liquid chromatography coupled to mass spectrometry
and gas chromatography coupled to mass
spectrometry (LC-MS and GC-MS). Methodology: The
urine and blood samples of the patient were stored at
- 8ºC and used for the investigation of the molecules
suspected by the cross-reactions identified on the
day of the analysis by GC-MS and LC-MS. In addition,
a chronological evaluation of the patient’s case
demonstrated the clinical evolution of the patient, the
main results of laboratory tests and the treatment
used. Results: The results obtained were:
- Immunogromatography test:
* Serum: positive for TCA.
* Urine: positive for PCP, BZO and TCA.
- GC-MS: No traces found in urine or serum;
- LC-MS:
*Serum: positive for Midazolam, Fentanyl and
Lidocaine
*Urine: positive for Midazolam, Fentanyl and
Lidocaine
Discussion and Conclusion: The results obtained
in the analysis of LC-MS showed the presence of
midazolam, fentanyl and lidocaine, the first two being
used by the hospital team at the patient’s entrance.
Lidocaine, however, was not used by the hospital
staff or by the patient. Lidocaine has two degradation
products (o-toluidine and 2.6 dimethylaniline) that
have structural similarities to phencyclidine and may
result in a false-positive immunoassay. The analysis
of the Tanimoto index and LC-TOF analysis will be
made to confirm this cross-reaction not studied in
the literature. Both analyses will be done before the
presentation of this work and will be on the final
poster. Acknowledgments: HUSM/UFSM and DACT/
UFSM
 117

Clinical-epidemiological profile of
elapid accidents in pediatric population
registered at CIATox/SC
Silva, Stephanie Soares1; Messias, Nayara Casagrande1,2; Bresolin, Nilzete
Liberato1,3; Silva, Denise Bousfield1,3; Santos, Claudia Regina1,2,4
1
 Federal University of Santa Catarina (UFSC), Brazil; 2 Poison Control Center of
Santa Catarina, Brazil; 3 Children’s Hospital Joana de Gusmão; 4 Department of
Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

Introduction: Snakes of the genus Micrurus are the
main representatives of the Elapidae family and
are distributed throughout the Brazilian territory.
Previous studies demonstrate that accidents
involving coral snakes are responsible for about 1 to
2% of all snakebites in Brazil. Although relatively rare
due to the non-aggressive behavior of these snakes,
the ophidian accidents caused by Micrurus genus
have great medical importance and are potentially
serious because of the neurotoxic effects of the
venom on the neuromuscular junction. However,
the incidence of these accidents and the behavior of
elapid venom among the pediatric population is still
poorly described in the literature. Objective: Describe
the epidemiological and clinical characteristics of
pediatric victims of Micrurus snakebites in the state of
Santa Catarina. Methods: A retrospective, descriptive,
cross-sectional, observational study was conducted
using data recorded by the Center for Information and
Toxicological Assistance of Santa Catarina (CIATox-SC)
of patients aged 0 to 18 years old who were victims of
accidents with venomous snakes in the state of Santa
Catarina during the period of 2014 to 2020. Results:
During the period of study, 41 cases involving snakes
of the genus Micrurus were registered in children and
adolescents, which corresponds to 7.1% of the total
number of ophidic accidents reported in the pediatric
population. The snake species was identified in 27
cases (18 M. corallinus and 9 M. altirostris) and in 14
cases only the genus Micrurus spp. was identified.
Most accidents occurred in the months of December,
February, March and April (63.4%), in males (63.4%),
at the habitual residence (68.2%) and the mean age
affected was 7 years old. The macro-regions of Santa
Catarina with the highest number of elapid accidents
were Grande Oeste (31.7%), followed by Vale do Itajaí
(19.5%) and Grande Florianópolis (19.5%). The most
frequently affected body site was the right foot
(29.2%). The average time elapsed between the event
and the first medical assistance was 1.25 hours. There
were reported 32 mild cases, 5 moderate cases, and
4 severe cases. Among the severe cases, 2 of them
required orotracheal intubation due to respiratory
failure and occurred in children under 1 year of age.
The main clinical manifestation observed was local
pain (39%). The symptoms of neurotoxicity, such as
drowsiness, paresthesia, altered olfaction/ palate,
ptosis, paralysis, occurred in 7 cases (17%). A total
of 20 patients (48.7%) were hospitalized and treated
with antielapid antivenom, with a mean time to
initiate antivenom therapy of 3 hours. Only 11 cases
were asymptomatic and all patients progressed to
cure with no sequelae outcome. Discussion: The
higher frequency of elapid accidents in the pediatric
age group compared to the general population is
consistent with the tendency of previous studies, and
it can be explained by the eye-catching color pattern of
coral snakes, which attracts the curiosity of children.
The clinical evolution of pediatric patients victims of
Micrurus snakebites is usually favorable; however, it
can lead to serious neurological manifestations and
respiratory failure, especially in children younger than
1 year old or who do not receive adequate medical
care. Therefore, preventive guidance for children to
avoid walking barefoot, prompt health assistance
in case of accident, and correct identification of the
snake are decisive measures to avoid potentially fatal
complications. Acknowledgments: We thank CIATox/
SC for providing the data. The authors have no conflict
of interest to declare.
 118

Determination of pesticides in blood

samples from patients after suicide attempt
by micro-QuEChERS and LC-MS/MS
Godoi, Alexandre Barcia1,2; Silva, Mariana Cristina1,2; Costa, José Luiz1,2
1
 Faculty of Medical Sciences, University of Campinas, Campinas, SP,
13083887, Brazil; 2 Campinas Poison Control Center, Faculty of Medical
Sciences, University of Campinas, Campinas, SP, 13083859, Brazil.

Background/Introduction: The widespread use of mL in methanol) and 500 μL of ice-cold acetonitrile,

pesticides has caused health problems and fatalities
worldwide, often due to occupational exposure
and accidental or intentional poisoning. According
to the World Health Organization (WHO), pesticide
poisoning is one of the main means used in suicide
attempts in development countries, especially those
with a high proportion of rural residents who work
in small-scale agriculture. In Brazil, between 2007
and 2015, rodenticides had the highest percentage
of intoxication among the pesticides, with more than
40% of cases, followed by pesticides for agricultural
use with 36.5%. In this context, cases of suicide
attempts stand out, representing 53.6% of all cases
of intoxication, which 5% evolved to death as a result
of exposure. The combination of choices of sample
preparation technique and detection method is of
paramount importance in order to obtain an adequate
methodology for an analysis of pesticides. In this
sense, this work analyzed the suitability of using
the combination between micro-QuEChERS sample
preparation technique and liquid chromatography-
tandem mass spectrometry (LC-MS/MS) to determine
pesticides in blood samples of patients after suicide
attempt. Objective: Evaluate the suitability of micro-
QuEchERS extraction method combined with LC-MS/
MS detection of the following pesticides in blood
plasma of patients after suicide attempt: aldicarb-
sulfone, mevinphos, aldicarb, atrazine, carbofuran,
fenthion, chlorpyrifos, 2,4-D and fipronil. Methods:
micro-QuEChERS technique was employed for sample
preparation, which 250 μL of blood were used, added
to 25 μL of internal standard (diazepam-d5, 200 ng/
in addition to 100 mg of QuEChERS salt (Q-sepTM,
Restek®). The tubes were closed, shaken for 10 min
and centrifuged at 14,000 rpm for 10 min. Lastly, 125
μL of the organic phase was transferred to another
tube containing 375 μL of the aqueous mobile phase.
Two microliters of the dilution were injected into a
LC-MS/MS system, model LCMS8060 (Shimadzu,
Kyoto, Japan). Three blood samples collected from
individuals over 18 years old who attempted suicide
and were treated at the emergency unit of Hospital de
Clínicas (UNICAMP) were analyzed with the developed
method. The samples were refrigerated (2-8 ºC) until
the moment of analysis. Results: The method was
able to quantify nine different pesticides in blood,
presenting linearity greater than or equal to 0.99 for
all the pesticides. The recovery for the analytes was
higher than 13.9% and the matrix effect observed
was less than 41.2%, except for the analytes fenthion
and chlorpyrifos. The detection limit for analytes
was defined as 5 ng/mL, except for 2,4-D, which was
five times higher (25 ng/mL). The quantification limit
presented a concentration value equal to the detection
limit. Discussion/Conclusion: micro-QuEChERS
extraction combined with LC-MS/MS allowed the
detection of different pesticides with different
chemical properties in a single methodology. Thus, the
analytical method developed can be used for analysis
of blood samples for detection and quantification of
the evaluated compounds. Acknowledgments: The
authors thank Fundação de Amparo à Pesquisa do
Estado de São Paulo – FAPESP (process number 2020-
10809-5).
 119

Determination of tricyclic antidepressants

in whole blood by liquid chromatography
with diode diarray detector employing
dispersive liquid-liquid microextraction
Berlato, Dener Gomes1; Rosa, Victória Gomes2; Pacheco, André Lucas Bezerra2; Santos, Rachel2;
Ugalde, Gustavo Andrade1; Cardoso, Leonardo Corrêa1; Reginato, Fernanda Ziegler1; Chimendes, Nayomi
Andrade2; Santos, Lara Celestina2; Nascimento, Marcelo Henrique Santana2; Bairros, André Valle1
1
 Nucleus Applied to Toxicology, Postgraduate Program in Pharmaceutical Sciences,
UFSM; 2 Nucleus Applied to Toxicology, Course of Pharmacy, UFSM.

Introduction: Psychoactive drugs are the main agents
involved in cases of intoxication according to the
Toxicological Information Center of Rio Grande do
Sul (CIT-RS) and the National Toxic-Pharmacological
Information System (Sinitox). Within the classes of
drugs involved, the class of tricyclic antidepressants
(ADTs) stands out, generating severe effects in
cases of intoxication, with cardiotoxic effects,
which can be fatal. Objective: The objective is to
develop an analytical method by dispersive liquid-
liquid microextraction (DLLME) to determine
tricyclic antidepressants and their respective
biotransformation products in whole blood using
liquid chromatography with a diode array detector (LC-
DAD). Methods: The extractive technique was based
on dispersive liquid-liquid microextraction (DLLME)
using hexane as low density extracting solvent and
methanol as dispersing solvent. Whole blood was the
biological matrix of choice for the determination of the
tricyclic antidepressants amitriptyline, imipramine
and doxepin and their respective biotransformation
products nortriptyline and desipramine. The samples
were analyzed by liquid chromatography with a
diode array detector, the wavelength for the analysis
was doxepin, amitriptyline and nortiptyline at 239
nm, imipramine and desipramine at 249 nm and the
internal standard medazepam 255 nm. Results: The
limit of quantification was 10 ng/ml for amitriptyline
and doxepin, 20 ng/ml for nortriptyline and 30 ng/
ml for imipramine and desipramine. The mobile
phase was constituted by KH2PO4 buffer (pH 2.5)
and methanol (60:40) with a flow rate of 1.2 ml/min.
Conclusion: The DLLME developed in whole blood
for the determination of ADTs developed proved
to be a simple, reliable, robust and reproducible
method that can be used in toxicology laboratories.
Acknowledgements: We wish to thank CAPES for
providing financial support.

Development and validation of a method
to measure Tenofovir concentrations
in urine of HIV-positive patients
Introduction: In the early 1980s, some patients presented
symptoms of a pathology with an unknown cause.
Identified in 1983, about 37 million people live with HIV,
and it’s estimated that 27 million are on antiretroviral
treatment (UNAIDS, 2020). Once the HIV treatment
introduction in the 80s, it’s known that adequately
treatment infected individuals can present undetectable
viral load (SIEDNER; TRIANT, 2019). However, there’s no way
to perform therapeutic monitoring directly, and currently
the therapy is monitored based on viral load, pill counts,
and patient self-reporting (GARBIN; GATTO; GARBIN, 2017).
Tenofovir is one of the most used drugs in combination
therapy regimens and the adherence to the treatment
is fundamental to reach undetectable viral load. A valid
strategy to evaluate adherence to tenofovir treatment is
through urine concentrations of the drug. Methodology:
Urine samples were prepared by simple dilution in purified
water (1:10, v/v). After centrifugation, a 2 µL aliquot of
the diluted sample was injected into an Acquity I-Class
liquid chromatography system associated with a Xevo
TQD triple quadrupole mass spectrometer, both from
Waters (Milford, USA). Chromatographic separation was
performed on an Acquity HSS T3 column (100 x 2.1 mm,
d.p. 1.7 µm), also purchased from Waters. Mobile phase A
was composed of purified water with 0.1% formic acid and
mobile phase B was composed of acetonitrile with 0.1%
formic acid. Elution was performed in gradient mode, with
an initial mobile phase composed of 98% mobile phase A,
maintained for 2 minutes, followed by a linear gradient to
70% A in 3.5 minutes, followed by a new linear gradient
to 30% of A in 4 minutes. This condition was maintained
until 4.5 min, with the return to initial conditions in 4.6
min. The mobile phase flow rate was 0.4 mL/min, and the
column was maintained at 30°C. Tenofovir was detected
by electrospray ionization in positive mode. The operating
conditions of the mass spectrometer were capillary
voltage of 5 kV, cone voltage of 50 V, source temperature
of 250 °C, desolvation gas flow of 1000 L/h and curtain
gas flow of 40 L/ H. The monitored mass transitions were
288/136 (quantification) and 288/176 (qualification),
with a collision energy of 25 V. The method was validade
120
Santos, Rafaela Knak; Linden, Rafael
Feevale University/RS.
according to FDA bioanalytical guidelines and applied to
urine samples from 21 patients treated with tenofovir.
Results: Tenofovir retention time was 1.75 minutes.
Chromatographic run time was 6 min. The method was
linear between 100 to 50,000 ng/mL. The precision and
accuracy of the assay were tested at concentrations of 100,
300, 5000 and 30000 ng/mL. The intra-assay precision
was from 2.64 to 4.56%, the inter-assay precision was from
2.33 to 5.15% and the accuracy presented values between
94.97 and 103.27%. The matrix effect, tested in urine
from 10 volunteers, was -11.9 to 13% at a concentration
of 300 ng/mL and from -4 to 13.7% at a concentration of
30000 ng/mL. Tenofovir was quantified in 18 samples, at
concentrations between 8011 and >50000 ng/mL. Patients
with undetectable concentrations had poor adherence
according to the Morisky-Green questionnaire. Discussion
and Conclusion: In previous studies, Koenig et al. (2017)
but current adherence measurements are inadequate
for real-time adherence monitoring. We developed
and validated a urine assay to measure tenofovir (TFV
demonstrate that through urinary concentrations of
tenofovir it is possible to indirectly evaluate how long ago
the patient used the last dose of the drug, being a marker
of therapeutic adherence. Tenofovir urine testing is a non-
invasive strategy to evaluate adherence, with high patient
acceptance, and does not require prior preparation, with
the sample collection performed at any time of the day.
It’s an accessible way to monitor the treatment, allowing
pharmaceutical interventions to increase adherence to
therapy and control of viral spread.
REFERENCES
GARBIN, C. A. S.; GATTO, R. C. J.; GARBIN, A. J. I. Adesão à terapia
antirretroviral em pacientes HIV soropositivos no Brasil: uma revisão da
literatura. Archives of Health Investigation, v. 6, n. 2, p. 65–70, 2017.
KOENIG, H. C. et al. Urine assay for tenofovir to monitor adherence in real
time to tenofovir disoproxil fumarate/emtricitabine as pre-exposure
prophylaxis. HIV Medicine, v. 18, n. 6, p. 412–418, 2017.
SIEDNER, M. J.; TRIANT, V. Undetectable = Untransmittable and Your
Health: The Personal Benefits of Early and Continuous Therapy for HIV
Infection. Journal of Infectious Diseases, v. 219, n. 2, p. 173–176, 2019.
UNAIDS. Estatísticas mundiais sobre o HIV. Aidsinfo.Unaids.Org, p. 1–6,
2020.
 121

Early adverse reactions to different types

of anti-poison serum in the period from
2017 to 2021: a descriptive analysis
Priedols, Gustavo Abud1; Alves, Jonas Alher Meira1; Oliveira, Jordana
Meirelles1; Girotto, Edmarlon2; Guidoni, Camilo Molino2
1
 Medical student at the State University of Londrina - UEL - Londrina (PR), Brazil and CIATox
trainees Londrina (PR); Brazil; 2 Professor at the Department of Pharmaceutical Sciences at
the Health Sciences Center at the State University of Londrina - UEL - Londrina (PR), Brazil.

Introduction: Antivenom serum therapy consists of
the administration of concentrated heterologous
serums of immunoglobulins, usually from equine
hyperimmune plasma, being the main treatment in
poisoning by venomous animals. It is a procedure
intrinsically related to the possibility of occurrence of
systemic hypersensitivity reactions. Early reactions
can occur during the infusion of antivenom and within
two hours and are classified as common and severe.
Severe early reactions are uncommon but are of great
importance as they carry a risk of complications and
occasionally lead to anaphylaxis. Objective: To analyze
the frequency of administration of different types of
antivenom serum and the association between type
of antivenom serum and early reactions. Method: This
is a cross-sectional and descriptive study with cases
of poisoning by venomous animals treated at the
Information and Toxicological Assistance Center of
Londrina (CIATox-Londrina) of the University Hospital
of the State University of Londrina. Data were collected
from the Brazilian System of Intoxication Records
of Toxicological Information and Assistance Centers
(DATATOX). The study period comprised the visits
performed from January/2017 to December/2021, for
which the administration of antivenom serum therapy
was indicated. The CIATox-Londrina advises that the
serums be diluted and administered after infusion
of pre-serotherapy medication, which includes
antihistamines and corticosteroids. The frequency
of administration of antiarachnidic, antibothropic-
crotalus, antibothropic, anticrotalic, antielapidic,
antiscorpionic, antilonomic and antiloxoscelic serums
was quantified and the prevalence of early reactions
to each type of serum was verified. Furthermore, it
was observed that the antivenom serum was diluted
and the use of pre-serum therapy medication was
used. For data analysis, the Epi Info® program, version
7.2.5.0 was used. Results: 808 cases of antivenom
administration were analyzed, observing the following
frequency: 491 (60.77%) antibothropic, 197 (24.38%)
anticrotalic, 51 (6.31%) antiscorpionic, 33 (4.08% )
antiarachnidic, 26 (3.22%) antiloxoscelic, 18 (2.23%)
antibothropic-crotalus, 6 (0.74%) antilonomic and 3
(0.37%) antielapid. Dilution occurred in 805 (99.63%)
antivenom serum infusions and pre-serum medication
was administered to 780 (96.53%) patients. As for early
reactions, it was observed that there were 72 (8.91%)
cases. Of these, there were early reactions in 23.53%
of the anti-bothropic-crotalic serum infusions, 14.29%
of the anti-crotalic, 6.15% of the anti-bothropic, 3.85%
of the antiloxoscelic and 2.04% of the antiscorpionic
serum. In the analysis of the relationship between
antivenom serum therapy versus early reactions, a
statistically significant association was identified in
cases that received anticrotalic serum (p=0.0002).
Discussion/Conclusion: It was observed that, among
the accidents with venomous animals that required
antivenom serum therapy, snakebites were the most
prevalent. With regard to early reactions, some
factors favor their occurrence, which are related to the
antivenom serum (dose, type of serum, concentration
of proteins and immunoglobulins and infusion rate),
the form of administration (with or without dilution;
with or without pre-serotherapy medication), and
to the patient (atopy and prior sensitization to
the horse or equine products). In the literature, an
association could be found between administration
of anticrotalic serum and early reactions. In this case,
early reactions were more frequent and severe in
children who received the anticrotalic serum, when
compared to children who received the antibothropic.
Therefore, studies and analyzes are needed to
investigate and deepen evidence of this relationship.
Acknowledgments: We thank CIATox-Londrina, as an
institution that promotes care and knowledge sharing
in clinical toxicology for the population.
 122

Epidemiological variables and anti-

poison serotherapy: a descriptive
analysis of early adverse reactions
Alves, Jonas Alher Meira1; Priedols, Gustavo Abud1; Oliveira, Jordana
Meirelles1; Girotto, Edmarlon2; Guidoni, Camilo Molino2
1
 Medical Student at the State University of Londrina - UEL - Londrina ( PR),
Brazil and trainee CIATox Londrina (PR), Brazil; 2 Professor at the Department of
Pharmaceutical Sciences at the Health Sciences Center of the State University of
Londrina - UEL - Londrina (PR), Brazil and on-duty CIATox Londrina (PR), Brazil.

Introduction: Antivenom serum therapy is the main
treatment for poisoning with venomous animals. On
the other hand, there are risks related to systemic
hypersensitivity reactions, highlighting early
reactions during serum infusion, which occur within
two hours after infusion of antivenom serum therapy.
Objective: To analyze the frequency of administration
of antivenom sreum therapy, prevalence of early
reactions and factors associated with early reactions.
Method: This is a cross-sectional and descriptive
study with cases of poisoning by venomous animals
treated at the Information and Toxicological Assistance
Center of Londrina (CIATox-Londrina) of the University
Hospital of the State University of Londrina. Data were
collected from the Brazilian System of Intoxication
Records of Toxicological Information and Assistance
Centers (DATATOX). The study period comprised
the visits for which the antivenom serum therapy
administration was guided from January/2017 to
December/2021. A descriptive analysis was performed,
with calculations of frequencies, and association,
using early reactions as the dependent variable and
the independent variables sex, age, pregnant woman
(in the case of females), skin color and need for
antivenom serum therapy complementation. For data
analysis, the Epi Info® program, version 7.2.5.0 was
used. Results: Of the 808 cases of antivenom serum
therapy administration, there were more indications
for men (n=602; 74.50%), adults (n=669; 82.80%)
and whites (n=598; 74.01%). Only two (0.24%)
pregnant women received antivenom serum therapy.
The most common outcome for cases that required
antivenom serum therapy was cure (n=787; 97.40%)
and antivenum serum therapy complementation
occurred in the minority of times (n=108; 13.37%). As
for the early reactions, the occurrence was recorded
in 64 (7.99%) initial administrations and 8 (0.99%)
additions of the antivenom serum therapy. No
statistically significant factors associated with early
reactions were found during serum administration.
Discussion/Conclusion: As for sex and age, the data
analysis refers to residents of rural areas, where
males are more abundant in the state of Paraná, and
who perform agricultural work, being more exposed
to venomous animals. As for the most prevalent
color, the data coincide with the ethnic distribution
of the state of Paraná. Cure, in turn, as the most
common outcome, demonstrated the effectiveness
of antivenom serum therapy in the management of
accidents with venomous animals and in reversing
the toxicity of their venoms. Finally, regarding the
administration of the antivenom serum therapy or the
complementation, although not significantly related
to the variables analyzed, it was shown that the early
reactions are infrequent. Acknowledgments: We
thank CIATox-Londrina, as an institution that promotes
care and knowledge sharing in clinical toxicology for
the population.
 123

Estimation of hematocrit values by

potassium quantification in capillary
dried blood spots (DBS)
Silva, Laura Cé; Alves, Pedro Adolfo Pereira; Linden, Rafael; Antunes, Marina Vezon

Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo - RS, Brazil.

Background: Dried blood spots (DBS) are minimally
invasive samples increasingly used for therapeutic
drug monitoring. Despite the advantages, there
are still some challenges related to this technique,
such as the hematocrit (HCT) measurement. The
variability of patients HCT and the impact of it on
blood viscosity and assay accuracy, as well as the
differences on blood to plasma ratios, are obstacles
for the implementation of this technique in clinical
toxicology and therapeutic drug monitoring. In this
study, potassium (K+) was used as a marker of HCT,
considering that 98% of the total body K+ is located
within the intracellular compartment. Objective:
To evaluate the applicability of an electrode-based
method for the quantification of K+ and estimation
of HCT in capillary DBS samples. Method: Quality
controls and calibrators were prepared by adding or
removing plasma from a blood sample with known
HCT. Calibration curve was performed with HCT 20,
25, 30, 35, 40, 45, 50, 55 and 60% and controls HCT
25, 40 and 55 %. Precision and accuracy were tested
in triplicate within 3 days. Capillary and venous blood
samples were collected from 32 healthy volunteers.
Capillary blood was collected from finger prick and
applied to Whatman 903® paper card, dried for at
least 3 hours until analysis. The venous blood was
collected with venipuncture and stored in EDTA
containing tubes. The K+ was measured in DBS from
volunteers and calibrators after extraction of a 6 mm
punch with 300 uL of ultrapure water incubated for
40 minutes at room temperature and 1000 rpm. The
aqueous extract was analyzed into the K+ LAQUAtwin
(HORIBA) compact meter for ion quantification.
The HCT of venous blood samples was quantified
using the Sysmex Kx-21n hematology analyzer.
Results: Precision (CV%) ranged from 2.4 to 2.7%,
while accuracy ranged from 94.6 to 111.8%. The HCT
measured in venous blood ranged from 34 to 43.5%,
with a mean standard deviation (SD) of 40.11 ± 2.8%.
While capillary HCT estimated from K+ measurement
in DBS resulted in K+ ranging from 22.36 to 35.9%, with
a mean SD of 28.2 ± 3.35%, representing 72 ± 7% of
the values from venous blood. Passing-Bablok linear
regression performed with a confidence level of 95%
demonstrated the existence of a systematic error,
thus the equation DBS HCT = -24.837358 + 1.389067
venous HCT was applied to estimate HCT values in
DBS. The estimated values ​​of HCT from capillary
blood ranged from 33.97 to 43.71% with a mean SD of
38.18 ± 2.4%, representing 98 ± 6% of the venous HCT,
with no proportional errors. Discussion/Conclusion:
The quantification of K+ in DBS samples can be used
to estimate the HCT as long as a correction factor is
applied.
 124

Evaluation of cytotoxic potential of

polyhydroxylated phenylchromones in
human osteosarcoma in vitro models
Oliveira, José Miguel P. Ferreira1; Proença, Carina1; Santos, Raquel1,2; Moreira, Beatriz1,3;
Rufino, Ana T.1; Freitas, Marisa1; Ribeiro, Daniela1,4; Fernandes, Eduarda1
1
 LAQV, REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty
of Pharmacy, University of Porto, 4050-313 Porto, Portugal; 2 Department of Biology, Faculty of
Sciences, University of Porto, 4169-007 Porto, Portugal; 3 Department of Chemistry and Biochemistry,
Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal; 4 Faculty of Agrarian Sciences
and Environment, University of the Azores, 9700-042 Angra do Heroísmo, Açores, Portugal.

It is estimated that, in 2020, cancer was associated
with approximately 10 million deaths worldwide.1
Among children, the most common types of cancer
include leukemias and lymphomas, central nervous
system tumors, kidney cancer and malignant bone
tumors.2 The most common childhood bone cancer,
osteosarcoma (OS), is treated with a sequence of
neoadjuvant therapy, tumor surgical resection and
postoperative adjuvant therapy. However, since
therapy for recurrent OS is limited by poor survival
rates, novel therapeutic agents are urgently required.3
In recent years, chromone-related compounds have
been described to inhibit several processes related to
cancer and to OS in particular.4 The aim of the present
study was to evaluate the potential toxicity of a group
of polyhydroxylated phenylchromones to OS cells,
using in vitro models. For this, human OS cell lines -
MG-63, Saos-2, HOS, and 143B - were incubated for
48 h with 10, 20, 40, 80 and 160 µM phenylchromones
with diverse hydroxy substituents at positions C-3,
C-5, C-6, C-7, C-8, C-3’, C-4’, and C-5’. Subsequently,
sulforhodamine B (SRB) assay was used to determine
cell growth inhibition, followed by spectrophotometric
measurement at 510 nm. Moreover, cell viability was
investigated upon incubation with WST-8 reagent,
followed by spectrophotometric measurement at 450
nm. The obtained results suggest that the presence
of hydroxy substituents simultaneously at C-3 and
C-8 of C-ring improve the in vitro cytotoxic activity
of the tested compounds in OS. A higher cytotoxicity
was also observed in compounds with hydroxy
substituents simultaneous at positions C-3’, C-4’ and
C-5’ of the B-ring combined with hydroxylation at C-3
and/or C-7 of the C-ring. These preliminary results
enable the study of the potential application of these
compounds in OS therapy. Acknowledgments: This
work received financial support from the European
Union (FEDER funds through COMPETE POCI-01-0145-
FEDER-029243) and National Funds (FCT, Fundação
para a Ciência e Tecnologia) through project PTDC/
MED-QUI/29243/2017 and from PT national funds
(FCT/MCTES) through grant UIDB/50006/2020.
ATR and CP thank to FCT for the funding through the
project PTDC/MED-QUI/29243/2017. JMPFO thanks
FCT for funding through program DL 57/2016 –
Norma transitória (ref. SFRH/BPD/74868/2010). MF
acknowledges her contract under the CEEC Individual
(2020.04126.CEECIND/CP1596/CT0006).
REFERENCES
1. H. Sung et al., CA Cancer J. Clin. 2021, 71(3), 209-249.
2. R.L. Siegel et al., CA Cancer J. Clin. 2021, 71(1), 7-33.
3. M.E. Anderson, Orthop. Clin. North Am. 2016, 47, 283-292.
4. J. M. P. Ferreira de Oliveira et al., Pharmaceuticals (Basel).
2021, 14(7), 640.
 125

Evaluation of experimental conditions for

sample preparation to identify the untargeted
metabolomic profile of plasma by GC-MS.
Nolasco, Daniela M.; Pires, Sumaia Araújo; Souza, Tainá Brumate; Souza, Mirna
Maciel D'Auriol; Paiva, Maria José Nunes; André, Leiliane Coelho

Toxicological Analysis Laboratory, Department of Clinical and Toxicological Analysis,

College of Pharmacy - Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.

Introduction: Metabolites are biomolecules of low
molecular weight with different organic functions,
which are formed as intermediates in biochemical
reactions. The metabolites profile and concentrations
may be related to the physiological state of an
organism. Gas chromatography coupled with mass
spectrometry (GC-MS) has been extensively used
in metabolome analysis due to its high separation
efficiency, which can resolve very complex biological
mixtures. However, the limitation of GC-MS is that
the analytes must be volatile to be separated on a
gas chromatography column. Therefore, analysis of
biological samples by GC-MS requires derivatization
so that non-volatile or thermally unstable species can
be detected. Objective: In this study, the optimization
of sample preparation was performed by variation of
some factors in order to analyze how the experimental
variables interact and which condition generates
the largest number of molecular features. Results
and Discussion: A pool of plasma from 10 healthy
individuals was prepared and stored at -80°C. The
sample preparation was based on existing protocols in
the literature and the experimental variables were: 1)
different solvents used in the deproteinization step; 2)
methoximation reaction time and 3) silylation reaction
time. Proteins were precipitated before derivatization
of the sample, using 100% acetonitrile or a ternary
mixture of isopropanol:acetonitrile:water (3:3:2)
followed by a clean-up step, adding acetonitrile:water
(50:50). The supernatants collected were evaporated
to dryness using a SpeedVac Concentrator System.
Methoxymation was performed by adding 10 µL of
O-methoxyamine hydrochloride in pyridine to each
sample and incubated at room temperature for 16 h
or 18 h or at 70°C for 90 min. For silylation reaction,
10 µL of BSTFA with 1% TMCS was added and left for
1 h at 70°C with or without agitation of the samples.
Then 100 µL of C18:0 methyl ester (10 ppm) in-
heptane was added as an instrumental internal
standard and analyzed by GC-MS. The analysis was
performed with a GC instrument 7890A coupled to
an inert mass spectrometer with triple-Axis detector
5975C from Agilent Technologies. Chromatography
and MS parameters followed the protocol proposed
by Fiehn et al. (2016). Samples were injected (2 µL)
with the injector temperature set at 250°C in a split
mode (1:10), using helium as a carrier gas at 1.1 mL/
min flow. The capillary column used was a DB5-
MS 30 m length, 0.25mm i.d. and 0.25 m (Agilent
Technologies®) with temperature programming
set at 60°C for 1 min, then raised by 10°C/min until
it reached 325°C and maintained for 10 min before
cooling down. MS parameters were: transfer line
a 280°C, filament source at 230°C, quadrupole at
150°C, electron ionization energy of 70eV, mass
range of 50-600 m/z. The data were acquired using
the Agilent MSD ChemStation Software. The analysis
was performed in triplicate, resulting in 21 samples,
plus five quality controls and two blanks. The data
processing was performed using the R software to
obtain the matrix of results (number of ion peaks).
Conclusion: statistical calculations showed that
the sample preparation condition that provided
the identification of a greater number of molecular
features was achieved when the samples were
submitted to two extraction steps (deproteinization
followed by sample clean-up), a methoximation
reaction time of 16 hours at room temperature and
the silylation reaction at 70°C with agitation for 1 hour.
Keywords: Metabolomics profile, GC-MS, untargeted
metabolomics. Acknowledgement: To CNPq.
 126

Exclusive therapy of N-acetylcysteine in

accidental butanox intake: toxicological
analysis of methyl ethyl ketone peroxide
Santos, Rachel1; Bairros, André V.1; Saldanha, Geovane A.1; Berlato, Dener G.1;
Moraes, Liliana S.1; Gündel, Augusto R.2; Carvalho, José A.M.2; Habib, Isabela A.3;
De Carli, Diego M.3; Oliveira, Tiago F.4; Oliveira, Sarah C.W.S.E.F.4
1
 Nucleus Applied to Toxicology, Center of Health Sciences, Federal University of Santa
Maria, Santa Maria, Brazil; 2 Laboratory of Clinical Analysis, University Hospital of Santa
Maria, Federal University of Santa Maria, Santa Maria, Brazil; 3 Gastroenterology Unit,
University Hospital of Santa Maria, Santa Maria, Brazil; 4 Mass Spectrometry Research
Group, Federal University of Health Sciences of Porto Alegre, Porto Alegre, Brazil.

Introduction: Accidental ingestion and consequent
methyl ethyl ketone peroxide (MEKP) poisoning during
occupational activity as well as due management
of the affected patient were reported. From the
perspective of the MEKP, which it is applied in the
industrial field, this colorless liquid causes oxidative
stress and is therefore considered a highly toxic
compound. Objectives: Delineate a rare accidental
poisoning during the period of occupational activity
by methyl ethyl ketone peroxide (MEKP), in addition
to treatment and toxicological analysis. Methods:
Oral administration of N-acetylcysteine (NAC)
with a loading dose of 20 sachets (600 mg/sachet)
with subsequent doses (10 sachets/4h) for three
consecutive days. Application of Dipyrone ampoules
(2 mL to 500 mg/mL) causing emesis. Biochemical,
endoscopic, hematological and toxicological tests
were performed. Results: Changes were observed
in creatinine (1.27 mg/dL), creatinine phosphokinase
(223 U/L), hematocrit (58.5%), hemoglobin (20 g/
dL), leukocytes (31420/µ L), rod leukocytes (22%),
urea (59 mg/dL). Ulcerative lesions (≥ 22 cm) in the
gastrointestinal tract with necrotic stains exhibited
by digestive endoscopy. Diagnosis of Zargar grade IIB
acid erosive esophagitis, as well as the presence of
Zargar grade IIIA acid gastric ulcers. The use of NAC
is unquestionably appropriate due to its high efficacy
in the elimination of free radicals, resulting from
the metabolization of MEKP. Regarding the reaction
between NAC and MEKP, (2S)-3-[(2-carboxy-2-
acetamidoethyl)dissulfanyl]-2-acetamidopropanoic
acid and 2-hydroperoxybutan-2-ol are then formed,
which will be subsequently converted into MEK and
thus excreted in the urine. The aspects of this case
were favorable to the exclusive oral administration
of NAC, even with the patient presenting emesis,
in which, in situations like this, the NAC is
contraindicated. It was collected, 24 hours after
the exposure, ethylene diamine tetra acetic acid
(EDTA)-blood samples (stored at 23ºC/48h), which
allowed the identification of MEKP (monomer/dimer)
with LC-QTOF/MS, thus demonstrating something
unprecedented. GC-FID (32.85 µg/mL) quantified
methyl ethyl ketone above the occupational limit (0.1-
10 µg/mL). During hospitalization, hepatic markers
were elevated and occupational exposure (10 years
without the use of PPEs) should be considered, since
Butanox® has dimethyl phthalate in its composition,
therefore, is an hepatoxic agent absorbed by the
respiratory and dermal pathways. In addition, with
a small working area (4m2) and no ventilation, in
which the individual was, this caused anosmia, which
culminated in the accidental ingestion of the solvent
in a potentially lethal volume (10-100 mL). Conclusion:
Determination, in the blood collected, of MEKP and
MEK. Under the perspective of oral administration of
NAC, success was achieved in the poisoning by MEKP.
However, using N-acetylcysteine as the exclusive
therapy is considerably critical, which requires proper
supervision and assistance to the patient during
hospitalization. Acknowledgments: We acknowledge
the Coordination for the Improvement of Higher
Education Personnel (CAPES) for the Master’s
scholarship provided. Keywords: Methyl ethyl ketone
peroxide; N-acetylcysteine; Occupational aspects;
Toxicological analysis.

Fast method of quantitative analysis of
serum vitamin A (retinol) using LC-MS/MS
Background: Vitamin A performs several functions
in the body. It is involved with the formation of
rhodopsin in the retina, acts as a bone growth factor
and is a cofactor in the synthesis of steroid hormones.
From animal foods, vitamin A is absorbed as retinol.
Vitamin A deficiency may be associated not only
with insufficient intake, but also with chronic lipid
malabsorption, liver disease, intestinal parasites,
and alcoholism. It also causes night blindness, skin
dryness, hair loss and in cases of severe deficiency,
irreversible blindness. Toxicity can occur due to
administration of high doses of vitamin A for long
periods, causing a hypervitaminosis. Acute symptoms
are tiredness, headache, nausea and vomiting, which
result in chronic disorders such as bone fractures,
intracranial hypertension, skin lesions, hepatotoxicity
and teratogenicity. The aim of the study was to
validate an easy and fast preparation method for
the analysis of retinol. Methods: Validation was
performed using a Xevo TQS micro tandem mass
spectrometry sensitive liquid chromatography (LC-
MS/MS). Analytical specificity is ensured using
multiple reaction monitoring with fragmented ions
that are exclusive to retinol, quantifier ion 269.15 >
93.00 and qualifier ion 269.15 > 83.00. The procedure
involves a small amount of 50 µL of serum, followed
by liquid-liquid extraction. The samples are then
submitted to reverse phase separation on a Zorbax
Eclipse Plus C8® analytical column (50.0 x 2.1 mm, 1.8
127
Soares, Marcela de Oliveira; Sugawara, Eduardo Kinio; Mazete,
Fernanda Pine S.; Ramadan, Debora R.; Tufik, Sergio
Associação Fundo de Incentivo à Pesquisa - AFIP.
μm). Mobile phase was A: 2.0 mM ammonium acetate/
0.1% formic acid (aq) B: 2.0 mM ammonium acetate/
0.1% formic acid/ MeOH, using a gradient with an
initial ratio of 40:60 (v/v) with a flow rate of 0.3 ml/
min. Quantitation is achieved by the comparison of the
responses from a given sample with the responses of
calibrators with known concentrations. Linearity was
observed in the expected concentration range from
2.0 to 64.0 mg/L and serum samples were evaluated
on six different concentrations and six times each at
the same time point of the study. Albumin was used
as a biological matrix for the study, retinol standard
(Sigma – Aldrich). Results. The linearity coefficient of
determination (R2) was 0.998746. The method showed
100% of selectivity and interference of residual effect
of less than 5%. In order to determine the average
inter-assay CV%, three different concentrations
were analyzed over three days and the results for
each low, medium, and high concentration level were
4.60, 5.72 and 4.24%, respectively. The accuracy of
the method was cheeked by analyzing samples of
known concentration and expressed as a percentage.
A retention time (RT) of 1.6 min was obtained and
the total analysis time was 3.5 min. Conclusion: The
method was fast and efficient for the determination of
serum retinol. The efficiency and selectivity combined
with the technical robustness can be used in the
diagnosis of nutritional and absorption disorders and
also in cases of toxicity.
 128

Finger-prick volumetric absorptive

microsampling (VAMS) for imatinib
therapeutic drug monitoring: method
development and validation
Guterres, Fernanda S.1; Krutzmann, Maria Eduarda2; Kohlrausch,
Ramona1; Linden, Rafael1,2; Antunes, Marina Vezon1,2
1
 Institute of Health Sciences, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate Program
on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil.

Background: Imatinib mesylate (IM) is a selective
tyrosine kinase inhibitor, considered first-line therapy
for the treatment of chronic myeloid leukemia (CML).
A significant portion of patients using imatinib
experience adverse events, up to 45% of patients
discontinue therapy after 8 years, 6% due to adverse
effects and 16% due to unsatisfactory therapeutic
effect. The relation between IM though plasma
concentrations and therapeutic response has been
demonstrated. Therapeutic drug monitoring (TDM) of
imatinib has been performed for improving treatment
outcomes and as a tool to evaluate adherence.
Also, norimatinib, a CYP mediated metabolite of
has similar pharmacological activity to imatinib
and represents approximately 20–25% of the
steady-state concentration of the original drug. Up
to date there is no report of the use of volumetric
absorptive microsampling (VAMS) for capillary blood
measurements of imatinib. It could help increasing
the access to TDM, with less invasive collection at
pharmacokinetically appropriate time, with high
stability and logistic advantages, once samples do
not need to be refrigerated. Objective: to develop
and validate a method for imatinib and norimatinib
quantification in capillary blood collected with VAMS
by LC-MS/MS. Methods: The 20 µl VAMS tip were
prepared by liquid extraction with 100 µL of water
and formic acid 0,1%, incubated for 20 min at 45 °C
and 1000 rpm. After the proteins were precipitated
with 300 µL acetonitrile 0,1% formic acid containing IS
(Imatinib-D8 80 ng/mL). After 5 min mixture, samples
were cooled at -20°C for 10 min and centrifugated for
10 min. The supernatant was transferred to vial and
5 µL injected into the LC-MS/MS with electrospray
ionization in positive mode. Source temperature was
350°C, capillary at 200 °C, auxiliar gas nitrogen at
15 arb and sheath gas at 50 arb. Chromatographic
separation occurred in an Acquity® UPLC BEH C18
(150 x 2.1 mm x 1.7 μm) at 40 °C. Mobile phase was
acid formic in water 0,1% (eluent A) acid formic in
acetonitrile 0,1% (eluent B) in initial gradient 15% to
60% (A:B, v/v) eluted at 0.25 mL/min -1. The following
transition were monitored as quantifier: imatinib
m/z 494 - 378, norimatinib m/z 480 - 394, and IS
m/z 502 - 378. The validation tests are following the
recommendations of FDA and EMA. Results: Total
analytical run time was 7 min, with retention time of
3.5 for norimatinib and 4 min for imatinib and IS. The
method was linear from 100 to 2500 ng/ml for both
analytes (r2 ≥ 0.99), accurate 89-103% for imatinib
and 92-112% norimatinib, precise with CV% 4.3-11.3%
for imatinib and CV% 5.9-13.5% for norimatinib. Matrix
effect was compensated by the use of the deutered
internal standard, from -14 to 3% for imatinib and
-12 to 10% for norimatinib. No significant effect
of hematocrit (Hct) was observed (85-115%), with
extraction yield ranging from 98% for Hct 25% to 85%
for Hct 55% for imatinib and 95% for Hct 25% and 87%
for Hct 55% for norimatinib. Discussion/Conclusion:
The method presented adequate performance for
imatinib and its metabolite norimatinib determination
in blood VAMS. The method is going to be applied in
samples from patients with CML for clinical validation.
Acknowledgments: Financial support FAPERGS and
CAPES.
 129

Garlic burn injuries: a clinical-epidemiological

profile of cases registered at CIATox/SC, Brazil
Cordeiro, Gabriela Batista Cavalcanti1,2; Arruda, Fernanda Wolff da Silva1; Petry,
Andrea2; Resener, Marisete Canello3; Santos, Claudia Regina1,2,3
1
 Federal University of Santa Catarina (UFSC), Brazil; 2 Poison Control Center of Santa Catarina,
Brazil; 3 Department of Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

Introduction: Garlic is widely used in cooking
as a seasoning for food. In popular culture it is
recommended in the treatment of skin conditions.
However, topical use may cause chemical burns on the
skin, which may have varying degrees, including the
appearance of blisters and vesicles at the injury site.
These burns are due to sulfuric substances present in
garlic. The Toxicological Information and Assistance
Center of Santa Catarina (CIATox/SC) has received
calls referring to patients exposed to garlic, mainly
when using this product for the “treatment” of injuries
caused by venomous or non-venomous animals.
Methods: This is an observational, cross-sectional,
descriptive and retrospective study of a series of
cases of patients registered in the DATATOX system
(DATATOX BI Pentaho / Saiku version 2.6) due to
CIATox/SC instructions. Data was collected from 2014
to 2021 and was analyzed in the Microsoft Excel®. The
variables were: age range, gender, circumstance, injury
site, time from exposure to attendance, symptoms,
severity, and outcome. Results: There were 47 cases
of garlic burns ranging from 01 to 79 years old,
predominantly between 60 to 69 years old (19,14%),
and with a female profile (51.06%). Most patients
(57.44%) applied garlic on the skin due to sting/bite/
contact with venomous animals or when doubting if
the animal was venomous or not, in order to relieve
the pain. Regarding the injury site, 42.55% were on the
lower limb, and 40.42% were on the upper extremities.
Also, 40.42% of patients searched for attendance 12
to 24 hours after exposure. The symptoms were: pain,
paresthesia, hyperemia, erythema, edema, itching,
burns, vesicles, and eruptions on the skin. Headache,
fever, nausea, and vomiting were also registered. All
of the cases were classified as mild and had good
outcomes. Conclusion: The use of crushed garlic as
a remedy for pain relief is especially frequent in the
eastern cultures. Its curative properties are widely
used for over 4000 years for a variety of conditions
such as headache, tumors and intestinal worms.
Therefore, its use in the skin is responsible for varying
degrees of irritant reactions, of which may be affected
by its concentration, freshness, the total duration of
exposure on the skin, the anatomical area of the body
applied, and individual’s skin sensitivity. The concept
of “natural remedy”, when applied incorrectly, might be
responsible for many injuries. These results show that
there is still part of the population that is not aware of
the irritant effects of garlic when applied on the skin.
Educational measures in schools, workplaces, social
media, and television programmes would be very
useful to make that clear to various groups of people.
 130

Hepatotoxicity risk assessment in suicide

attempts by acetaminophen with doses
between 7.5g and 10g at a reference
center in Santa Catarina, Brazil
Fonseca, Karoline Kuhnen1; Costa, Ana Carolina Conchon1; Cordeiro, Gabriela Batista
Cavalcanti1; Arruda, Fernanda Wolff da Silva2; Resener, Marisete Canello1
1
 Assistance and Toxicological Information Center in Santa Catarina;
2
 Medical Student in Federal University of Santa Catarina.

Introduction: Acetaminophen intoxication has
been considered the most common cause of liver
transplant indication for liver failure in the world and
the main one in the United States. Its toxicity comes
from increased production of the toxic metabolite
N-acetyl-p-benzoquinone imine (NAPQI). Used as an
antipyretic, it is easily acquired and considered by
many to be harmless. Despite a good safety profile
and antidote N-acetylcysteine (NAC), intoxication can
lead to severe liver damage when ingested in large
quantities as in suicide attempts (SA). Treatment with
NAC is indicated in cases that reach a toxic dose (TD) or
with serum levels of acetaminophen with potential or
high risk of hepatotoxicity. In the United Kingdom, the
stablished TD is above 7.5g, while the USA and some
other countries adopted a higher dose, with prediction
of lower toxicity below 200 mg/kg or 10g. Object:
Evaluate the risk of hepatotoxicity in intoxication with
acetaminophen by means of SA with doses between
7.5g and 10g that were treated at the Assistance and
Toxicological Information Center of Santa Catarina
(CIATox/SC), Brazil, between 2014 and 2021. Methods:
Retrospective analysis of SA with acetaminophen
between 2014 and 2021 recorded at the service’s
information data source (DATATOX BI Pentaho / Saiku
version 2.6). Were included patients with SA with
acute ingestion of acetaminophen between 7.5g and
10g who had records of laboratory tests 24 hours
after ingestion and/or serum acetaminophen dosage
available. Were excluded those who, after initial care,
had confirmed a dose outside the predicted range
or who lost follow-up. Results: Of the total of 224
records of SA with acetaminophen at doses between
7.5g and 10g in the years from 2014 to 2021, 173 met the
inclusion criteria. Of these, 101 (58%) had laboratory
tests 24h after the ingestion and 72 (42%) had serum
dosage of acetaminophen. 70% were women and
30% were men. The main age group was between 15-
39 years old (80%). 137 cases (79%) ingested doses
between 7.5g and 10g, while 36 cases (21%) ingested
a dose of 10g. Laboratory alterations 24h after
ingestion and/or the need to repeat the NAC regimen
were observed in 8.6% of those with an ingestion of
7.5g, 8.8% of those with intakes between 7.5g and 10g
and 19.4% of ingestion of 10g. 50% of the cases with
serum levels compatible with potential or high risk of
hepatotoxicity ingested 10g. Discussion/Conclusion:
In this retrospective analysis, we observed that
less than 10% of the cases evolved with laboratory
alterations or with potential/high of toxicity. Besides
that, we observed that ingestion of a dose of 10g have
a twofold increased risk of toxicity when compared
to those between 7.5g and 10g. More studies at a
national level are needed, as well as serum dosages
made available, in order to stablish the safest toxic
dose of acetaminophen to the Brazilian population
and to standardization of treatment protocols.
Acknowledgements: CIATox/SC for the partnership
and provision of data.
 131

Liver and kidney function at the

pesticide’s exposure of rural workers
from Casimiro de Abreu - RJ
Nunes, Rafaella Ferreira Nascimento1,2,3; Poça, Kátia Soares2,3; Cabral, Yngrid dos
Santos2,3; Aguiar, Gilberto Santos; Siqueira, Janas; Geraldino, Barbara Rodrigues;
Otero, Ubirani Barros2; Martins, Isarita1; Sarpa, Márcia Sarpa de Campos2,3
1
 Laboratório de Análises de Toxicantes e Fármacos (LATF)/ Universidade Federal de Alfenas
(UNIFAL-MG), Programa de Pós-Graduação em Ciências Farmacêuticas/Doutorado em Ciências
Farmacêuticas;. 2 Área Técnica Ambiente, Trabalho e Câncer- Instituto Nacional de Câncer José
Alencar Gomes da Silva – INCA; 3 Laboratório de Mutagênese Ambiental, Departamento de
Bioquímica, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro (UNIRIO);
4
 Programa de Saúde do Trabalhador, Secretaria Municipal de Saúde de Casimiro de Abreu.

Introduction: Pesticides exposure is considered (CAAE 64799217.3.0000.5274). Results/Discussion:

one of the main risk factors for human health. Such
substances can produce toxic effects in the several
body systems. In this context, the liver and kidneys
are target of these chemicals causing a disbalance
in the elimination process of xenobiotics. In addition,
due the relevance of these organs, the changes in
their functions increase the susceptibility. Objective:
The objective of the study was to verify changes in
liver and kidney functions as a possible result of the
exposure to pesticides of rural workers from Casimiro
de Abreu (RJ). Experimental: This is a cross-sectional
study with individuals residing in the municipality
of Casimiro de Abreu, Rio de Janeiro, Brazil, whose
study inclusion criteria were: both sexes, residing
in the rural area and aged above 18 years. Between
March and July (2021), biological samples were
collected from 95 workers/residents of Casimiro de
Abreu, being 75 rural workers and 20 urban workers
(comparison group). The samples were transported
from the collection site to the Clinical Pathology
Service of the HC1/INCA, in accordance with sanitary
regulations. The biochemical parameters evaluated
were creatinine, alkaline phosphatase, transaminases
(alanine aminotransferase - ALT and aspartate
aminotransferase – AST). The analysis of parameters
evaluated and their respective cut-off points took
into account the limit values ​​of the Clinical Pathology
Service of the HC1/INCA. From them, the subjects were
classified as having or not having alterations in the
parameters. The non-parametric Mann-Whitney test
was used for statistical analyses. This project was
approved by the Research Ethics Committee of INCA
Among the 75 individuals evaluated in the rural
area most were man (83.2%), aged between 36 and
60 years (58.0%). Regarding education, 67.9% had
elementary school, followed by 18.5% who reported
not knowing how to write. In additional 83.9% lived
in the municipality for more than 10 years and 9.9%
have lived for less than 5 years. The mean (SD) of
the biochemical parameters of each of the studied
groups was performed. The mean values (±standard
deviation) of ALT (21.90 (±11.10) U/L) and AST (21.72
(±7.84) U/L) for rural workers were higher than in
comparison group (21.00 (±4.58) U/L and 20.22
(±7.44) U/L, respectively), except to phosphatase
(30.05 (±17.53) U/L) that were almost 3 times lower in
rural workers than in urban workers (86.06 (±32.84)
U/L) . All observed results were in agreement with the
reference limits (ALT ≤ 40 UL; AST ≤ 41 UL, phosphatase
40 – 129 UL and creatinine 0,3 – 1,3 mg/dL), despite
the groups showing different mean values. Of the
parameters analyzed, only creatinine showed a
statistically significant difference (p value =0.024).
Conclusion: The results obtained allowed to conclude
that no relevant differences were observed when the
results of the two groups were compared and suggest
that there is a possible environmental exposure
unable to change the parameters of liver and kidney
functions. Therefore, the present study contributes
to the risk assessment to pesticides exposure and to
the health surveillance of rural workers in Casimiro
de Abreu, an agricultural region in the Rio de Janeiro
state.
 132

microRNAs as serum biomarker for Senecio

brasiliensis poisoning in cattle
Winter, Evelyn1; Cisilotto, Julia2; Goetten, André L.F.1; Veiga, Ângela1; Ramos, Adriano
T.1; Zimermann, Francielli C.1; Reck, Carolina4; Creczynski-Pasa,Tânia B.3
1
 Department of Agriculture, Biodiversity and Forest, PPGMVCI, UFSC, Curitibanos;
2
 PGFAR, UFSC, Florianópolis; 3 Department of Pharmaceutical Sciences, UFSC,
Florianópolis; 4 VERTÁ, Laboratory of Veterinary Diagnostic, Curitibanos.

Introduction: Senecio spp. is one of the most frequent
plant-related poisoning in cattle and its ingestion
generates the disease seneciosis, characterized by
serious hepatic damages. Liver biopsies and serum
liver markers dosage are tools used in seneciosis
diagnosis; however, a large number of cattle breeding
is undiagnosed. The microRNAs are small non-coding
RNA molecules, stable in biological fluids, and the
difference in expression levels may indicate the
presence, absence and/or stage of the poisoning.
Objective: Identification of a miRNA profile in cattle
serum intoxicated to Senecio brasiliensis. Methods:
Liver biopsy was performed in all the cows and the
histopathological analysis were performed in order to
confirm intoxication. Blood samples were collected to
evaluate miRNAs and liver biochemical parameters.
Expression of miR-21-5, miR-885-5p, miR-122-5p,
miR-181b-5p, miR-30a-5p, miR-378-5p, and let-7f-5p
were evaluated in serum of naturally exposed cattle,
by RT-qPCR. The normality of the variable distribution
was evaluated by the Kolmogorov-Smirnov test.
Continuous variables were compared using Mann-
Whitney test and the correlation was evaluated using
Spearman’s correlation. A ROC curve analysis was
performed and the AUC was calculated to evaluate the
diagnostic value. The optimal cut-off value, sensitivity
(SE), and specificity (SP) were determined by
calculating the Youden index.A p<0.05 was considered
statistically significant. Results: Twenty animals were
considered as intoxicated because presented one or
more histological change as fibrosis, megalocytosis,
ducts proliferation and binucleate cells in liver.
Control group (eight animals) did not presented any
alteration in histology of liver. In poisoned animals,
lower quantity of albumin (**p<0.01) and high activity
of aspartate aminotransferase (AST) and alkaline
phosphatase (ALP) (*p<0.05) were also detected. No
alteration was observed in γ-glutamyl transferase
(GGT) and alanine aminotransferase (ALT) activities.
MiRNAs miR-30a-5p (*p=0,0478), miR-378-5p
(*p=0,0358), miR-21-5p (****p<0.0001), miR-885-
5p (*p=0,0154), and miR-122-5p (**p=0,0026) were
significantly more expressed in intoxicated than in
healthy animals. Furthermore, miR-122-5p (SE and
SP 100%), miR-885-5p (SE 73.4% and SP 100%) and
mainly miR-21-5p (SE 71.4% and SP 100%) signatures
demonstrated high sensitivity and specificity based
in ROC curve, indicating a potential application for
detecting cattle poisoning by Senecio brasiliensis.
The miRNA concentrations were also correlated with
worse clinical parameters such as albumin and ALP.
Results demonstrated a predominance of degenerative
lesions, associated with mild proliferation of the
ductal epithelium and fibrous connective tissue,
according to hepatotoxicity induced by Senecio spp.
Discussion: Despite miR-21-5p was highly expressed
in cattle liver, no study demonstrated its role in liver
function. MiR-21-5p was described as identical in
humans being found to be upregulated in liver diseases
as hepatocellular carcinoma, hepatitis C virus and
autoimmune hepatitis. MiR-21-5p is upregulated in
many biological processes, including inflammation
and fibrosis and we hypothesized that high miR-21-5p
levels could occur to induce liver tissue regeneration.
Conclusion: Although, the present study has some
limitations, including the small sample size, it is
unprecedented and brings some insights related
to farm animals poisoning with plants containing
pyrrolizidine alkaloids as well as showing the
necessity of additional studies. Acknowledgments:
FAPESC
 133

Profile of drug-associated toxicological

events in women of reproductive age
Costa, Quezia dos Santos1; Priedous, Gustavo Abud2; Brunello, Giovanna Cristina Spagnuolo2; Alfieri,
Daniela Frizon3; Guidoni, Camilo Molino3; Alves, Jonas Alher Meira2; Girotto, Edmarlon3
1
 Pharmacy student at the State University of Londrina– Londrina/PR, Brazil; 2 Medical Student at the
State University of Londrina– Londrina/PR, Brazil; 3 Professor at the Department of Pharmaceutical
Sciences at the Health Sciences Center at the State University of Londrina– Londrina/PR, Brazil.

Introduction: Poisonings are events that produce
harmful effects on the physiological state of the
organism and drugs are identified as the main agents
involved in these events. Drug-associated toxicological
events (ETMs) in women of childbearing age have been
shown to be even more frequent, which are usually
associated with two or more drugs, especially those
that act on the central nervous system. Objective: To
characterize drug-associated toxicological events in
women of reproductive age. Methods: Cross-sectional
study with data from consultations at a Toxicological
Information and Assistance Center (CIATox) of all
cases of drug-associated toxicological events in
women of childbearing age (10 to 49 years). Data were
extracted from the Brazilian System of Intoxication
Registration of Information Centers, referring to
the period from 2017 to 2020. Variables related to
the patient, exposure and clinical conditions were
explored. The final outcome (negative - moderate
or severe manifestations, or death - or not) was the
dependent variable analyzed. Logistic Regression was
used for association analysis with calculation of the
Odds Ratio (OR), using the Statistical Package for the
Social Sciences (SPSS), version 19.0. Results: 3,304
cases of drug toxicological events were evaluated in
women of childbearing age, with a mean age of 26.0
(±10.2) years. Of the drug toxicological events, about
90.1% involved drugs exclusively, and it is noteworthy
that 5.5% of the events were associated with ethyl
alcohol. The suicide attempt occupied 89.0% of the
circumstances that led to these ETMs, followed by
self-medication (5.6%) and medication error (2.1%).
On average, 2.02 were used (±1.44) drugs per event
and 20.2% of these drugs are antidepressants, being
the most recurrent therapeutic class, followed by
the class of hypnotics and sedatives (17.1%). The
most frequent drugs were benzodiazepines (16%),
followed by serotonin reuptake inhibitors (10.4%).
In the association analysis, a higher prevalence of
negative final outcome was identified among cases
that involved suicide attempts (OR1.55; 95% CI 1.15-
2.09), four or more medications (OR 1.94; 95% CI 1.52-
2.48), with time between exposure and care of more
than 4 hours (OR 1.60; 95% CI 1.30-1.96) and that
occur during the night (00:00h – 05:59h) (OR 1.39;
95% CI 1.05-1.82). Discussion/Conclusion: In view
of the data presented, it is concluded that the drugs
most commonly used in drug-associated toxicological
events act on the central nervous system and,
considering that the main circumstance involved
in the events were suicide attempts, it is expected
that psychiatric disorders and/or or mental factors
were important motivations for the events identified.
In addition, considering that antidepressants are
among the main agents in toxicological events, it
is believed that depressive and anxiety disorders
are predominant in the population served. Finally,
the circumstance of the event, number of agents
involved, exposure time and occurrence shift were
factors associated with the negative final outcome,
which demonstrates the importance of urgency and
emergency services to give more attention to patients
who are involved in ETMs from suicide attempts and
with multiple medications. Also, that the management
of this patient is as fast as possible to minimize the
negative impact, especially when these events occur
during the night. Acknowledgments: We would like to
thank the entire team at the CIATox responsible for the
care.
 134

Profile of suicide attempts by self-poisoning in

the Rio Grande Sul, Brazil, between 2010-2020
and the influence of the COVID-19 pandemic
Santos, Bruno Pereira1,2; Eller, Sarah1; Gouveia, Giovanna Cristiano1; Borges, Gabriela
Ramos1; Sebben, Viviane Cristina3; Arbo, Marcelo Dutra2; Oliveira, Tiago Franco1
1
 Graduate Program in Health Sciences, Federal University of Health Sciences of Porto
Alegre (UFCSPA), Brazil; 2 Faculty of Pharmacy, Federal University of Rio Grande do Sul
(UFRGS), Brazil; 3 Toxicological Information Center of Rio Grande do Sul (CIT/RS), Brazil.

Background/introduction: Globally, suicide is more strongly in the age group of 20 to 39 years

responsible for more than 800,000 deaths every year,
being the second leading cause of death among the
young population. Suicide, suicide attempt or suicidal
ideation are multifactorial, mainly associated with
previous psychiatric disorders and demographic
characteristics. Consequently, it becomes important
to evaluate the relationship of the variables among
themselves and their influence on individuals, to
characterize the profiles of people who attempt
suicide and thus to contribute to the improvement of
public healthcare services. Objective: This work aims
to identify a profile of poisoning by suicide attempt
in Rio Grande do Sul, as well as evaluating the impact
of the COVID-19 pandemic on these cases. Methods:
Data were collected from cases of poisoning attended
by Toxicological Information Center of Rio Grande do
Sul (CIT/RS), registered between 2010 and 2020. Only
cases where the circumstance was suicide attempt
were selected. Statistical analysis was performed
using the IBM SPSS 28.0 software. Association
between variables and groups of toxic agents was
performed using Pearson’s Chi-Square test. For
temporal evaluation, AutoRegressive Integrated
Moving Average Model (ARIMA) was performed.
Results: The total number of suicide attempt cases
was 55,131. The most incident groups of toxic agents
were medicines (47,295 cases; 85.8%), chemicals
(3,034 cases; 5.5%), rodenticides (2,895 cases; 5.3%)
and pesticides (2092 cases; 3.8%). About 73% of
patients were women and 50% of the cases involved
patients between 20 and 39 years old. The gender
variable was associated with several groups, mainly,
female patients associated with the use of medication
(p<0.001), while male patients were associated with
drugs of abuse, pesticides, and chemical products
(p<0.001). As for the age group, the use of medication
was associated with adolescents and young adults
(p<0.001). The use of drugs of abuse was associated
(p<0.001), while the use of pesticides is associated
with the age group of 40+ years (p<0.001) and
rodenticides with 60+ years (p<0.001). The evolution
variable was associated with the medicines group
for cure (p<0.001) and pesticides and chemicals for
death (p<0.001). The association of these two groups
with death is mainly due to the lethality rate, being
3.6% for pesticides and 1.2% for chemicals. In the
temporal analysis, the best model was ARIMA (0,1,4),
with a good correlation between time and the number
of cases up to the first quarter of 2020 (R2=0.87).
Although it was expected an increase in the cases,
from the beginning of the pandemic in Brazil (end of
2020/1) an intense decrease in relation to predicted
cases was observed in the following three quarters of
2020, with drops respectively of -37.1%, -38.7% and
-28.8%, with all exceeding the confidence interval
(CI95%). Discussion/conclusion: The main profile
of suicide attempt is characterized by women, with
15 to 29 years-old, using medicines as a toxic agent,
as representing the majority of cases. An important
association between the variables can be seen,
especially from the type of the agent involved in the
intoxication with the sex and age of the patient, thus
being able to identify different patterns of use among
the groups. Considering the reduction in cases in 2020
with the pandemic, there are numerous hypotheses
for explain this data, such as a real decrease in
attempts, effectiveness of suicide, change of suicidal
method and/or underreporting of poisonings. From
this, other factors involved in suicide (attempt)
must be studied to synthesize a global panorama
and, thus, evaluate the real impact of the COVID-19
pandemic. The results of this study provide a basis for
toxicovigilance system, as well as the development
of mechanisms to prevent suicide attempts.
Acknowledgments: CAPES; FAPERGS.
 135

Services provided by the Nucleus Applied to

Toxicology (NAT) during the period 2019-2021
Reginato, Fernanda Ziegler; Bairros, André Valle; Santos, Lara Celestina; Rosa,
Victória Gomes; Chimendes, Nayomi de Andrade; Santos, Rachel; Pacheco, André
Lucas Bezerra; Cardoso, Leonardo Corrêa; Ugalde, Gustavo Andrade

Núcleo Aplicado a Toxicologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria.

Introduction: The Nucleus Applied to Toxicology
(NAT) is a service that helps the University Hospital of
Santa Maria (HUSM) performing toxicological analysis
of patients with suspected or proven intoxication, in
order to contribute to the proper diagnosis, monitoring
and treatment. Objective: To present the toxicological
occurrences in Santa Maria and region during the period
of 2019-2021 assisted by NAT. Methods: NAT advises
health professionals through telephone contact. The
analysis of biological matrices were performed by
means of immunoassays, spectrophotometry, HPLC-
UV/Vis or DAD and/or GC-MS, depending on the case.
Results: During the last three years, until October
2021, the NAT carried out 39 toxicological analysis,
in addition to 17 toxicological supports to health
professionals. Among the most detected substances
in the analysis are cocaine (found in 15 patients),
benzodiazepines (9) and cannabinoids (5). In 2019, four
immunochromatographic assays for drugs of abuse
and medications and a quantification of paracetamol
by spectrophotometry were performed. In 2020, in
addition to six immunoassays for drugs of abuse and
medications and two quantitative determinations
of paracetamol, two analysis related to pesticides
were performed: one case of suspected paraquat
intoxication, in which the colorimetric method was
performed with sodium dithionite, and in another
case, quantification of serum butyrylcholinesterase
was performed in order to estimate the degree of
intoxication by malathion and fusilade. In 2021, 23
immunochromatographic assays for drugs of abuse
and medications, two quantifications of paracetamol
and one of butyrylcholinesterase were performed, the
latter aiming to elucidate a suspicion of carbamate
intoxication. In this year, analysis in gaseous (GC)
and liquid (LC) chromatography began, aiming at the
detection and quantification of substances, in view
of the growing complexity of the cases. Since the NAT
does not have CG equipment and the LC equipment
has limited performance, the techniques were
developed in another laboratory, which has a high
demand for work, a fact that restricts the execution
of the NAT analyses. The CG-MS equipment was used
in the research of adulterants (levamizole) in cocaine
in a biological matrix, and determination of valproic
acid, quetiapine, ketamine and amitriptyline. LC-UV/
Vis was used in the analysis of trans, trans-muconic
acid, quetiapine, nortriptyline and amitriptyline
in biological matrices. Discussion/Conclusion:
Intoxication cases usually represent a defiance for the
health professionals, as they are difficult to define the
diagnosis, treatment, often there are several agents
involved and potential risk of fatal complications.
Although the NAT is not a Toxicological Information
Center and has a limited technological apparatus,
it has significantly contributed to the advice of
professionals and elucidation of toxicological
occurrences attended at the HUSM. In addition to
improving the patient’s health status, NAT contributes
to the economy of public spending. With the correct
identification of the agent causing the damage and
thus the proper treatment and monitoring, there is a
faster hospital discharge and there is often no need
to use an ICU bed, generating savings of R$ 1,600.00 /
patient / day, given that this is the amount that a SUS
patient at the HUSM costs the public coffers per day,
according to the AuditaSUS expenditure table in 2020.
Thus, the important role that NAT plays for Santa
Maria and the region is highlighted, both for health
and for the economy through its toxicological support
and analyses. Acknowledgments: UFSM.
 136

Suicide attempts in Santa Catarina,

Brazil, before and during Covid-19
pandemic: epidemiological profile
Costa, Ana Carolina Conchon1; Taruhn, Lillian Freitas2; Resener, Marisete Canello1
1
 Assistance and Toxicological Information Center in Santa Catarina;
2
 Medical Student in Federal University of Santa Catarina.

Introduction: Suicide is an important public health
problem, with impacts on society as a whole. According
to the World Health Organization (WHO), it is the fourth
leading cause of death for young people aged 15 to 29
years. A survey revealed that between 2010 and 2019,
the Southern region of Brazil had the highest suicide
rate among all regions, with Santa Catarina being the
second state with the highest rate in the country. The
data also show that poisoning corresponded to 60%
of the means of aggression recorded in this period,
followed by sharp objects (16.8%). The COVID-19
pandemic has exacerbated risk factors associated
with suicidal behaviors such as job or economic loss,
trauma or abuse, mental disorders, and barriers
to accessing healthcare. Thus, understanding the
characteristics of these individuals can contribute
to the planning of public policies to reduce the
impact of suicide attempts. Objective: Evaluate
the epidemiological profile of suicide attempts in
the state of Santa Catarina before and during the
Covid-19 pandemic, assessed from 2014 to 2020, and
to evaluate whether the pandemic had any influence
on suicide attempts. Methods: Data regarding
attempts of suicide of adult patients older than 15
years, both man and woman, with suicide attempts
by poisoning, were analyzed for this work. The data
regarding suicide attempts were extracted from the
open source DATATOX BI Pentaho / Saiku version
2.6, used in the service routine of the Assistance and
Toxicological Information Center of Santa Catarina
(CIATox/SC), while data regarding deaths by suicide
by means of poisoning were extracted from Datasus,
following the same criteria, from the years 2014 to
2020. Statistical analysis was performed at GraphPad
Prism (version 9.0). Results: Based on the data from
CIATox/SC, there was an increase in suicide attempts
from 2014 to 2019, with a decrease in 2020. This
decrease, although not significant when compared
to 2019 (p=0.88), is significant when compared to the
growth observed throughout these years (p=0.01).
The majority of patients who attempted suicide
belong to the age groups of 20-29 (29.3%) and 30-39
(23.3%). The toxicant group with the highest attempts
was medicaments (77%) and women had a higher rate
of suicide attempts than men (69.2% vs. 30.8%). As
for the number of deaths by suicide in Santa Catarina,
extracted from Datasus, no statistical significance
was observed between 2020 and 2019 (p=) or between
2020 and the previous years (p=0,99). Discussion/
Conclusion: A decrease in attempts of suicide in the
State of Santa Catarina among adult patients in the
year of 2020 was observed, when compared to the
growth in these cases during the past years. Besides
that, we observed that there was no increase or
decrease in deaths by means of poisoning during the
year of 2020 when compared to the previous years.
Since the number of deaths in 2020 remains within the
normal rates from the past years, we can infer that the
decrease observed in the suicide attempts in 2020 in
the state of Santa Catarina does not reflect the deaths
by poisoning, which could be explained by the fear of
Covid-19, which made patients not seek health care
services in suicide attempts. Acknowledgements:
CIATox/SC for the partnership and provision of data.
 137

Suicide attempts in adolescents assisted by

a toxicological information and assistance
center due to toxicological events
Brunello, Giovanna Cristina Spagnuolo1; Alves, Jonas Alher Meira1; Costa, Quezia dos Santos2;
Priedous, Gustavo Abud1; Alfieri, Daniela Frizon3; Guidoni, Camilo Molino3; Girotto, Edmarlon3
1
 Student of Medicine at the State University of Londrina – Londrina/PR, Brazil; 2 Pharmacy
Student at the State University of Londrina – Londrina/PR, Brazil; 3 Professor, Department of
Pharmaceutical Sciences, Health Sciences Center, State University of Londrina – Londrina/PR, Brazil.

Introduction: Suicide is a critical public health issue
and one of the leading causes of death in adolescence,
a period marked by significant physiological,
psychological, and social transformations. Because
this phenomena is multifaceted, additional research
and regional assessments are needed to intervene
in suicide prevention and enable more suitable
treatments in those at risk. Objective: To investigate
the clinical and epidemiological aspects of adolescent
suicide attempts related to toxicological events
assisted by a Poisoning Information Center. Methods:
From 2017 and 2020, a cross-sectional and descriptive
study was conducted utilizing data from the Londrina
Poisoning Information Center (CIATox-Londrina),
involving adolescent patients aged 10 to 19 who were
treated for attempted suicide. Variables relating
to the patient, their exposure, and their clinical
conditions were investigated. The dependent variable
was the ultimate outcome (negative - mild, severe
manifestations, or death - or not), which was analyzed
using Logistic Regression and the computation of
the Odds Ratio (OR) with the Statistical Package for
the Social Sciences (SPSS), version 19.0. Results:
We analyzed 1,462 cases of toxicological events
due to suicide attempt in adolescents, whose mean
age was 16.0±2.1 years. Suicide attempts were more
common in females (79.8%), as well as white and
yellow people (76.0%). The events happened most
frequently in October and November (10.2% and 10.4%,
respectively), particularly on Mondays (17.8%), with
the periods between 6:00pm and 11:59pm being the
most common (37.9%). More than half of the suicide
attempts (52.9%) were carried out with a single
agent, and the most common strategy was ingestion
of medication (87.9%), followed by drugs of abuse
(4.8%), domestic cleaning/chemical products (4.8%),
and rodenticides (4.1%). Furthermore, depression was
the main reason of suicide attempts (14.1%), followed
by problems with romantic relationships (10.5%) and
family issues (10.4%). Almost all of the cases (99.8%)
were treated or remained asymptomatic, with the
remaining cases resulting in death (three deaths).
In the association analysis, a higher prevalence of
negative final outcome was identified among cases
involving males (OR 1.45; 95% CI 1.08-1.95), with
time between exposure and care equal to or greater
than 300 minutes (OR 1.80; 95% CI 1.24-2.62), which
took place in January and December (OR 1.89; 95%
CI 1.61-2.56), and charcoal administration activated
(OR 1.54; 95% CI 1.19-1.96). Discussion/Conclusion:
Although adolescent females try more suicides,
young males have a more serious outcome, possibly
because they employ more hazardous drugs and
more severe self-harming methods. The findings
show that the presence of depression symptoms
is the most common reason for suicide attempts.
Additionally, in recent years, these incidents have
resulted in a more serious outcome, with the shorter
time between exposure and treatment serving as a
key prognostic factor. As a result, emergency services
must be prepared to identify risk factors and manage
toxicological incidents appropriately in order to ensure
more humane health procedures and contribute to the
reversal of this extreme public health scenario.
 138

Variation of cholinesterasès activity and

the importance of the involved toxic agent
identification on the acute intoxication
evolution by an acetylcholinesterase
inhibitor – Case Report
Bauermann, Lauren1; França, Liana Conrado2; Nogueira, Janer Alves2; Paula,
Eliza Bianchini1; Marchioni, Camila3; Santos, Claudia Regina3
1
 Federal University of Santa Catarina (UFSC), Brazil; 2 Laboratory of Toxicological
Research - Unit II - Clinical Analysis Unit - HU-UFSC-EBSERH; 3 Department of
Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

Introduction: Rodenticide and pesticides are chemical
products mainly used on agriculture and may trigger
severe even lethal intoxication in patients who
come into intentional or accidental contact, topically
or orally. In 2020, Assistance and Toxicological
Information Center of Santa Catarina (CIATox/SC)
recorded 559 cases of poisoning involving pesticides.
Among these records, an expressive number of
suspected toxic agent was unknown. In cases of
cholinesterase inhibitor poisoning, when it involves
“chumbinho”, as it is sold as an illegal rodenticide, the
composition may variate and contain carbamates or
organophosphates. Although both cause (reversible
or irreversible) inhibition of cholinesterases, the
enzymes responsible for degrading acetylcholine. In
clinical practice the evolution of the two cases may
be different, in terms of management and severity.
Thus, although not always available, recognize the
toxic agent helps to confirm the diagnostic suspicion,
justify prolonged antidotal treatment and identify
the composition commercialized. This strategy, as it
involves chromatographic analyses, is more restricted,
being most common the determination of the activity
of cholinesterases, usually butyrylcholinesterase.
Objective: Report a clinical case of acute intoxication
by a cholinesterase inhibitor, along with the medical
management, evolution of the patient’s clinical
condition and toxicological analyzes performed
during hospitalization. Case Report: Male patient,
49 years old, taken unconscious by a family member
to the Health System, after swallowing “chumbinho”.
On admission, he presented Glasgow 7 on the coma
scale, normotensive, with sweating and tremors only,
with no other symptoms of the cholinergic syndrome
being described. The patient quickly received doses
of atropine, an antidote used to relieve and control
the patient’s condition. One day after ingestion,
erythrocyte (AChE) and plasma (BChE) cholinesterase
activity was determined, with 19.9 U/mmol Hb and
1000 U/L, respectively, far below the reference
values (352-779 U/mmol Hb and 7000 - 19000 U/L,
respectively). The patient continued to be treated with
atropine, sometimes requiring a new bolus dose. The
patient was followed up for 14 days with recurrent
analyzes of both cholinesterases and it was observed
that the greatest inhibition was recorded one day after
ingestion for BChE and 2 days for AChE. There was a
slow and progressive reduction in inhibition, with AChE
at 47.77 U/mmol Hb and BChE at 6000 U/L in 14 days.
The patient continued receiving atropine infusion until
the 15th day and was discharged from the hospital 21
days after ingestion. During hospitalization, analysis
was performed to identify cholinesterase inhibitors
in serum, by Gas Chromatography with Mass
Spectrometry (GC-MS), using a validated method.
The result made it possible to detect the presence
of Terbuphos and Chlorpyrifos organophosphates,
proving and emphasizing the severe clinical condition
with slow evolution. Conclusion: The intoxications
caused by “chumbinho” always deserve attention,
since it depends on the involved agent, the evolution
is slow and demands a sufficient atropine for the
adequate treatment of the patient. Both AChE and
BChE are significantly inhibited in acute intoxication,
being the most rapid reversal of BChE. The
identification of the Terbuphos and Chlorpyrifos in
this case was extremely important for understanding
the evolution, the necessary management and the
appropriate recording of intoxication. Keywords:
“chumbinho”; acute intoxication; acetylcholinesterase,
butyrylcholinesterase, GC-MS, Terbuphos,
Chlorpyrifos.
 04 
DRUGS OF ABUSE

A green dispersive liquid-liquid microextraction
for quantitative analysis of synthetic
cathinones in urine and postmortem blood
Background/Introduction: Synthetic cathinones
(SC), one of the most popular groups of New
Psychoactive Substances (NPS), are closely related
to amphetamines and marketed as legal alternatives
to such conventional illicit stimulants. These new
chemicals pose a threat to public health, as little
is known of their toxicological properties, such as
potency, adverse effects, and metabolism; moreover,
they are usually not detected by conventional
analytical methods, thus the development of
new ones applicable to SC is of high importance
in forensic toxicology. Furthermore, the concept
of Green Analytical Chemistry (GAC) is gaining
attention in toxicological sciences with the search for
environmentally benign approaches during method
development. Objective: The present work aimed at
developing and validating a quantitative method for
the analysis of 15 SC in urine and postmortem blood
while applying the principles of GAC. Methods: A
dispersive liquid-liquid microextraction (DLLME)
was developed and the Design of Experiment (DOE)
statistical tool was used to find optimal conditions for
extraction of SC from both matrices. Full factorial and
Central Composite designs were chosen to analyze
the solvent for precipitation, pH of extraction, ratio
and volume of extraction and dispersive solvents.
Analysis of Variance (ANOVA) was used to find the
solvent yielding the best analyte recovery. The ANSI/
ASB Standard 036, 1st edition 2019 international
guidelines were used for method validation. All
analyses were performed using a UPLC-MS/MS
with the multiple ion monitoring (MRM) approach.
Ethical Committee approval nº 46404121.8.3001.0067.
Results: Hazardous solvents, such as chloroform, are
commonly employed in DLLME, which are not fit when
sustainable applications are intended. Thus, ethyl
acetate (EA) was proposed as a greener alternative
to chlorinated solvents in DLLME. First, a comparison
140
Fabris, André Luis; Yonamine, Mauricio
Department of Clinical & Toxicological Analyses, School of
Pharmaceutical Sciences, University of Sao Paulo.
between EA and chloroform was performed and
equivalent, when not higher, analyte recovery for SC
was achieved using EA (p < 0.05). In the following
optimization step, it was observed that 200 µL of a
EA:ACN mixture (1:2.5) yielded higher SC recovery,
while the system was maintained at pH 8.0. Once
optimized, the method was validated according to
international guidelines. For blood: limit of detection
(LOD) and quantitation (LOQ) of 0.25 and 1 ng/mL,
respectively; linearity 1-100 ng/mL; whereas for urine:
LOD and LOQ of 1 and 10 ng/mL, respectively; linearity
10-1000 ng/mL. For both matrices: no carryover was
observed within linearity range; bias and precision
(> 85%); analyte recovery varied from 27.4-60%; and
selectivity studies have confirmed the absence of
interfering peaks due to endogenous and exogenous
substances at the retention times of all analytes.
In addition, non-labeled internal standards were
not identified as an impurity. Curiously, the matrix
effect (ME) was higher than 100% for all analytes.
Finally, the method was applied to the analysis of
two authentic samples. Discussion/Conclusion:
We demonstrated for the first time that EA is suited
to replace chloroform in DLLME, at least for SC, as
higher – when not equivalent – analyte recovery was
observed. Moreover, although the overall analyte
recovery was not high, the sensitivity sufficed for the
intended applications. In this regard, even with a high
ME, acceptable precision and bias using only 200 µL
of sample was achieved. Furthermore, this is the first
method including N-ethyl Heptedrone. We successfully
developed and validated an analytical method for SC,
while incorporating multiple GAC concepts, e.g. low
sample and solvent volumes (i.e. miniaturization of
extraction techniques), use of less hazardous organic
solvents, reduced generated residues, and simple
sample treatment. Acknowledgements: CNPq –
142056/2020-0; CAPES/INSPEQT Nº16/2020.

A novel workflow for the analysis of drugs of
abuse in oral fluid samples using disposable
pipette extraction technologies (DPX-XTR)
Background/Introduction: Analysis of oral fluid
generally requires some type of sample preparation
to concentrate the analyte and reduce matrix effects
for sensitive and reproducible analyses. The use of
disposable pipette extraction (DPX) tips has been
previously shown to offer advantages over other
solid-phase extraction (SPE) in terms of speed and
feasibility with automation. Most SPE methods for
analyzing drugs of abuse in oral fluid incorporate
a strong acid in order to achieve high recoveries of
basic drugs using strong cation exchange sorbent.
Moreover, there have been reports that this acid
may lead to conversion of cannabidiol (CBD) to
tetrahydrocannabinol (THC). In this research, the use
of mixed mode DPX-XTR tips without the addition
of a strong acid to avoid potential conversion of
CBD to THC was evaluated. The study focused on
analyzing 41 drugs of abuse commonly found in
driving-under-the-influence (DUI) cases. The analysis
used a single sample extraction method and liquid
chromatography - tandem mass spectrometry (LC-
MS/MS). An additional methodology is also presented
for differentiation of delta-8 and delta-9 THC (for
positive THC cases). Objectives: The objective of
this study was to develop a DPX based methodology
for analyzing comprehensive drugs of abuse in oral
fluid for driving-under-the-influence casework. The
use of mixed-mode sorbent is evaluated to provide
reproducible and high recoveries of acidic, neutral
and basic drugs commonly found in DUI case work. A
separate LC method from the comprehensive method
is used in order to differentiate delta-8 THC from
delta-9 THC. Methods: Synthetic negative saliva
buffer solutions (500 µL) were fortified with 50 µL of a
comprehensive drug standard (45 drugs) that includes
opiates, opioids, benzodiazepines, barbiturates, THC
(delta-8 and delta-9, as well as CBD), antidepressants,
amphetamines, and cocaine. Concentrations ranged,
depending on the analytes, as low as 0.1 ng/mL to
as high as 500 ng/mL. An automated liquid handler
141
Cabrices, Oscar G.
G-Flo Scientific Laboratory Testing & Consultants (USA).
was used to perform all extractions of oral fluid. The
extraction involved the steps of conditioning with 800
µL 50% methanol in water, aspirating and dispensing
the solution 5 times, washing with 800 µL of water,
and elution with 500 µL of 48/48/4% acetonitrile/
methanol/ammonium hydroxide. The solutions
were subsequently evaporated and reconstituted
in 125 µL 10% methanol in water. All analyses were
performed using LC-MS/MS using mobile phases of
pH 3.6 ammonium formate in water and methanol. For
comprehensive analysis, a biphenyl column (50 x 3.0
mm, 2.6um) was used. For selective analysis of CBD,
delta-8 and delta-9 THC, a fluorophenyl column (2.7
µm, 100 x 2.1 mm) was used. Results: The extraction
studies included the analysis of 24 samples extracted
weekly for 2 months, for a total of 192 samples.
The extractions using the DPX-XTR tips in average
took less than 10 minutes to complete (i.e., for 96
samples simultaneously). Almost all of the drugs
had recoveries greater than 80%. Drugs fortified at
suggested cutoffs (from the literature) were readily
detected at less than 8% C.V. The comprehensive
LC-MS/MS method was used to detect THC and CBD,
but a separate methodology was used to quantify
delta-8 and delta-9 THC at levels as low as 0.5 ng/
mL. Conclusion/Discussion: A novel sample analysis
workflow using DPX-XTR technologies was developed
for the analysis of drugs of abuse oral fluid samples
often collected DUI cases. 45 drugs and metabolites
were efficiently extracted (>%80 recovery for
all drugs) using mixed-mode DPX-XTR tips as an
alternative to traditional SPE columns, which can help
laboratories to enhance throughput while maintaining
drug quantification requirements. Acknowledgments:
The author would like to acknowledge William E.
Brewer, Kaylee Mastrianni from DPX Technologies and
Karl Scheidweiler from Immunalysis for the technical
development and method implementation of these
assays.
 142

Alcohol and alcohol combined with

texting: effects on speed and braking
behaviours in a closed-course section.
Costa, Bruno Ruiz Brandão1; Freitas, Bruno Toledo2; Bigão, Vítor Luiz Caleffo
Piva1; Perdoná, Gleici da Silva Castro3; De Martinis, Bruno Spinosa2
1
 Faculdade de Ciências Farmacêuticas de Ribeirão Preto – Universidade de São Paulo.
Avenida do Café, s/nº Ribeirão Preto - SP 14040-903, Brazil. 2 Faculdade de Filosofia, Ciências
e Letras de Ribeirão Preto - Universidade de São Paulo. Av. Bandeirantes, 3900, Ribeirão
Preto - SP, 14040-900, Brazil. 3 Faculdade de Medicina de Ribeirão Preto – Universidade
de São Paulo. Av. Bandeirantes, 3900, Ribeirão Preto - SP, 14049-900, Brazil.

Background: Despite its well-known harmful effects
to the society, drinking and driving remains a serious
public health concern worldwide. Another issue
that has recently raised concern is the risk posed
by associating alcohol intoxication and cell phone
use while driving. Objective: To examine the driving
impairment effects of alcohol and of alcohol combined
with texting. Methods: Fifteen licensed drivers with
similar drinking habits completed a lap in a closed-
course section in six different situations: (I) sober;
(II) sober and while replying to a cell phone message
via WhatsApp; (III) 30 minutes after ingesting a
moderate dose of ethanol (0.50 g/kg); (IV) 30 minutes
after drinking and while texting; (V) 60 minutes after
drinking, (VI) 60 minutes after drinking and while
texting. Driving performance was analyzed by means
of mean speed (measured as time to complete one
lap in the course, LT) and maximum speed (Smax);
braking time (BT) and braking distance (BD); and off-
course excursions (i.e., when the drivers hit a traffic
cone or exceeded the boundaries of the course). For
the evaluation of LT, Smax, BT, BD a non-parametric
Friedman test was performed. Pairwise comparisons
using the Bonferroni-Dunn post hoc test were
performed when significant main effects were present
(p < 0.05). Off-course excursions were evaluated by
means of a Cochran Q test. The ethanol levels were
measured at 30 and 60 minutes after drinking by using
a breathalyzer. Results: Paired sample t-test showed
no statistically significant differences between the
blood alcohol levels (BAC) in 30 and 60 minutes after
drinking. All participants presented BAC levels above
0.020 g/dL in both periods. Therefore, according
to the Brazilian legislation, they would have their
drivers’ license retained for one year. Additionally,
60% of the participants (N=9) presented BAC levels
above 0.06 g/dL in at least one period of time. In a real
situation, they would have been arrested. Regarding
LT, the Friedman test result showed statistically
significant difference between the treatments. The
post hoc comparisons revealed that Situation III (30
minutes after drinking) and Situation V (60 minutes
after drinking) were statistically different from
every other conditions, except when comparing one
to another. Comparing with the baseline (Situation
I, LT median value of 48 seconds), the Situations
III and V presented a decrease of 8 and 11 seconds
on the median, respectively. Concerning Smax, the
pre-alcohol treatments were statistically different
from the post-alcohol conditions. The median Smax
values for Situations 1 and 2 were 30 and 31 km/h,
respectively; whereas for Situations 3, 4 and 5, the
median Smax increased to 40 km/h, and for Situation
6 was 39 km/h. Neither alcohol intake nor texting
had a significant effect on the evaluated braking
parameters. None of the participants exceeded the
boundaries of the course. However, one third of the
participants (N=5) knocked down cones at least in
one condition and, interestingly, this happened only
in the driving and texting experiments. Discussion/
Conclusion: Our findings strengthen the hypothesis
that even moderate alcohol doses significantly
impair the driving performance by increasing the
probability of risky driving decisions, which highlights
the importance of lowering the BAC legal limit to
reduce the risk of traffic crashes. On the basis of the
parameters evaluated, alcohol is the main driving
impairment factor in speed behaviours, whereas
texting directly influenced the ability to control the car.
Therefore, alcohol and texting have complementary
effects on driving impairment, and their combination
represents a significant risk factor for accidents.
Acknowledgments: This work was supported by
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES).
 143

Analytical evaluation of four

screening devices for drug detection
in Brazilian traffic enforcement
Scherer, Juliana N.1,2; Borges, Gabriela Ramos1,3; Santos, Bruno Pereira1,3; Dalanhol,
Carolina Silveira1; Gouveia, Giovanna1,3; Viola, Patrícia Pacheco1; Carlson, Renato Romera1;
Govoni, Bruna1; Mello, Raissa1; Vasconcelos, Mailton1; Pechansky, Flavio1
1
 Center for Drug and Alcohol Research, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; 2 Post-
Graduate Program in Collective Health, Universidade do Vale do Rio dos Sinos, São Leopoldo, Brazil;
3
 Pharmacosciences Department, Federal University of Health Sciences of Porto Alegre (UFCSPA), Brazil.

Background/Introduction: In recent years, Brazil cannabinoids, devices presented sensitivity ranging

has foreseen the possibility of including drug
screening devices as a traffic enforcement tool for
the detection of impaired drivers. However, these
devices have a high level of heterogeneity in terms
of accuracy, and must be evaluated according to
local needs before implementation. Looking for
the development of an evidence-based policy,
a large-scale study was conducted to evaluate
different point-of-collection testing devices for
drug detection aiming their application on Brazilian
traffic enforcement scenarios. Objective: To evaluate
the reliability of point-of-collection testing devices
for the detection of psychoactive substances in
oral fluid. Methods: Samples of drivers stopped at
roadblocks in 10 different Brazilian locations were
screened by trained highway police officers using
one of the four different devices available. Paired oral
fluid samples were collected in all positive screened
samples and in around 15% of the negative samples
through Quantisal®, and confirmatory analyses were
performed by a validated HPLC-MS/MS method for
the detection of cocaine, cannabinoids, amphetamine,
and methamphetamine. Sensitivity, specificity and
accuracy of each device for each drug class were
calculated according to each device’s cutoffs. Results:
Of the 8.990 samples screened by the devices, 5.9%
were positive for cannabis, 3.1% for amphetamine,
2.7% for cocaine, and 0.9% for methamphetamine.
Sensitivity for cocaine among the four devices ranged
between 76.9 - 97.0%; specificity between 96.5 -
98.8%; and accuracy between 96.5 - 98.2%. For
between 0 - 96.8%, specificity between 90.2 - 98.1%;
and accuracy between 87.2 - 97.0%. For amphetamine,
devices presented sensitivity between 72.0 - 100.0%,
specificity between 93.2 - 97.8%; and accuracy
between 93.4 - 97.7%. For methamphetamine, devices
presented sensitivity ranging 72.0 - 100.0%, specificity
between 97.4 - 99.6%; and accuracy 96.9 - 99.7%.
Discussion/Conclusion: The D2 was the only device
that presented the minimum of 80% for all reliability
parameters for the detection of all classes of drugs. The
D4 device showed sensitivity of 72% for amphetamines
and methamphetamines; the parameters for cocaine
and cannabinoids were greater than 80%. D3 and D1
devices showed very low sensitivity for cannabinoids
(0% and 12.1%, respectively). The point-of-care
testing devices analyzed in our study presented
acceptable reliability for detecting most substances,
showing the applicability of the devices as point-of-
care testing for screening proposes. As seen in other
studies, the sensitivity for cannabinoid detection is
the critical parameter of most devices, and therefore
this analytical limitation must be considered and
evaluated within public policy contexts. The low values
of positive predictive value highlights the importance
of performing mandatory confirmatory analysis
considering the legal scenario of traffic enforcement.
Acknowledgments: We thank the Secretaria Nacional
de Políticas sobre Drogas for their institutional and
financial support, and the Brazilian Federal Highway
Police for their institutional support. We also thank all
the volunteers who participated in the study.
 144

Assessment of possible neurotoxic effects

of oxycodone in SH-SY5Y cell lineage
Lima, Luiza Siqueira1,2; Costa, Nayara de Souza1,2; Pereira, Meire Ellen1,2; Almeida, William3,4;
Cestari, Marta Margarete3,4; Irioda, Ana Carolina1,2; Oliveira, Cláudia Sirlene1,2
1
 Programa de Pós-Graduação Strictu Sensu em Biotecnologia Aplicada à Saúde da
Criança e do adolescente, Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, PR,
Brazil; 2 Faculdades Pequeno Príncipe, Curitiba, PR, Brazil; 3 Programa de pós-graduação
em Ecologia e Conservação; 4 Universidade Federal do Paraná, Curitiba, PR, Brazil.

Opioids are analgesic drugs, used to control moderate
and severe pain, including cancer pain. This class
of drugs is derivate from opium, which is extracted
from poppy (Papaver somniferum L.). Opioids act by
regulating pain in the descending pathway that can be
associated with the release of serotonin, through their
binding to G protein-coupled receptors, mu, delta, and
kappa in the central and peripheral nervous systems.
Its action can be presynaptic, inhibiting the release
of neurotransmitters from primary afferent endings
in posterior horn neurons, or postsynaptic, reducing
the excitability of dorsal horn neurons. Opioids cause
changes in pyramidal physiology, attenuating the
excitatory response, so that the longer the exposure
to the drug, more difficult will be to control over their
ingestion. There is a reduction in metabolic activity in
the frontal cortical regions, associated with changes
in the expression and function of G protein-coupled
channels, harmful to neuronal activation, however,
when activated, it is more difficult for them to return to
basal levels, these are also associated with changes,
increased of impulsivity, cognitive inflexibility, memory
and concentration impairment, in addition, can lead to
euphoria and generate hallucinations, reasons which
lead to its illicit use. These drugs can cause addiction
and are illegally used for recreational purposes. In
addition to being associated with changes in sleep
parameters, leading to apnea and consequently
hypoxia. Oxycodone is a potent opioid drug, classified
as semisynthetic because it is derived from thebaine,
it is a mu receptor agonist, which also has activity
at the kappa receptor, used for the treatment of
acute and chronic pain. Therefore, the objective of
this work was to evaluate the toxicity of oxycodone,
through in vitro studies using neuroblastoma (SH-
SY5Y) cell lineage. Cells were exposed to oxycodone
at concentrations of 1, 10, and 100 µM for 24 hours.
The drug effect was evaluated by the following tests:
cell viability by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) test; DNA
damage was analyzed using the alkaline comet assay;
cell death by apoptosis and/or necrosis detection
method with the combination of the compounds
Annexin V (ANX) and 7-amino-actinomycin D (7-AAD).
For the tests, at least three independent experiments
were performed. Data were statistically analyzed
using Prisma Graphpad software, version 6.0, using
the Kruskal-Wallis test followed by Dunn’s test and
presented as median ± interquartile range. Effects
were considered significant when p<0.05. Kruskal-
Wallis test showed no effect for oxycodone in the cell
viability test at any concentrations tested. For the
comet assay, no statistically significant difference
was observed when the concentrations tested were
compared to the control, however, the damage caused
to the DNA was ~7 times greater in the cells exposed
to 100 µM when compared to the control group (non-
exposed cells), being possible to observe an alteration
generated to the cellular DNA from exposure to
oxycodone. For the death and apoptosis test, the
Kruskal-Wallis test was not reveal statistically
significant difference, in any of the concentrations
tested, when compared to the control group. At the
concentrations and time tested, no toxic effects were
observed. In the future, models that mimic an acute
exposure to dopaminergic neuron-like will be tested.

Everton Boff1; Daniel, Caroline2; Sá, Clodoaldo Antônio2; Brugnera, Débora Schmitz2;
Silva, Maria Isabel Gonçalves2; Marcon, Scheila2; Corralo, Vanessa da Silva2
1
 Universidade do Oeste de Santa Catarina - UNOESC; 2 Universidade
The consumption of alcoholic beverages can be
considered one of the main factors responsible for
the high incidence of traffic accidents with fatal
victims, becoming a public health problem. Low
doses of ethanol in the body can cause changes
such as euphoria and relaxation, which can lead to
imprudent driving. The objective was to evaluate the
relationship between the toxicological examination
of alcohol and road traffic accidents (ATT) with fatal
victims in Chapecó/SC and region. This is a descriptive
observational epidemiological study, developed at
the Médico-Legal Institute (IML) of Chapecó, with the
General Institute of Expertises of Santa Catarina (IML-
IGP/SC). Data were obtained from criminal reports,
autopsies and records of fatal victims from traffic
accidents in the city of Chapecó and region, in the
period between 2014 and June 2019. For data analysis,
descriptive statistics, rate, standard deviation and
frequency distribution (%) were used. The association
between the variables was analyzed using Pearson’s
Chi-square test or Fisher’s exact test. The significance
level adopted was 5% (p < 0.05). Considering the
437 criminal reports of fatal victims of road traffic
accidents evaluated, 76.0% were men, 25.4% were
aged between 21 and 30 years, 87.9% were white
and 47.1% had fundamental school completed. The
145
Blood alcohol levels in fatal
victims of traffic accidents
Comunitária da Região de Chapecó - UNOCHAPECÓ.
afternoon was the period with highest occurrence
of traffic accidents with fatal victims (32.3%). The
months with the highest frequency were June and
February, with 10.8% and 10.5% respectively, being
2017 the highest number of cases. Most victims were
motorcyclists (18.8%), followed by drivers of motor
vehicles with four wheels or more (18.3%). As for the
results of the toxicological test, 27.2% had positive
blood alcohol levels and 5.0% of the cases were
positive in the detection of narcotics. Among the fatal
victims of ATT with positive alcohol toxicology, 41.0%
were men, 46.4% were between 21 and 30 years old,
36.3% were white and 42.3% were drivers of motor
vehicles with four wheels or more. Road accidents
in individuals who tested positive for alcohol were
associated with being male (p = 0.000) and aged
between 21 and 30 (p = 0.005). From the results, it
is concluded that there was a high percentage of
ATT with fatal victims presenting positive blood
alcohol levels. In this sense, greater inspection is
suggested through existing laws, in order to prevent
accidents and raise awareness among the population.
Keywords: ethanol, road traffic accidents, fatal
victims. Acknowledgments: We thank all participants
and collaborators of this research, as they contributed
to a scientific and social well-being.
 146

Determination of a comprehensive set of

drugs of abuse, metabolites and human
biomarkers in wastewater using passive
sampling followed by UHPLC-MS/MS analysis
Hahn, Roberta Zilles; Bastiani, Marcos Frank; Lizot, Lilian de Lima Feltraco; Linden, Rafael

Universidade Feevale, Brazil.

Introduction: Human urine and feces contain
chemical markers of consumed products, and the
concentration of these markers in residual waters
found in wastewater treatment plants (WWTP) can
be used for the estimation of the consumption of
specific products by a given population, a strategy
named wastewater-based epidemiology (WBE).
However, obtaining representative samples for
analysis is challenging. The most used sampling
strategy in WBE studies is the use of active sampling,
with specialized devices resulting in composite
samples. Usually, active sampling studies are limited
to short sampling campaigns and characterized by
low temporal representativeness and high variability.
Alternately, the use of passive samplers can provide
integrated temporal estimates with a smaller cost.
Among the passive samplers, the polar organic
chemical integrative sampler (POCIS) is used for
the measurement of concentrations of hydrophilic
compounds such as drugs of abuse. Sampling with
POCIS is usually performed over several days and
result in a time-weighted average concentration (CTWA).
The capability of the POCIS to accumulate compounds
present in the wastewater is characterized by its
sampling rate (Rs). Rs values are not consensual and
must be defined by calibration experiments. The
target compounds of this study were biomarkers
of tobacco, cocaine, amphetamines, cannabis,
and caffeine consumption. Objective: Develop an
analytical strategy integrating passive sampling
with the quantification of drug of abuse consumption
biomarkers by ultra-high performance liquid
chromatography associated with mass spectrometry
(UHPLC-MS/MS). Methods: POCIS samplers were
constructed as sandwiched structures containing
Oasis® HLB sorbent between two polyethersulfone
membranes in triplicate one day before the exposure.
POCIS are replaced every two weeks at the inlet of
the WWTP that was monitored for 1 year. The sorbent
was removed from the POCIS and extracted with
methanol and the methanolic extract was submitted
to double solid-phase extraction (SPE). The extracts
were injected in UHPLC-MS/MS, with separation
in reverse phase and with electrospray ionization
in positive and negative modes. Matrix effects
were evaluated by the post-extraction addition
method. The efficiency of the POCIS extraction
procedure was evaluated in quintuplicate. Rs values
for each target compound were calculated by the
water concentration decrease method. The CTWA in
wastewater of the target compounds were calculated
using Rs values determined in the experiments.
Results and Discussion: In this study, an analytical
method integrating POCIS sampling, selective SPE of
POCIS extracts, and analysis by UHPLC-MS/MS was
developed and validated for 12 biomarkers of substance
use. Chromatographic separation was achieved in
11 min. The selective SPE procedure, combining both
cation and anion exchange cartridges, resulted in
low matrix effects (-19.3 to 22.8% for compounds
with a deuterated analog internal standard) and high
extraction yields (93.4 to 106.4%). The lab made POCIS
sampler was adequately calibrated for 9 compounds
(4 drugs of abuse, 4 drug metabolites, and 1 human
non-drug lifestyle markers). The method was applied
in a WWTP in Brazil, with continuous monitoring
for 392 days in 2020-2021. The highest calculated
wastewater concentration among drugs of abuse
and metabolites was found for benzoylecgonine
(431.5±190.6 ng L-1), followed by 11-nor-9-carboxy-
∆9-THC (58.3±21.1 ng L-1), biomarkers of cocaine and
cannabis consumption, respectively. Conclusion:
Passive sampling of wastewater is an interesting
and cost-effective strategy for the quantification of
biomarkers of drugs consumption in epidemiological
studies. Particularly when combined with a sensitive
analytical method as UHPLC-MS/MS, POCIS sampling
allows the long-term monitoring of a wide range
of analytes. Acknowledments: Feevale University,
CAPES

Determination of levamisole in cocaine
samples seized in the south of Santa Catarina
Bombazar, Amanda1; Martins, André Bittencourt2; Steiner, Bethina Trevisol1; Ávila, Ricardo Andrez Machado1
1
 University of the Extreme South of Santa Catarina; 2 Santa Catarina Scientific Police.
Introduction: Cocaine adulterated with levamisole as a cause
of purpura rectiformis progressing to full-thickness skin
necrosis was first documented in 2003 and currently comprises
over 200 reported cases. Agranulocytosis in cocaine users is
a newly recognized worldwide condition, this novel cutaneous
vasculitis syndrome can be recognized by its characteristic rash
and skin pathology, along with leukopenia and autoantibody
production. Certain clinical features can be attributed to the
adulterant levamisole, although cocaine may also play a role
in its pathogenesis. Objective: To characterize the seized
samples for the presence of cocaine and levamisole through
the association of thin layer chromatography (TLC) and Mass
Spectrometry (GC-MS) tests in the samples seized in the south
of Santa Catarina in the period of March, April and May of 2020.
Methods: SAMPLES – The samples used were provided by
the Division of Forensic Analysis of Criciúma of the scientific
police of Santa Catarina, which covers the entire southern
region of Santa Catarina, classified into mesoregions. The
Criciúma mesoregion includes Balneário Rincão, Cocal do Sul,
Criciúma, Forquilhinha, Içara, Lauro Muller, Morro da Fumaça,
Nova Veneza, Orleans, Siderópolis, Treviso and Urussanga;
Araranguá mesoregion includes Araranguá, Balneário Arroio
do Silva, Ermo, Maracajá, Meleiro, Morro Grande, Timbé do Sul
and Turvo; Sombrio mesoregion includes Balneário Gaivota,
Jacinto Machado, Passo de Torres, Praia Grande, Santa Rosa do
Sul, São João do Sul and Sombrio; Tubarão mesoregion includes
Armazém, Braço do Norte, Capivari de Baixo, Grão Pará, Gravatal,
Jaguaruna, Pedras Grandes, Rio Fortuna, Sangão, Santa Rosa de
Lima, São Ludgero, São Martinho, Treze de Maio and Tubarão.
THIN LAYER CHROMATOGRAPHY (TLC) – In the months of
March, April and May, an N of 239 samples of white substance
in the form of powder was obtained, which were admitted to
the laboratory of the scientific police. For the analysis of the
selected samples, 1 mL of methanol was added for each 1mg of
white matter in powder form. These were shaken and submitted
to the physicochemical TLC method. A drop of the mixture was
applied to the plate under the following operating conditions:
stationary phase on silica gel G60F254; mobile phase methanol
100 mL, plus 1.5 mL of ammonium hydroxide; main comparison
standards: cocaine and levamisole. Afterwards, they were
allowed to dry naturally, the acidified iodoplatinate reagent was
sprayed on the plate and the color and retention factor (Rf) of
the stains were observed, comparing them with the samples
and reference standards. GAS CHROMATOGRAPHY COUPLED TO
MASS SPECTROMETRY (GC-MS) – From the N of 239 samples,
8 samples were positive for cocaine and levamisole with the
TLC method. Sample preparation consisted of weighing 1 mg of
147
cocaine, adding 1 mL of ethanol, both in a microtube. They were
shaken for 5 seconds and the microtubes were transferred to a
microcentrifuge, centrifuged for 3 minutes at 10000rpm. After
centrifugation, 500µL of the sample supernatant was transferred
to vials, coupled to a support, which were accommodated in
the chromatographic equipment and analyzed according to the
standards of the scientific police. Agilent Technologies® 7890A
GC coupled to the Agilent Technologies® 5975C mass selective
detector, with electron impact ionization source (70 eV), and
Agilent Technologies® CTC GC sampler 80 autosampler. HP-5MS
column (30m x 0.25mm x 0.25µm) was used. The software used
for data acquisition was Agilent MassHunter Data Acquisition.
Injections were performed in split mode with a ratio of 1:10. The
injection volume was 1 µL. The injector temperature was set at
280 °C. The oven was programmed with an initial temperature
of 200°C for 0.5 minutes, followed by linear heating from 40°C/
min to 230°C and then linear heating from 30°C/min to 290°C,
being maintained at this temperature for 1.5 minutes. Helium
was used as the mobile phase with a flow rate of 1 mL/min. The
transfer line was set at 280°C. Under these conditions, the total
running time was 6.0 minutes. The substances were collected
by the equipment and identified in the SWGDRUGS version 3.8
library. Results: From the analysis of the samples seized from
March, April and May 2020, when added up, an N of 239 samples
was observed, these went through the TLC method and when
comparing the bands, it was identified that 3.35% contained
not only cocaine, as well as the substance of interest in the
work, levamisole. These were sent to the GC-MS method, which
confirmed the result already seen by TLC and, in addition, the
technique observed other peaks of other substances, caffeine,
lidocaine and tetracaine and the alkaloids benzoylecgonine
and the cis and trans isomers of cinnamoilocaine. Discussion/
Conclusion: From the analysis of the samples seized between
March and May 2020, it was observed that out of an N of 239
samples, 3.35% contained levamisole adulteration. Based on the
results obtained in this research, we confirm that the substance
is present in the region and that it will serve as a basis for future
studies, so that the incidence of cases related to adulteration by
levamisole can be investigated. In conclusion, the methods used
in this work proved to be effective in determining the presence
of levamisole in samples of cocaine seized in the south of Santa
Catarina in the period of March, April and May 2020. In addition,
with the CG-EM it was possible to detect other adulterants such
as caffeine, tetracaine and lidocaine and the benzoylecgonine
alkaloids and the cis and trans isomers of cinnamoilocaine, from
the extraction process.
 148

Developing a drug checking service in

Brazil and the potential of harm reduction
strategies to reduce intoxication risks
Maluf, Ana Cristhina Sampaio1,2,4; Costa, José Luiz1,2,3
1
 Campinas Poison Control Center, University of Campinas (UNICAMP), Campinas, SP, Brazil;
2
 Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil;
3
 Faculty of Pharmaceutical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil;
4
 ResPire Harm Reduction Project, Centro de Convivência É de Lei, São Paulo, SP, Brazil.

Background: Variations in the illegal drug market
increase overdose risk. Drug checking is a public
health intervention that allows people who use drugs
to submit substances for content analysis, receive
information and counseling. It is also useful for
obtaining information like patterns of use, drug effects
and adverse reactions, that would not be obtained
from other sources such as seizures. Drug checking
has been used as a harm reduction strategy for over
50 years in other countries and has proven to be an
effective tool in monitoring changes in the drug market,
including the emergence of NPS. However, in Brazil, it
is only emerging and there are no scientific studies in
this field. Objective: The main objective was to start
to set the basis for the development of a community-
based drug checking service in Brazil and study the
impacts of this intervention. Methods: Data collection
was carried out in an electronic music party held in
December 2021. Prior authorization and the access to
the party was based on a collaboration with a harm
reduction organization that has extensive experience
in health education in party contexts. The area
reserved for harm reduction offered various services
such as psychological support, rapid hepatitis test,
distribution of informational material and there was
also a specific space for drug checking. This service
used field techniques such as colorimetric reagents
(Marquis, Mecke, Simon, Mandelin, Liebermann, Scott,
Erlich, Hoffman) and Thin Layer Chromatography
(TLC). Participants also answered a self-administered
questionnaire on sociodemographic data, pattern
of substance use and reactions to the result of
the analysis. Results: A total of 22 samples were
analyzed. Among tablets, 5 were positive for MDMA,
3 were negative and 1 contained MDMA with an
unidentified mixture. Among crystal samples, 4 were
positive and 3 negatives for MDMA. Negative samples
were suggestive of containing MDA. One blotter paper
was positive for LSD. Among powdered samples, 1 was
positive for cocaine, 1 contained cocaine mixed with
ketamine, 2 contained cocaine with an unidentified
mixture and 1 was positive for ketamine. In summary,
approximately 27% of the samples did not contain the
expected substance and 18% contained some type
of mixture. The questionnaire analysis showed that
13% of participants gave up using the substance after
knowing the test result, another 36% reported that
they would use less amount of the substance. 91% of
respondents stated that they always or almost always
plan consumption in advance. 92% of respondents
rated the service as very useful. Discussion/
Conclusion: This preliminary data suggests that
electronic music party attendees positively engage
with harm reduction services when offered. The
high rate of adulteration represents a great risk to
drug users and shows the potential of drug checking
services to prevent the inadvertent use of unknown
substances, to reduce the amounts ingested and
thereby decreasing the risk of intoxication. The
fact that the vast majority of respondents plan
consumption in advance demonstrates that there is
an opportunity for interventions. As future directions,
we intend to compare the results obtained by
colorimetric reagents and TLC with analysis in the
laboratory. Acknowledgments: The authors thanks
CAPES by the financial support

Development and validation of a highly
sensitive method for detection of Synthetic
Cannabinoid JWH-018, AB-CHMINACA, and their
metabolites in hair samples by LC-MS/MS
Background: Synthetic cannabinoids, originally
developed for research of CB1 and CB2 cannabinoid
receptors, were first identified in herbal incense in
2008. These herbal incenses, usually called ‘spice’,
have since gained global popularity because they are
sold on the Internet and in many head shops under
the disguise of normal herbal products. JWH-018
was shown to have psychoactive effects similar to
those of strong agonists of cannabinoid type 1 (CB1)
receptor, for example, Δ9-tetrahydrocannabinol
(THC). AB-CHMINACA, a recently introduced synthetic
cannabinoid, was first reported in Japan in 2013 and has
spread rapidly worldwide. It is a highly potent agonist
of the CB1 receptor and can cause serious adverse
health effects. Hair analysis is useful for monitoring
exposure to drugs, and is a unique material for the
retrospective detection of drug consumption, due to
its large detection window, and it is easy to collect,
store and transport. Objective:This study aimed to
develop and validate a sensitive LC-MS/MS method
for the quantification of JWH-018 and AB-CHMINACA
and their principal metabolites in hair samples.
Methods: In this method, hair samples were cut with
scissors to fragments smaller than 3mm, and then 15
mg was weighted in 2.0 mL polypropylene microtubes.
The samples were decontaminated sequentially with
dichloromethane and methanol and were dried at
ambient temperature. Then, 0.75 mL of methanol and
a labeled internal standards solution were added. The
microtubes were capped and incubated overnight at
40°C. The supernatant was transferred to a 2.0 mL
glass vial and dried at 40°C with a nitrogen sample
concentrator. The samples were resuspended with
149
Paulo, Breno F. Pereira; Zauli, Danielle Alves Gomes Breno
Group Hermes Pardini - Vespasiano, Brazil.
0.15 mL of methanol and 10.0 uL were injected into an
LC-MS/MS system. Chromatographic separation was
performed on an Agilent 1290 Infinity II coupled with
a Sciex 6500+ QTrap mass spectrometer. The system
was equipped with a HALO Biphenyl column and used
a mobile phase constituted by water and methanol
with 0.1% of formic acid. Results: The present method
was successfully validated for carryover, linearity,
precision, recovery, detection and quantification
limits. The chromatographic method is suitable to
analyze all of the analytes with a baseline separation,
principally the isomers JWH 018 N-(4-hydroxypentyl),
JWH 018 N-(5-hydroxypentyl) metabolites. The
analytes JWH-018 and metabolites (JWH 018 N-(4-
hydroxypentyl), JWH 018 N-(5-hydroxypentyl), and
JWH 018 N-pentanoic acid) achieved the linearity
of 1.25 – 50.00 pg/mg, the detection limit of 0.09 -
0.19 pg/mg, and 1.25 pg/mg of quantification limit.
The precision was less than 6%, and recovery was
between 91 – 113% for all analytes. The analytes AB-
CHMINACA and metabolite AB-CHMINACA M2 achieved
the linearity of 2.50 – 100.00 pg/mg, the detection
limit of 0.44 and 0.34 pg/mg, and 2.50 pg/mg of
quantification limit. The precision was less than 5%,
and recovery was between 93 – 101% for all analytes.
Conclusion: We develop and validate a sensitive and
precise method to detect and quantification synthetic
cannabinoids in hair samples. The method does not
need additional steps for clean-up the samples to
achieve sufficient sensibility to detect these drugs at
very low concentrations and can be used at routine
analysis.
 150

Epidemiological aspects of suicide

in the State of São Paulo
Souza, Karla Aparecida de Oliveira1; Gianvecchio, Victor Alexandre Percinio2; Gianvecchio,
Daniele Muñoz2; Jorge, Maria Helena Prado de Mello3; Costa, José Luiz1,4
1
 Faculty of Medical Sciences, University of Campinas, (UNICAMP), Campinas, SP, Brazil;
2
 Superintendence of the Technical-Scientific Police, Institute of Legal Medicine, SãoPaulo, SP,
Brazil; 3 Faculty of Public Health, University of São Paulo (USP), São Paulo, SP, Brazil; 4 Faculty
of Pharmaceutical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil.

Introduction: Suicide is a serious public health
problem that affects people of almost all ages
and has several social implications. However, its
causes are preventable, where a comprehensive
multisectoral strategy is necessary in order to revert
to improvements in the health of the population.
Objectives: To analyze suicide in the State of São
Paulo using a new data source, the Public Security
Secretariat (SSP) and the Legal Medical Institute (IML),
and to compare it with the Mortality Information
System of the Ministry of Health (SIM/MS). Method: a
database related to suicide cases that occurred in 2015
was assembled from two databases: one from the SSP
and the other from the IML, both in the state of São
Paulo. The variables related to the characteristics of
the victim, the event, injuries, presence of alcohol and/
or drugs at the time of death and possible motivation
for suicide were studied. Data were compared to SIM/
MS data. Results and Discussions: 2469 cases were
obtained, 7.5% higher than the SIM/MS data (2297
cases) showing an under-enumeration of suicide
cases from the official data. Males predominated
over females (ratio: 3.8) and, in terms of age, the
highest frequency occurred in the group between
20 and 39 years old. Regarding suicide methods,
hanging represented 60.2% of the total, followed by
self-poisoning (16.1%), fall from heights (9.3%) and
gun shooting (7.2%), there was a 92.5% decrease
among unspecified suicides in relation to SIM/MS
database, resulting in a data quality enhancement.
Regarding intoxications, the highest frequency was
due to the ingestion of medications, in which the
cases resulting from the use of antidepressants
stand out. Regarding ingestion of other substances,
there was a greater number of poisonings by
pesticides. The use of chemical substances for the
practice of suicide is common and can be seen by the
significant gain in relation to official data from SIM/
MS (from 100 to 172 in drug intoxications and from
155 to 225, when intoxication by other substances
is analyzed). Toxicological tests were positive in
51% of the cases performed, with 902 substances
found. The highest occurrence was ethanol (31%),
highlighting blood alcohol concentration ≥ 0.8 g/L
(82.1%), leading to the conclusion that the majority
of suicides with a toxicological test for ethyl alcohol
were drunk at the time of the act; the second group
of substances corresponded to drugs (37%), where
positivity in women was almost three times that
found in men. Cocaine was detected in 75 cases, being
more frequent in adult men. It was found in 55.2% of
positive exams in cases of hanging, in 50% of suicides
by sharp instruments and firearms, and in 42.9% of
suicides by precipitation from a high place. Pesticides
were detected in 93 cases, with carbamate being
the most frequent followed by organophosphates.
Carbon monoxide and liquefied petroleum gas were
found in 20 cases, being higher among adolescents.
Conclusion: Data from SIM/MS, regarding suicides,
are still incomplete, since, for various reasons, deaths
are under-enumerated, sometimes not specifying
the correct cause of the event. The methodology of
this research allowed quantitative and qualitative
gains in relation to suicide cases. These results
point to the importance of working together, data
from death certificates (SIM/MS) and from police
information and autopsy reports. It is possible to
estimate the appreciable gain that public safety data
would generate in health data and, consequently,
in prevention measures. Keywords: suicide,
epidemiology, mortality, health information systems.
Acknowledgments: FAPESP, CNPq.
 151

Exogenous poisoning by drug abuse in Brazil:

before and during the COVID-19 pandemic
Baccule, Nicole Santos1; Scanferla, Deborah Thais Palma2; Lini, Renata
Sano2; Marchioni, Camila3; Mossini, Simone Aparecida Galerani1,2
1
 Laboratory of Toxicology, Department of Basic Health Sciences, State University of Maringá, Paraná,
Brazil; 2 Postgraduate Program in Bioscience and Physiopathology, State University of Maringá,
Paraná, Brazil; 3 Department of Pathology, Federal University of Santa Catarina, Florianopolis, Brazil.

Introduction: The high number of intoxications by
drugs of abuse treated by the Sistema Único de Saúde
(SUS) contributes to the overload of the system.
Measures established to restrict movement during
the Corona Virus Disease pandemic (COVID-19) have
altered social dynamics, with possible impacts on
mental health. In this context, understanding the
profile of cases reported before and during the
pandemic can help in planning prevention strategies.
Objective: To analyze epidemiological data on
intoxications by drugs of abuse reported in Sistema de
Informação de Agravos de Notificação (SINAN), in the
previous period and during the COVID-19. Methods:
Exploratory, descriptive, and retrospective study
with analysis of secondary data obtained from SINAN,
available on the DATASUS website, referring to the
previous period and during the COVID-19 pandemic
(2018-2019 and 2020-2021). Results: In Brazil, in
the analyzed period, 66,889 intoxications by drugs
of abuse were registered, 44,191 (66.07%) cases in
the first period (2018 and 2019). The circumstance
“abusive use” and male gender were similar in the
analyzed periods, with a mean occurrence of 78.5%
and 73.3%, respectively. Suicide attempt (SA) was
reported in 3.6% of cases in 2018-2019 and 4.9% in
2020-2021. The age group with increased records
corresponded to 20-39 years, first and second period,
55.1% and 56.3%, respectively. Regarding education,
the data were similar in both periods, with the most
prevalent being ignored data 54.0% (2018-2019) and
49.8% (2020-2021). In both periods, a low percentage
of cases underwent clinical and laboratory diagnosis,
2.7 and 2.5%, respectively. Single-acute exposure
was the most recorded in both periods (33.7% and
31.06%). Most notifications in the first period came
from the Southeast region (62.2%), the city of São
Paulo with the highest percentage of notifications
(31.2%), followed by the Northeast region (14.7%). In
the second period, most of the notifications were also
registered in the Southeast (59.5%), followed by the
South region (15.8%, São Paulo maintained its position
(35.2%). The highest percentage of evolution was
cure without sequelae, 68.8% (2018-2019) and 71.8%
(2020-2021); 20.5% (2018-2019) and 20.8% (2020-
2021) with interrupted or unreported follow-up.
The number of deaths was 589 (2018-2019) and 320
(2020-2021). Discussion/Conclusion: The pandemic
did not change the profile of reports of intoxication
by drugs of abuse, despite the 32.1% reduction,
possibly resulting from underreporting, explained by
fear of contamination and overcrowding of services.
Between 2020 and 2021, there was an increase (1.2%)
in cases of SA, which may indicate a correlation with
the pandemic. In both periods, the majority of patients
were male, with an increase (1.5%) in the most recent
period. The prevalent age group was young adults,
which indicates the male adult stage as the main
target for prevention actions. A high percentage of
cases with no schooling data was observed, making
discussion unfeasible and demonstrating the need to
improve the training of professionals responsible for
collecting and recording data. Data show that clinical
laboratory diagnosis could be more applied in the
future as a confirmation criterion for drug use. The type
of acute-repeated exposure was more prevalent, with
an increase of 3.4% between the analyzed periods. The
Southeast region, which ranked first in both periods,
needs better prevention strategies, specifically the
city of São Paulo, which showed an increase of 3.9%
in notifications. Although most of the reported cases
evolved to a cure without sequelae, there was a
significant number of deaths. Thus, the relevance of
knowledge of the epidemiology of cases of exogenous
intoxication is evidenced so that prevention and
intervention actions can be thought and carried out to
reduce cases. Keywords: Intoxication, Epidemiology,
Drugs. Acknowledgments: The State University of
Maringá, Postgraduate Program in Biosciences and
Pathophysiology (PBF/UEM), CAPES e CNPq.
 152

Identification of synthetic drugs seized in the

region of Balneário Camboriú using ATR-FTIR
Costa, Karina Oliveira1; Bagio, Jéssica2
1
Biochemical Criminal Expert - Scientific Police Superintendence in Balneário
Camboriú; 2 Pharmacy graduate students- Vale do Itajaí University - UNIVALI.

The city of Balneário Camboriú is internationally
known for its tourist attractions and electronic parties
where there is great consumption of illicit drugs.
Tourism is the main economic activity in the city, which
is known, not only for its natural beauty, but also for
its real estate market, which attracts large financial
investors to the region. New psychoactive substances
(NPS) or designer drugs comprise a group of modified
drugs developed by changing molecular structures of
known illegal substances such as cannabis, cocaine,
lysergic acid diethylamide (LSD), and phenethylamine
derivatives (MDA, MDMA, MDE, etc). NPS are illegally
produced in a variety of preparations (powder, tablets,
or capsules), and they may be injected, ingested orally,
snorted, or smoked. Infrared spectroscopy is widely
employed as a qualitative and quantitative method
for the determination and structural analysis of drugs.
Fourier Transform Middle Infrared Spectroscopy
(FTIR) analysis can be a particularly useful tool for the
rapid screening of seized drugs tablets and powders.
Attenuated Total Reflectance (ATR) accessory for FTIR
allows direct sample measurement with minimal
preparation and with the potential to recover the
sample if necessary. This study aimed to analyze
and identify the content of synthetic drugs seized
in the region of Balneário Camboriú using ATR-
FTIR. The seized material was first subjected to
colorimetric analysis using the reagents Marquis,
Simon and Mecke, and then analyzed using (FTIR)
with Attenuated Total Reflectance (ATR), featured
by the following elements: detector with element in
Deuterated Triglycine Sulfate (DTGS), Zinc Selenide
(ZnSe) Beam Splitter and spectral range from 500
to 4000 cm-1. The sampling interface used was
Monolithic Diamond and thirty-two scans were
collected at a resolution of 4cm-1. Most of the synthetic
drug seizures in the region were marked in the form
of tablets in various shapes and colors. The most
common substances found in its composition were
3,4 methylenedioxymethamphetamine (MDMA) and
alpha-methyl-3,4-methylenedioxy-phenethylamine
or tenamphetamine (MDA) with prevalence of MDMA
in the form of crystallized powder and of MDA in the
form of tablets. In 2020, however, the first seizures
of methamphetamines began to appear in the region.
Keywords: New Psychoactive Substances; Balneário
Camboriú; ATR-FTIR. Acknowledgments: Chemical
Laboratory team of the Forensic Analysis Institute
– Scientific Police Superintendence in Balneário
Camboriú.
 153

In silico evaluation of biological,

therapeutical, and toxicological properties
for five groups of NPS: amphetamines,
cathinones, benzodiazepines, synthetic
opioids, and synthetic cannabinoids.
Santos, Christiano; Bruni, Aline Thais

Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.

The emergence of New Psychoactive Substances (NPS)
is observed worldwide. These compounds correspond
to changes in the chemical structures of the original
prohibited substances to create alternatives to
consumption and legislative prohibition. The rise of
these new drugs represents a challenge for identifying
and depicting their main attributes. This study aimed
to evaluate NPS by using silico methods to assess the
principal characteristics by predicting their biological,
therapeutical, and toxicological properties. We used
the MetadrugTM software for calculating seventy-five
variables among these properties for five different
groups of compounds collected from the literature:
amphetamines (15 molecules), benzodiazepines (13
molecules), cathinones (16 molecules), synthetic
opioids (14 molecules), and synthetic cannabinoids
(14 molecules). We calculated structural data, CYP450
QSAR Models, Protein binding QSAR Models, ADME
QSAR Models, the predicted therapeutic activity,
and predicted toxic effects. This tool is part of the
MetaBaseTM API to access the knowledge base behind
all systems biology products of Clarivate Analytics
(https://www.cortellislabs.com/page/?api=api-
MB). Multivariate analysis was applied to verify the
behavior of these groups of substances. Unsupervised
approaches PCA (Principal Component Analysis)
and HCA (Hierarchical Cluster Analysis) was used to
verify which variables better describe each group.
Observed clusters were submitted to supervised
learning through SIMCA (Soft Independent Modeling
of Class Analogy). We observed that the five different
groups were classified through PCA and HCA. SIMCA
confirmed these classes, and no misclassification was
found. Different characteristics were found for each
category, as follows: Class 1. Amphetamines.
We found four variables with high modeling power
among those with the prediction of therapeutic
activity: potential antiallergic, antianginal, and
antihypertensive activities besides action against
skin diseases. However, among the prediction
of toxic effects, we found potential for inducing
carcinogenicity in rats and mice, kidney necrosis,
and pulmonary toxicity. Class 2. Benzodiazepines.
This class was modeled for variables related to the
prediction of therapeutic activity, showing potential
for antifungal, analgesic, antiviral and antiallergic
activities. Besides, Protein binding QSAR Models
showed potential to bind the androgen receptor and
human hERG (human ether a-go-go-related gene)
channel inhibition. The potential for inducing kidney
necrosis was also found as a predicted toxic effect.
Class 3. Cathinones. The main discriminant variables
for this group were: human serum protein binding %
(ADME QSAR Models); potential to be metabolized by
CYP1A2 (CYP450 QSAR Models); potential for inducing
hepatotoxicity (Prediction of toxic effects); potential
anticancer activity (Prediction of therapeutic action).
Class 4. Synthetic opioids. For Protein binding QSAR
Models, these molecules presented the potential to
bind to Estrogen receptors at 100 uM or less. Among
the possibility of toxic effects, we found as variables:
growth inhibition of MCF7 cell line (human Caucasian
breast adenocarcinoma), pGI50; potential for inducing
carcinogenicity in male mice, and cardiotoxicity.
Finally, this group presented potential therapeutic
activity against skin diseases and antifungal action.
Class 5. Synthetic cannabinoids. Besides antifungal
action, this group presented potential therapeutic
activity against skin diseases and heart failure.
Regarding the toxic effects, the potential for inducing
carcinogenicity in male rats was an essential variable
for modeling this class. This study was adequate to
show different properties regarding different groups.
All groups presented both potential for therapeutic
and toxic effects. We thank the Brazilian Agencies
Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq, grant 465450/2014-8) and
Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES, Finance Code 001) for financial
support.
 154

In silico parameters and chemometrics

applied to the study of NBOMes and
amphetamine-type stimulants
Mariotto, Lívia Salviano; Rodrigues, Caio Henrique Pinke; Bruni, Aline Thais

Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.

NBOMe is an abbreviation for compounds that contain
an N-methoxybenzyl group. They are considered
NPS (New Psychoactive Substances), used as
alternatives to LSD (Lysergic Acid Diethylamide).
ATS is the abbreviation for amphetamine-type
stimulants, synthetic NPS used to mimic the effects
of classic controlled substances such as MDMA. Both
groups of NPS are compounds that present similar
structures once NBOMes can give an amphetamine
partial structure (https://analyticalsciencejournals.
onlinelibrary.wiley.com/doi/10.1002/dta.1889). These
compounds’ pharmacologic and biological properties
are unknown and might be elucidated. However,
the growth of NPS can cause many challenges in
understanding the properties of these drugs. The
main goal of this study is to use in silico methods
for investigating the biological and toxicological
behavior of these substances. For this study, we
applied the MetadrugTM tool, part of the MetaBaseTM
API, to access the knowledge base behind all systems
biology products of Clarivate Analytics (https://
www.cortellislabs.com/page/?api=api-MB). We used
MetadrugTM software for calculating seventy-eight
variables for 21 amphetamines, 21 cathinones, and
24 NBOMes. SMILES were used to simulate structural
properties, CYP450 QSAR Models, Protein binding
QSAR Models, and ADME QSAR Models, predicting
therapeutic activity and toxic effects. We evaluated
the influence of the variables on the samples by
multivariate tools. We used PCA (Principal Component
Analysis) as an unsupervised approach and SIMCA
(Soft Independent Modeling of Class Analogy) to
observe classification effectiveness. PCA results
showed there are mainly two classes regarding
NBOMes and ATS. However, it is possible to observe
some differentiation between ATS amphetamines and
cathinones structures. SIMCA evaluation confirmed
three different classes, but significant similarities
between amphetamines and cathinones. Mouse
carcinogenicity showed a higher modeling power for
NBOMEs. The retention time for the affinity to human
serum albumin was the most crucial variable in
modeling the ATS class. Our findings showed a great
potential of the in silico tools to foresee the properties
and provide valuable information about unknown
substances. We found that even NBOMes with
amphetamine core structures differ in behavior from
ATS. The computational approach helped to promote
an understanding of the biological and toxicological
characteristics of these NPS. It can support decision-
making in public policies and law enforcement. We
thank the Brazilian Agencies Conselho Nacional
de Desenvolvimento Científico e Tecnológico
(CNPq, grant 465450/2014-8) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES,
Finance Code 001) for financial support.
 155

Intoxication case series by N-ethylpentylone

(N-ethylnorpentylone or ephylone)
Denkena, Isadora Locilento1,2; Cunha, Kelly Francisco2; Lanaro,
Rafael2; Cunha, Ricardo Leal3,4; Costa, Jose Luiz1,2
1
 Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo 13083-859, Brazil;
2
 Campinas Poison Control Center, Faculty of Medical Sciences, University of Campinas, Campinas,
São Paulo 13083-859, Brazil; 3 Institute of Chemistry, Federal University of Bahia, Salvador, Bahia
40170-115, Brazil; 4 Institute of Analysis and Forensic Research, Aracaju, Sergipe 49100-000, Brazil.

Context: New psychoactive substances (NPS)
continue to proliferate in recreational drug markets
worldwide. Synthetic cathinones is one of them.
After administration of high doses or repeated use,
they can produce serious medical complications,
including tachycardia, hypertension, agitation,
psychosis, aggressive behavior, seizures and, in some
cases, death. N-ethylpentylone is a newly-emerging
synthetic cathinone, but little information is available
about its toxicology and pharmacology. Objective:
Characterize the analytical quantification and describe
the clinical presentation of analytically-confirmed
cases of N-ethylpentylone intoxication. Materials
and Methods: The quantification of N-ethylpentylone
in blood obtained from human cases was carried out
using liquid chromatography coupled to tandem mass
spectrometry (LC-MS/MS), and the clinical symptoms
exhibited by the intoxicated individuals who were
taken to the hospital emergency room for medical care
and laboratory are described. Results: Our LC-MS/MS
method assayed N-ethylpentylone concentrations
with limits of detection and quantification of 1 and
5 ng/mL, respectively. Quantitation was linear from
5 to 500 ng/mL, and the method displayed excellent
specificity and reproducibility. Five intoxication
cases were monitored by Campinas Poison Control
Center, men and women aged 18 to 35 years reported
circulating concentrations of N-ethylpentylone
ranging from 7 to 170 ng/mL and symptoms included
palpitations, tachycardia, agitation, hallucinations,
coma and death. Discussion/Conclusion: Until the
present study, only two published articles reported the
amount of N-ethylpentylone. We present a validated
method to quantify N-ethylpentylone in human case
work. All cases accompanied by laboratory results
showed high levels of creatine kinase, a symptom often
associated with synthetic cathinone intoxication. The
two highest concentrations of N-ethylpentylone that
we measured were found in a fatal overdose case
(ie, 170 ng/mL) and a severe intoxication with long-
lasting cerebrovascular complications (ie, 149 ng/
mL). Importantly, N-ethylpentylone was the only
psychoactive compound detected in our fatal case,
confirming that the drug can have lethal consequences,
as well as a variety of symptoms, including
palpitations, tachycardia, agitation, aggression,
hallucinations, and coma. Acknowledgments: The
authors thank Fundação de Amparo à Pesquisa do
Estado de São Paulo – FAPESP (process number
2015/10650-8 and 2018/00432-1) and Conselho
Nacional de Desenvolvimento Científico e Tecnológico
– CNPq (process number 830525/1999‑8).
 156

MDA in silico toxicity assessment

suggests low hepatotoxicity and calls
for further pre-clinical investigations
Oliveira, Arthur Lima1; Miranda, Raul Ghiraldelli2; Dorta, Daniel Junqueira1
1
 Universidade de São Paulo (USP), Faculdade de Filosofia, Ciências e Letras de Ribeirão
Preto, Departamento de Química, Ribeirão Preto, SP, Brazil; 2 University of São Paulo (USP),
Faculty of Pharmaceutical Science of Ribeirão Preto, Ribeirão Preto, SP, Brazil.

Introduction: 3,4-methylenedioxyamphetamine was not predicted as hepatotoxic. As of metabolism,

(MDA) is an entactogen often used in music
festivals, mostly sold as MDMA, that showcases
similar effects as the last, including euphoria, self-
awareness, compassion and acceptance for self
and others. Nevertheless, little is known about its
pharmacokinetics in humans and data about its
hepatotoxicity is scarce. Following the resurgence
of MDA in the streets and the prevalence of ecstasy
tablets containing both a mixture of MDA and
MDMA or just MDMA - according to police reports-,
insights on its pharmacology and dose response for
a safer use under the perspective of harm reduction
is imperative. Similarly, considering its entactogen
properties and the advances in psychedelic research
aiming to find alternatives to treat neurological
disorders, pre-clinical information about MDA could
enable further investigation of the drug as a potential
therapeutic candidate. Objective: The aim of this
study was to assess the toxicity of MDA in silico to
provide more information about its pharmacology and
sustain further pre-clinical studies. Methods: Toxicity
assessment was run through statistical and machine
learning-based IS-TOXTM platform (Altox Ltda, funded
by FAPESP process number 2016/08322-5) under the
following softwares: iS-LiverTM, to predict in vitro and
in vivo hepatotoxicity; Acute-ToxTM, for acute toxicity;
DevTox-IsTM, for developmental toxicity prediction;
Pred-CYP2DTM, for metabolites prediction; Pred-OralTM,
for bioavailability and permeability prediction; and
lastly Genotox-IsTM, to predict genotoxicity; Results:
Assessment of the molecule has returned structural
alerts regarding potential DILI and action as agonist of
the Aryl Hydrocarbon Receptor (AhR). The cytotoxicity
assay using HepG2 cells predicted a weak-moderate
toxicity (above 4,8 mg/L). MDA was classified as
hepatotoxic, producing DILI in rodent but not humans.
Its main metabolite, alpha-methyldopamine however,
MDA is a substrate of the following CYP (cytochrome
P450) isoforms: 2B6, 1A2, 3A4, 2C19, 2E1, 2A6, 2C9
and 2C8; acting as an inhibitor of the isoforms 2B6,
2D6, 1A2 and 3A4. The assessment of developmental
toxicity predicted MDA as toxic in vivo, but not toxic
in terms of reproductive toxicity. High permeability
was predicted using Caco-2 cells (23,3 x 10-6 cm/s)
and Paralell Artificial Membrane Permeability (PAMP)
was moderate (1,2 x 10-6 cm/s). Rat oral acute toxicity
was estimated in 125,8 mg/kg (OECD Category 3).
MDA was not considered genotoxic. Discussion/
Conclusion: Based on the predictions obtained MDA
produces acute toxic effects in lower doses compared
to its analogue and parent molecule MDMA. The toxic
concentration predicted does not seem to pose serious
risks to most users in controlled environments, once
most tablets contain less than 120 mg of MDA, which
would not be enough to produce hepatic damage.
Although the data regarding its toxicity both in vitro
and in vivo are scarce, set and setting seem to play an
important role in MDA-induced toxicity, as for MDMA,
once its use can cause hyperthermia, increased
blood pressure and heartbeat rate. Considering the
consumption pattern features of MDA it is likely that
the adverse effects reported are due to secondary
events concerning recreational use and not due
to its intrinsic pharmacological properties. Sales
of ecstasy tablets allegedly containing MDMA but
being in reality MDA or a mixture of both might also
contribute to missdosage among inexperienced users
which may lead to scenarios of increased anxiety and
panic attacks. Therefore, novel pre-clinical studies
with MDA under therapeutic/recreational doses are
encouraged not only to support medical assistance in
cases of intoxication but to potentially bridge its use
in assisted psychotherapy. Acknowledgments: None.
 157

MDMA in silico toxicity analysis reinforces

need of mimicking consumption patterns
towards translational reliability
Oliveira, Arthur Lima1; Miranda, Raul Ghiraldelli2; Dorta, Daniel Junqueira1
1
 Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão
Preto, Universidade de São Paulo; 2 University of São Paulo (USP), Faculty of
Pharmaceutical Science of Ribeirão Preto, Ribeirão Preto, SP, Brazil.

Introduction: 3,4-methylenedioxymethamphetamine
(MDMA) is an entactogen widely used in music fes-
tivals owing to its effects of increased compassion
and acceptance for oneself and others, interpersonal
closeness and changes in emotions recognition. How-
ever, MDMA is thought to cause drug-induced liver
injury (DILI) and numerous reports of intoxications
and fatalities related to ecstasy tablets containing
MDMA have arisen in the past couple of decades. In
contrast, no clinically relevant adverse events were
found in controlled administration of the drug, which
raises the hypothesis of toxicity induced by second-
ary events such as hyperthemia, lack of attention to
somatic cues, hyponatremia, pre-existent cardiac and
metabolic disorders. Set and setting are also funda-
mental to assess whether the consumption scenario
may offer risks of adverse effects, since most of them
include uncontrolled environments associated with
vigorous exercise, poly-drug use and lack of hydra-
tion. Nevertheless, many studies have linked the ad-
verse effects of MDMA alone to a net of events that
do not necessarily owe to its pharmacological prop-
erties. Objective: The aim of this study was to assess
the toxicity of MDMA in silico as a mean of predicting
its toxicity in vivo. Methods: Toxicity assessment was
run through statistical and machine learning-based
IS-TOXTM platform (Altox Ltda, funded by FAPESP pro-
cess number 2016/08322-5) under the following soft-
wares: iS-LiverTM, to predict in vitro and in vivo hepa-
totoxicity; Acute-ToxTM for acute toxicity; DevTox-IsTM,
for developmental toxicity prediction; Pred-CYP2DTM,
for metabolites prediction; Pred-OralTM, for bioavail-
ability and permeability prediction; and lastly Geno-
tox-IsTM, to predict genotoxicity. Results: Assessment
of the molecule has returned structural alerts regard-
ing potential DILI and action as agonist of the Aryl
Hydrocarbon Receptor (AhR). The cytotoxicity assay
using HepG2 cells predicted a weak-moderate toxic-
ity (above 9,4 mg/L). MDMA was classified as hepa-
totoxic, producing DILI in both rodent and human. Its
main metabolite, MDA, was also considered hepato-
toxic and weighed in the final toxicity result. In terms
of metabolism, MDMA is a substrate of the following
CYP (cytochrome P450) isoforms 2B6, 1A2, 3A4, 2C19,
2E1, 2A6, 2C9 and 2C8; acting as an inhibitor of the iso-
forms 2B6, 2C19, 2D6, 1A2 and 3A4. The assessment
of developmental and reproductive toxicity predicted
MDMA as toxic in vivo. High permeability was predict-
ed using Caco-2 cells (11,2 x 10-6 cm/s) and Paralell
Artificial Membrane Permeability (PAMP) was also
high (11,9 x 10-6 cm/s). Rat oral acute toxicity was esti-
mated in 179 mg/kg (OECD Category 3). MDMA was not
considered genotoxic, which is consistent with exper-
imental findings. Discussion and Conclusion: Based
on the toxic concentration prediction MDMA does
not seem to represent serious health risks under the
common recreational dose (100-125 mg) which would
result in a peak plasmatic concentration of around
225 ng/mL, unlikely to produce liver injury. This find-
ing, nevertheless, contradicts clinical reports of DILI
after MDMA intake and studies using animal models,
which might be explained by difficulties in translation
of the results obtained in pre-clinical studies owing to
its non-linear pharmacokinetics. Additionally, as sup-
ported by the predictions obtained there seem to be
factors influencing intoxications and fatalities – that
were not considered in silico –, other than MDMA’s in-
trinsic pharmacology which could be better assessed
mimicking the real consumption pattern, to raise
awareness on how environmental and secondary so-
matic features might affect adverse outcomes. In this
context, designing experiments to better mimic rec-
reational use with models that recreate human phys-
iology settings such as 3D cell culture might present
itself as an alternative to increase translational relia-
bility. Acknowledgments: None.
 158

Profile of new psychoactive substances (NPS)

in seized materials analyzed in the Rio de
Janeiro State during the COVID-19 pandemic
Oliveira, Adriana Sousa1; Almeida, Fernando G.1; Bhering, Cecília de A.2; Antonio,
Ananda da S.2; Wurzler, Gleicielle T.2; Costa, Gabriela Vanini2
1
ICCE - Instituto de Criminalística Carlos Éboli/PCERJ, Rio de Janeiro, RJ, Brasil,
20060-050; 2 UFRJ - Universidade Federal do Rio de Janeiro, Instituto de Química,
NAF – LADETEC / IQ – UFRJ, Rio de Janeiro, RJ, Brasil, 21941-909.

Introduction: New psychoactive substances (NPS)
have similar effects to “classic” drugs, such as cocaine,
“ecstasy”, THC and lysergic acid diethylamide (LSD),
and they have become a global phenomenon over the
last decade. Up to August 2020, over 1000 individual
NPS have been reported to the United Nations Office
for Drugs and Crime (UNODC) Early Warning Advisory.
Brazil reported the identification of 116 NPS, being
stimulants and classic hallucinogens the main groups.
In Rio de Janeiro State (RJ/Brazil), NPS sold as blotter
papers were mostly related to phenethylamine (NBOMe
and NBOH families) between 2006 and 2019. The lack
of knowledge about the side effects and toxicity of
NPS, makes its use a risk to society, representing a
public health problem. Objective: The goal of this
work is to conduct a retrospective study of seized
materials containing NPS, analyzed by the Police Civil
of the Rio de Janeiro state (Brazil) during the COVID-19
pandemic, between March 2020 and February 2022.
Methods: 38 drug samples representing a total
of 3164 blotter or infused papers obtained from
different regions of the Rio de Janeiro state were
analyzed by gas chromatography coupled with
mass spectrometry (GC-MS), high-resolution mass
spectrometry (Orbitrap-HRMS) and attenuated total
reflectance Fourier transform infrared spectroscopy
(ATR-FTIR). Results: From the data obtained, synthetic
cannabinoids (SC) were the predominant group in this
study with 5 different substances identified in the
samples. Among these, ADB-BUTINACA compound
was the main constituent, discovered in 37.1 % of the
infused papers seized in the Rio de Janeiro state. Two
of the evaluated samples presented both MDMB-
4en-PINACA and ADB-BUTINACA. In 2021, the ADB-
4en-PINACA and 4F-ABUTINACA were identified
for the first time in 0.3% and 1.3% of the materials
seized, respectively. These substances are already
controlled in the country, through Ordinance SVS/MS
no 344, of May 12, 1998, being framed in Structure B10
of the structural classes of SC. During the COVID-19
pandemic, phenethylamines were the second group
with the highest number of seizures (n=13). The NBOHs
(25I-NBOH, 25C-NBOH, 25B-NBOH and 25E-NBOH)
were discovered in 373 blotter papers (12.9% of
the materials seized). The 25E-NBOH was the most
frequent derivative in these periods (9.8%). A fentanyl
analog (furanylfentanyl or an isomer thereof) was
observed in a single seized with 35 blotter papers
in the Green Coast region in 2020. In this study,
synthetic cathinones were not identified. Discussion/
Conclusion: The market for illicit synthetic drugs
remains complex and very dynamic, although it goes
through periods of innovation and stagnation. During
the COVID-19 pandemic, new substances continued
to emerge; especially substances from the SC group
seized in prisons in the metropolitan region of the
state of RJ. Prior to the pandemic period, there were
only one report of the seizure of SC. Quite remarkably,
the second most frequently encountered compounds
belonged to the class of phenethylamines. The ongoing
COVID-19 pandemic and related response measures
have affected existing synthetic drug markets by
promoting changes in the types of synthetic drugs
seized. Mapping trafficked drugs is essential because
the information obtained from monitoring drug
seizures is important to alert the judicial authorities
and to help the legislative effort to ban NPS and
promote the updating of analytical methods by
forensic laboratories. Acknowledgments: Carlos
Chagas Filho Foundation for Supporting Research
in the State of Rio de Janeiro (FAPERJ) and National
Council for Scientific and Technological Development
(CNPq).
 159

Risk factors associated with deaths by

multiple drugs of abuse intoxications
Messias, Nayara Casagrande1,2; Silva, Stephanie Soares1; Resener, Marisete Canello1,2; Albino, Danielle
Bibas Legat1,2; Barotto, Adriana Mello1,2; Santos, Claudia Regina1,2,3; Marchioni, Camila1,2,3
1
 Federal University of Santa Catarina (UFSC), Brazil; 2 Poison Control Center of Santa Catarina,
Brazil; 3 Department of Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

Introduction: The use of drugs of abuse is a global
risk factor for disability and premature deaths,
accounting for over 585,000 lives lost prematurely
each year. Previous studies estimate that the national
prevalence of illicit drug use is approximately 10%,
and alcohol is, directly or indirectly, the drug most
associated with the risk of death. Although the health
harms of drug of abuse are well researched, studies
are mostly concerned with the effects of specific
substances, with limited attention to the concomitant
use of drugs such as alcohol and cocaine. Estimating
the prevalence and morbimortality associated with
the use of multiple drugs of abuse is important to
quantify the extent and severity of drug use and
to refocus public health interventions. Objective:
Describe the epidemiological profile of users
intoxicated by multiple drugs of abuse associated with
death registered at the Assistance and Toxicological
Information Center of Santa Catarina (CIATox/SC) and
identify the specific drugs most frequently involved in
the deaths. Method: Observational, cross-sectional,
descriptive and retrospective study of a series of
cases registered between 2014 to 2021 at CIATox/SC.
The data were obtained from the attendance records
of cases of intoxication by drugs of abuse with death
outcome related to the event. The following variables
were evaluated: sex, age group, agent, route, type of
exposure, and initial classification. Results: During
the period of the study, 96 cases of death associated
with drug abuse were registered, 44 deaths (45.8%)
occurred by the use of a single type of drug of abuse,
18 deaths (18.75%) were caused by the use of multiple
drugs of abuse, 20 deaths (20.8%) occurred by the
association of drugs of abuse and medicines, 12 deaths
(12.5%) by the combination of drugs of abuse and
pesticides and 2 deaths (2.08%) by the concomitant
use of drugs of abuse, medicines and pesticides. From
the majority of the deaths related with a single type
of drug, cocaine/crack was the main drug of abuse
consumed (86.36%), followed by alcohol (7.89%) and
N-Methyl-3,4-methylenedioxyamphetamine (MDMA)
(7.89%). Among the deaths involving intoxication by
multiple drugs of abuse, the use of cocaine was present
in 16 cases (89%), and the combined use of cocaine
and alcohol was the main association found, being
present in 7 cases (38.8%). The profile of deaths from
multiple drug abuse was predominantly male (66.6%)
in the age between 20-29 years old (55.6%). As for the
circumstance, all 18 cases were classified as substance
abuse and were admitted to the health service with
a severe condition due to acute exposure. The main
route of exposure was nasal and oral, which occurred
in 12 cases (66.6%). Conclusion: In the current study,
cocaine/crack was the drug responsible for most
deaths when consumed isolated or in combination
with other substances. Poly-drug use occurs mainly
by association of cocaine, alcohol, and/or marijuana,
being related with the intention of enhancing the
desired effects and reducing undesirable effects,
such as withdrawal syndrome. Simultaneous use
of cocaine with alcohol produces a new compound,
cocaethylene, which is pharmacologically active
and related to potentially unfavorable outcomes. In
previous studies, the use of multiple drugs of abuse
predominated among younger (<30 years old) male
users. The pattern of drug usage in this study showed
the same tendency. Keywords: illicit drugs; alcohol
intoxication; drug overdose; epidemiology, drug
abuse. Acknowledgments/conflict of interest: We
thank CIATox/SC for providing data. The authors have
no conflict of interest to declare.

Study of NBOMes using MetadrugTM
Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.
NBOMes, also known as “N-Bombs” or “N-Bomb”
consist of substituted class 2C phenethylamine
derivatives. These molecules are already cataloged
by the Convention on Psychotropic Substances
by UNODC, present in Schedule I of 1971. As well
as LSD, it has potent hallucinogenic effects; its
consumption has increased over the years, despite
being a recently discovered substance and having
adverse effects linked to its toxicity. This work
targeted the use of in silico methods for investigating
the biological and toxicological behavior of these
substances. We employed the MetadrugTM software
for calculating seventy-eight variables for 23
NBOMes. We simulated structural properties, CYP450
QSAR Models, Protein binding QSAR Models, ADME
QSAR Models, the predicted therapeutic activity,
and predicted toxic effects. This tool is part of the
MetaBaseTM API to access the knowledge base behind
all systems biology products of Clarivate Analytics
(https://www.cortellislabs.com/page/?api=api-
MB). We used multivariate analysis to evaluate
the behavior of these substances. PCA (Principal
Component Analysis) and HCA (Hierarchical Cluster
Analysis) were used as unsupervised approaches.
We performed Supervised learning through SIMCA
(Soft Independent Modeling of Class Analogy). PCA
and HCA results showed that NBOMes were divided
into four main classes, according to the similarity in
structures. SIMCA confirmed these classes. Different
properties were found for each category, as follows:
Class 1. Molecules: 2C-B, 2C-B-BZP, 2C-C, 2C-E, 2C-
T-2, 2C-T-7, 2C-TFM. We found three variables with
160
and Chemometrics
Mariotto, Lívia Salviano; Bruni, Aline Thais
higher modeling power are related to the prediction
of therapeutic activity: potential activities against
heart failure and Parkinson’s disease. They also
showed antithrombotic activity. Class 2. Molecules:
4-MA-NBOMe, 4-MMA-NBOMe, 25-APB NBOMe. This
class was modeled mainly for variables related to the
prediction of therapeutic activity, showing potential
for antidiabetic, anti-osteoporosis, and antithrombotic
activities. Besides, CYP450 QSAR Models showed
the potential to inhibit CYP1A2 at 10 uM or less. The
potential for inducing epididymis toxicity was also
found as high modeling power. Class 3. Molecules:
25B-NBOMe, 25C-NBOMe, 25H-NBOMe, 25N-NBOMe,
25T-NBOMe, 30C-NBOMe. This class showed high
modeling power for potential activity against arthritis
and heart failure. However, they have the potential
for inducing carcinogenicity in rats and mice. Class
4. Molecules: 25D-NBOMe, 25E-NBOMe, 25G-NBOMe,
25P-NBOMe, 25T2-NBOMe, 25T4-NBOMe, 25T7-
NBOMe. This group presented a potential for inducing
cardiotoxicity. liver lipid accumulation, nephrotoxicity,
epididymis toxicity. In conclusion, we have observed
that even considering the same type of NPS; these
substances presented different activities according to
the structure. Particular attention should be given to
those molecules which showed predicted therapeutic
properties. We thank the Brazilian Agencies Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq, grant 465450/2014-8) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES,
Finance Code 001) for financial support.

Study of synthetic cannabinoids regarding
their metabolites: an in silico approach
Castro, Jade Simões; Rodrigues, Caio Henrique Pinke; Bruni, Aline Thais
Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.
New Psychoactive Substances (NPS) consist of
structurally modified chemical compounds that
intend to mimic the effects of the controlled classic
substances besides circumventing prohibition.
The growth of NPS can cause many challenges in
understanding their behavior regarding detection,
identification, and biological properties1. This work
studies synthetic cannabimimetic (SCB) substances
that aim to mimic the THC (Tetrahydrocannabinol)
chemical structure regarding their metabolites. THC
metabolism is mainly at the hepatic level. It involves
phase I reactions: aliphatic hydroxylation, oxidation of
alcohols to ketones and acids, β-oxidation, degradation
of the pentyl side chain, epoxidation, decarboxylation,
and conjugations. Given the large variety of reported
structures, in silico tools can be an alternative to get
information about the metabolism of SCBs2. SMILES
structures were used to simulate the metabolites
of 44 synthetic cannabinoids, using the iS-Tox®
platform (In Silico Toxicology Platform) developed by
the company Altox®3. The computational tool Pred-
CYP2D was used. It combines artificial intelligence
and is based on knowledge of models to predict the
molecular metabolism network (MMN). We analyzed
the metabolic reactions, as well as the formation rate
and structures of the metabolites predicted. The three
most likely metabolites for 44 cannabinoids were
selected. We calculated information on molecular,
distribution and toxicological properties using the
Cambridge University Pkcsm platform, which results
in 14 (fourteen) variables4. The molecules were
divided into six different classes according to the
structural similarities among the SCB. We evaluated
the influence of the variables on the samples by
multivariate tools. We used Principal Component
Analysis – PCA as an unsupervised approach and
161
Soft Independent Modeling of Class Analogy –
SIMCA to observe classification effectiveness5. PCA
results showed there is some pattern among the
structures. SIMCA evaluation confirmed this pattern
but also showed that metabolites for different
substances can present similar properties. The
Blood-brain Barrier Permeability showed to be the
most discriminant variable for discriminating the
differences in structures. According to the results,
we concluded that in silico tools could generate the
metabolites for synthetic cannabinoids. It can help
indicate experimental determinations. For the studied
variables, our findings showed that they have a kind
of pattern depending on the structures. Still, there is
a need for an in-depth investigation of new variables
using different software. WethankAltox®for the
partnership and access to the platform, theBrazilian
Agencies Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq, grant 465450/2014-8)
and Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES, FinanceCode 001) for financial
support.
REFERENCES
1. United Nations. UNODC World Drug Report 2019. in.
2. United Nations Office on Drugs and Crime. Recommended
Methods for the Identification and Analysis of Cannabis and
Cannabis Products. (2013). doi:10.18356/1e8e4f16-en.
3. ALTOX. IS-TOX PLATFORM An artificial intelligence platform
developed by the Brazilian company Altox will avoid using
animals in tests. https://is-tox.com/publications-and-
news/84-an-artificial-intelligence-platform-developed-by-
the-brazilian-company-altox-will-avoid-using-animals-in-
tests.
4. Pires, D. E. V., Blundell, T. L. & Ascher, D. B. pkCSM: Predicting
small-molecule pharmacokinetic and toxicity properties using
graph-based signatures. J. Med. Chem.58, 4066–4072 (2015).
5. Ferreira, M. QUIMIOMETRIA. Conceitos, métodos e aplicações.
(2015).
 162

Tracking methamphetamine surges

in the Joinville region (Santa
Catarina) from 2014 to 2021
José, Gustavo Pinheiro Coelho1; Pericolo, Suellen1; Parabocz, Gisele Chibinski1;
Costa, Karina Oliveira1; Marchioni, Camila2; Ramos, Silvia Aparecida3
1
 Polícia Científica de Santa Catarina; 2 Universidade Federal de
Santa Catarina; 3 Universidade da Região de Joinville.

Introduction: Methamphetamine is a stimulant was obtained from Sirsaelp (Sistema de Registro

substance widely used as a recreational drug, but
also used medically for the treatment of attention
deficit hyperactivity disorder and obesity. It is
highly addictive and has established itself in mighty
consumer markets like the United States. Surveillance
of methamphetamine trade presented a real challenge
due to easy to make synthetic routes with readily
available over the counter (OTC) reagents which turns
control very difficult. Most recently, UNODC reported
continued growth in the supply of methamphetamine
on East and Southeast Asia. Meanwhile, Afghanistan
which has been a fertile ground for heroin production
appears to have shifted to the methamphetamine
business. This scenario of emerging consumer markets
and new producers shine light onto Brazil, an avid
market for drugs of abuse which has been notoriously
distant from the methamphetamine commerce. The
Brazilian Federal Police reported in 2020 that there
have been few cases of methamphetamine since
2017 but showed worry due to two recent meth labs
that were found that year inside Brazilian territory.
The report also suggests other evidence that points
to domestic production aiming the national market.
One is that states were seizing small quantities of
the drug in plastic bags which indicates it was packed
for use. Also, the Federal Police seized methyl-2-
phenyl acetoacetate, a precursor for phenyl-2-
propanone (methamphetamine precursor). The
Federal Police would not be able to indicate whether
methamphetamine is being sold and used in the
national drug scene, since the Federation states are
responsible for handling the “street cases”. Therefore,
it is relevant that the states study and publish their
data to show new trends in Brazilian drug markets.
Objective: Evaluate methamphetamine seizures in
the Joinville region (Santa Catarina), from 2014 to
2021. Methods: Descriptive and retrospective study
of a series of seizures between 2014-2021 at Polícia
Científica de Santa Catarina (Joinville region). Data
de Solicitações, atendimentos e emissão de Laudos
Periciais) and processed on an Excel® spreadsheet.
Results: During the observed period, there had been
few cases of methamphetamine seizures. There were
4 cases in 2017, including a large seizure of 1108
pills, and 2 cases in 2018. Before (since 2014) or after
these years, methamphetamine had been completely
absent from police seizures in Joinville region.
Then, since june 2021, methamphetamine seizures
have been progressively increasing, totalizing 18
in 2021, an 800% increase in cases. While in 2017
and 2018 pills contained only methamphetamine,
mixtures became very common in 2021. Substances
detected with methamphetamine included
caffeine, 3,4-Methylenedioxyamphetamine (MDA),
3,4-methylenedioxymethamphetamine (MDMA)
and even Alprazolam in one case. Discussion/
Conclusion: The 2021 surge of methamphetamine
cases in Joinville region points to a new trend in the
regional drug market. Although there had been cases
in the past, in 2017 and 2018, these were few; all
seizures involved pills and no other substance was
detected. In 2021, however, there were a sudden rise
of cases involving methamphetamine, most being
seizures of small amounts which possibly indicate
use. Another difference in the recent scenery is that
methamphetamine is being found in different forms,
such as crystal and powders. It is also a novelty that
it is being mixed with other drugs and adulterants.
Particularly, the mixture of methamphetamine with
classical ecstasy drugs like MDMA and MDA has
become common. That is worrying as it exposes
occasional users of ecstasy to a highly addictive drug.
The variation in forms of presentation and mixtures
corroborate with the evidence pointed in the federal
police 2020 report that indicate a market being
established in Brazil. Keywords: Methamphetamine;
recreational drugs; illicit drugs seizures
 163

Use of psychedelics in the treatment of anxiety

and depression disorder - literature review
Silva, Luíza Madureira1; Holanda, Wiron Pimentel2; Honório Júnior, José Eduardo Ribeiro3
1
 Academic of Nursing in the University Center Christus, Laboratory of Neurociência Translacional-
Neurocit; 2 Academic of Biomedicina in the University Center Christus, Laboratory of Neurociência
Translacional-Neurocit; 3 Doctor in Biotechnology for the RENORBIO/UFC. Professor in the University
Center Christus, Coordinator of the Laboratory of Neurociência Translacional-Neurocit.

Introduction: The World Health Organization currently
estimates that about 300 million people suffer from
depression and about a third of the population does
not respond appropriately to at least three different
antidepressants. The available antidepressants
currently possess profile of effectiveness and similar
mechanisms of action, act in the modulation of
monoamino the cerebral ones and lead up to 2 weeks
to start to be efficient. The psychedelic medicines
as psilocibina, dietilamida of acid LSD (LSD) and
ayahuasca, induce modified states of conscience
acting in the receivers 5-HT2A of the brain, beyond
the 3,4-metilenodioximetanfetamina (MDMA) that it
presents effect of “alteration in the mind” by means of
different neurochemicals ways, given the number of
works reporting on the effects of psychedelics as an
aid in the treatment of anxiety and depression, we saw
the need to produce this article. Objectives: To revise
the advances of clinical studies on the effectiveness
of the psicodélicos in the treatment of the depression
and anxiety. Methodology: Descriptive narrative-type
study of the literature, selected articles published in
the last five years (2017-2022), the database used was
PUBMED, with the following descriptors: LSD, anxiety,
data depression, ayahuasca, psilocybin, MDMA. The
inclusion criteria for this study were clinical trials and
controlled clinical trials, in English and Portuguese.
A total of 42 articles were found, 4 of which were
used for this review and those that did not meet the
inclusion criteria were excluded. Results: According
to the study by Bershad et al (2020), the effects of
micro-dose LSD increases amygdala connectivity
with other brain areas correlated with positive mood
after the drug. However, psilocybin, another studied
psychedelic, has positive, lasting and faster effects
compared to existing pharmacological therapies and
with fewer side effects. Palhano-Fontes et al., states
that ayuhasca, when compared to placebo, has a
greater antidepressant effect and a high response to
treatment with only a single dose of the psychedelic
and low adverse effects. MDMA, on the other hand, was
well tolerated in all sessions and brought significant
improvement compared to participants who ingested
the placebo, but when comparing MDMA to other
psychedelic therapies, it has greater side effects
and can last up to 7 days. after use. Conclusion:
It is concluded that psychedelic therapy (PD) can
bring benefits to patients with anxiety disorder
and depression refractory to other unsuccessful
pharmacological therapies. More clinical trials with
larger samples and a diverse population should be
carried out to better assess the effects of PD.
 05 
ENVIRONMENTAL
SAFETY AND HEALTH
 165

Acute toxicity of Diuron, DCA and

DCPMU in the early life and adultohood
of the zebrafish (Danio rerio)
Sales, Bianca Camargo Penteado1,2; Andrade, Ítalo Bertoni Lopes1,2; Peixoto, Paloma
Vitória Lima1,2; Camargo, João Lauro Viana1,2; Pereira, Lilian Cristina2,3
1
 São Paulo State University (Unesp) Medical School, Botucatu; 2 Center for
Evaluation of Environmental Impact on Human Health (TOXICAM); 3 São Paulo
State University (Unesp) School of Agriculture, Botucatu, SP, Brazil.

Introduction: Over the past decades, the zebrafish
(Danio rerio) has emerged as a cost-efficient model
to be used in toxicological assays which may support
risk assessment of environmental contaminants.
Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is
an herbicide commonly used in Brazil, particularly in
sugar cane cultivation. Following application, diuron
is degraded in the environment to some metabolites,
including DCA (3,4-dicloroaniline) and DCPMU
(3-(3,4-dichlorophenyl-1-methylurea). There are
studies with rodents reporting the hazardous potential
of these substances, but few studies have shown
their toxicity to aquatic organisms. However, changes
induced by pollutants in fish are among the most
sensitive indicators of environmental disturbances.
Objective: To assess the potential for inducing
harmful alterations, embryos, larvae and adult male
zebrafish were exposed to a doses’ range of Diuron,
DCA or DCPMU from 0.5 to 100 µM, which included
Brazilian environmentally relevant concentrations.
Methodology: This study was approved by the local
Ethics Committee on the Use of Animals (CEUA,
Certificate No. 1304/2019). Embryos and larvae
were generated at this laboratory; adult zebrafish
were supplied by an external breeder (Powerfish
pisciculture, Itaguaí/RJ, Brasil). Embryo’s evaluation
followed the OECD 236 protocol “Fish Embryo Acute
Toxicity (FET) Test” slightly modified: instead of a 96-
hour post fertilization (hpf) evaluation period, the
study was extended up to 144 hpf. For evaluation of
adult animals, the OECD 203 “Fish Acute Toxicity (FET)
Test” protocol was used. Results: At concentrations of
0.5 – 10 µM Diuron, DCA and DCPMU induced teratogenic
effects in the zebrafish embryos. Exposures above
10µM were letal to embryos, larvae and adult animals.
There were no conspicuous histological alterations in
the brain, heart, spleen, and liver of adult zebrafish
exposed to the different test chemicals for 96 hours.
However, groups exposed to 10 µM of Diuron, DCA or
DCPMU presented mild cytoplasmic vacuolization of
hepatocytes. Discussion/Conclusion: Identification
of adverse outcomes induced by any chemical agent is
a first step for risk evaluation and regulatory decision.
Our results indicate that during the early phases of
their development (embryo and larvae) the zebrafish
is susceptible to the toxic effects of Diuron, DCA or
DCPMU. In the adult zebrafish tissues alterations were
considered not relevant, but the meaning of hepatocyte
vacuolation is under evaluation. The current data also
indicate an age-dependent zebrafish susceptibility to
diuron and its main metabolites. Keywords: Diuron,
DCA, DCPMU, zebrafish, development, histological
parameters. Acknowledgement: Financial support:
FAPESP – Proc. No. 2017/2542-5; TOXICAM.
 166

Blood total mercury levels of preschool

children from Sao Paulo, Brazil,
and associated risk factors
Olympio, Kelly Polido Kaneshiro1; Salles, Fernanda Junqueira1; Pereira, Elizeu Chiodi1;
Oliveira, Allan Santos1; Costa, Eric Augusto Caravaggio2; Costa, Brenda Natasha Souza3;
Pereira, João Paulo Goes3; Jesus, Iracina Maura3; Queiroz, Thais Karolina Lisboa3; Lima,
Marcelo de Oliveira3; Silva, Agnes Soares4; Cardoso, Maria Regina Alves5
1
 Departamento de Saúde Ambiental, Faculdade de Saúde Pública, Universidade de São Paulo, FSP/USP;
2
 Centro de Engenharia, Modelagem e Ciências Sociais Aplicadas, Universidade Federal do ABC, SECS/
UFABC; 3 Seção de Meio Ambiente, Instituto Evandro Chagas, SEAMB/IEC; 4 Communicable Diseases
and Environmental Determinants of Health (CDE), Pan American Health Organization, PAHO/WHO;
5
 Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, FSP/USP.

Background: Environmental exposure to mercury
in preschool children can impact their health by
increasing their blood levels, which can cause negative
health outcomes. However, we do not have reference
values for Brazilian children. Objective: This study
aimed to set the reference value for blood mercury
in preschool children from a megacity in Brazil, and
investigating associated risk factors. Methods: Blood
samples were collected of 2,463 children and the
present study reports preliminary results from 1,020
1-4-year-old children attending 29 daycare centers
(DCC) located in São Paulo, Brazil, 2013. Guardians
answered sociodemographic and potential risk
factors questionnaires. DCC and metal contaminated
sites (MCS) were georeferenced (QGIS™). Blood total
mercury levels (BTM) were determined by Cold Vapor
Atomic Absorption Spectrophotometry (CV-AAS). BTM
was dichotomized (low/high levels) in a cut-off point
of 1.06 μg.L⁻1, which represents the 95th percentile
reference value for total mercury established by the
U.S. Centers for Disease Control and Prevention (CDC).
Summary data and multiple logistic regression were
performed (p<0.05). Results: The geometric mean
(n=1,020) for BTM was 1.71 μg.L⁻1 (95%CI: 1.64-1.78
μg.L⁻1) and the 95th percentile was 5.43 μg.L⁻1 (95%CI:
4.89-6.26 μg.L⁻1). DCCs located in the Northwest
geographic zone were associated with high BTM
when the model was adjusted for fish intake, age, sex,
geographic zone, mother schooling, and amalgam
(p=0.018). No associations were found between high
BTM and fish consumption, amalgam teeth fillings,
and the georeferenced vicinity of the DCC to MCS. We
also concluded BTM analyses for 2,438 children (total
sample) and we will present the complete statistical
analyses for the whole sample at the XXII CBTOX
Congress. For the total sample, the geometric mean
for BTM was 1.87 μg.L⁻1 (95%CI: 1.82-1.92 μg.L⁻1) and the
95th percentile was 5.50 μg.L⁻1 (95%CI: 5.18-5.92 μg.L⁻1),
results very similar to the partial analyses, which
included multiple logistic regression. Conclusions:
BTM in Brazilian preschoolers (95th percentile) was
more than five times higher than U.S. children’s
levels. Even the Brazilian geometric mean was higher
than the U.S. BTM 95th percentile. More studies are
necessary to identify potential mercury exposure
sources for preschool children. Nevertheless, these
results showed the need to formulate public health
policies, intending to better understand and eliminate
mercury sources. Acknowledgments: Funded by
Fapesp (2011/13076-0, 2011/23272-0, 2012/21840-4,
2018/18391-0) and Capes.
 167

Determination of biomarkers of
exposure to pythroid pesticides through
wastewater-based epidemiology
Lizot, Lilian de Lima Feltraco; Bastiani, Marcos Frank; Hahn, Roberta Zilles; Linden, Rafael

Feevale University.

Background: Pesticides are active substances
potentially toxic to humans and their consumption and
production have increased significantly. Therefore,
monitoring exposure to these pesticides is extremely
important for public health. As an alternative
way to assess exposure to pyrethroid pesticides,
wastewater-based epidemiology (WBE) is a tool
capable of providing epidemiological information
through the analysis of wastewater, through the
quantification of endogenous metabolism products.
Commonly the sampling performed for WBE is single
point collection, but to minimize the limitations of
this type of sampling, the passive sampling, with
Polar Organic Chemical Integrative Sampler (POCIS)
is an attractive tool, with better cost-benefit, used to
estimate exposure to the polar compounds of interest.
Objective: Develop and validate analytical strategies
to determine the concentration of biomarkers of
pyrethroid pesticides exposure in wastewater using
POCIS. Methods: The biomarkers of pyrethroid
exposure monitored were 3-PBA, cis, and trans-DCCA.
The POCIS constructed in-house was assembled in a
sandwich format, with 200 mg of Oasis® HLB sorbent
between polyester sulfone membranes. The sorbent
was extracted with methanol, and after the methanol
extract, it underwent solid phase extraction (EFS)
using an ion-exchange cartridge (Oasis® MAX). The
extracts were analyzed in liquid chromatography
coupled to a triple quadrupole mass detector (LC-
MS/MS), as an electrospray ionization source in
negative mode. Collections took place every fortnight
and in triplicate, from FEB/2020 to MAR/2021, at
a wastewater treatment plant in Novo Hamburgo,
which serves about 5 thousand inhabitants. Results/
Discussion: A method for determining biomarkers of
exposure to pyrethroid pesticides has been developed
and is sensitive for detecting the compounds of
interest. In total there were 24 collection cycles. POCIS
proved to be efficient for collecting the compounds
of interest, being possible to detect them in all
collections. The amount of residual sorbent ranged
from 151-195 mg, which represents a maximum of
24.5% loss. The EFS protocol was efficient, allowing
high recoveries, ranging from 95.7-100.4% with
variation between replicates of 1.9-9.6%. The results
obtained in ng g-1 of POCIS for 3-PBA, cis-DCCA, and
trans-DCCA, were in the range of 208.6-2456.9, < Limit
of quantification-331.3 and 57-494,4, respectively.
To define the sampling rate, an experiment that
assesses the decay of the substance was used, and
only for 3-PBA was it possible to determine it. The
sampling rate for 3-PBA was 0.096 L day-1, so the
values found in sewage in ng L-1 ranged from 24.3
to 335.2. Conclusion: In all cycles, it was possible to
detect the presence of the analytes of interest, thus
showing the sensitivity of the method developed, as
well as proving the efficiency of the POCIS sampling
device, assembled in-house. The fact that we were
only able to define the sample rate for 3-PBA does
not represent a limitation, as this marker represents
at least 20 different pyrethroids. To our knowledge,
there is no other study that determines biomarkers
of exposure to pyrethroids using POCIS sampling.
Acknowledments: Feevale University, CAPES
 168

Embryonic exposure to genistein

induces persistent anxiolytic and
antisocial behaviors in zebrafish
Freddo, Natália1; Menegasso, Aloma Santin1; Soares, Suelen Mendonça2; Fortuna, Milena2; Maffi, Victoria
Costa3; Mozzato, Mateus Timbola3; Barcellos, Leonardo José Gil1,2,3; Rossato-Grando, Luciana Grazziotin1
1
 Programa de Pós-graduação em Bioexperimentação, Universidade de Passo Fundo,
Passo Fundo, Rio Grande do Sul, Brazil; 2 Programa de Pós-Graduação em Farmacologia,
Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil; 3 Curso de
Medicina Veterinária, Universidade de Passo Fundo, Passo Fundo, Rio Grande do Sul, Brazil.

Background/Introduction: Genistein is a (CEUA) of the University of Passo Fundo, UPF, Passo

phytoestrogen, which is structurally similar to
17β-estradiol. It is present in plants, food, and as a
contaminant in effluents. Danio rerio fish, known
as zebrafish, are small vertebrate, teleost fish of
freshwater. They were chosen as a model organism
in this work due to their rapid development, ease
of maintenance, availability of acquisition and
applicability. Objective: to demonstrate the effects of
embryonic exposure to three different concentrations
of genistein (10 μg/L, 40 μg/L, and 80 μg/L) using
the zebrafish model. Methods: Zebrafish eggs were
exposed to genistein (10 μg/L, 40 μg/L, and 80 μg/L)
during the first 72 h post fertilization (hpf). Heart
rate was evaluated at 48 hpf and mortality rate was
assessed during the first 72 hpf. The Light/Dark (LDT)
and Open Field (OFT) behavioral tests were applied
to the larvae (6 dpf), and the Novel Tank (NTT), Social
Preference (SPT), Light/dark (LDT), and sexing tests
were performed on adult fish (90 dpf). This study
was approved by the Animal Use Ethics Committee
Fundo, RS, Brazil (protocol n°: 041/2019). Results:
Embryonic exposure to genistein causes anxiolytic-
like behavior in larvae. These effects are persistent,
since were also observed when fish reach adulthood.
Adult fish also presented hypermotility and antisocial
behavior in the concentration of 40 μg/L. There was
an increase in the mortality rate in all concentrations
when compared to the control and an increase in
heart rate at the concentration of 80 μg/L. Exposure
to 10 μg/L generated a higher frequency of females
when compared to the control group. Discussion/
Conclusion: Our results show that exposure to
genistein during the embryonic phase provoke
damage in the short and long term as it increases the
mortality rate and leads to behavioral disorders both
in the larval stage, with persistence until adult stage.
Acknowledgements: This study was supported
by Postgraduate Support Program for Community
Higher Education Institutions - PROSUC, attached to
Ordinance No. 149, of August 1, 2017.
 169

Energetic metabolism and antioxidant

system modulation after fish chronic
exposure to glyphosate and imidacloprid
Sobjak, Thaís Maylin1; Zazula, Matheus Felipe2; Macarini, Leanna Camila3;
Rizzo, Elizete1; Guimarães, Ana Tereza Bittencourt⁴
1
 Departmento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de
Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; 2 Departamento de Biologia Celular,
Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, Brazil; 3 Programa
de Pós Graduação em Conservação e Manejo de Recursos Naturais, Universidade Estadual
do Oeste do Paraná, Cascavel, Paraná, Brazil; ⁴ Programa de Pós Graduação em Biociências
e Saúde, Universidade Estadual do Oeste do Paraná, Cascavel, Paraná, Brazil .

Population growth demands food production
increase, which has resulted in an exponential
increase in the world’s consumption of pesticides.
Among the most used pesticides worldwide are
glyphosate and imidacloprid. Assessments of the
sublethal effects resulting from exposure to these
pesticides are generally carried out in experiments
with animal models, and the results are often
used in extrapolations relating to human health.
Poecilia reticulata is an easy-to-handle fish, whose
characteristics favor research on such themes. Thus,
the aim of this work was to evaluate changes in
energetic metabolism (EM) and antioxidant system
(AS) of P. reticulata after chronic exposure to these
pesticides. Twelve 10 L aquariums containing 10 fish
each were divided into groups: Control (dechlorinated
water) and three groups, each treated with Glyphosate
(G), Imidacloprid (I), and Glyphosate+Imidacloprid
(GI). The animals were kept at a temperature of
25±2°C, constant aeration, and a photoperiod of
12h/12h. We exposed these animals for 6 months
(September 2020 to February 2021). The muscle from
8 animals in each group was collected and prepared
for enzymatic analyses (Hexokinase-HK, Malate
Dehydrogenase-MDH, Aspartate Transaminase- AST,
Creatine kinase-CK.MB, Pyruvate Kinase-PK, Lactate
Dehydrogenase-LDH, Citrate Synthase-CS) and AS
(Glutathione peroxidase-GPx, Glutathione reductase-
GR, Glutathione transferase-GST, Thiols). The variable
matrices were analyzed using principal component
analysis (PCA), and the factor loadings of the first
dimension of both matrices were related through
linear models for each experimental group. In all
statistical tests, the significance level used was 0.05.
In relation to the control group, homeostasis of the
antioxidant system and balance of energy metabolism
and ATP synthesis were observed. The group exposed
to imidacloprid showed a greater imbalance in
antioxidant system, and greater consumption of
ATP. The group exposed to glyphosate also showed
a greater imbalance in its antioxidant system but
resulting in a lower production of ATP. In the GI group
we observed a marked production of ATP, indicating
a metabolic alteration with increased activity of LDH,
CS, and MDH. Some studies have reported neurological
and behavioral effects in fish after exposure to
pesticides. Imidacloprid interacts with nicotinic ACh
receptors, preventing ACh binding, causing their
constant stimulation, and constant consumption
of ATP (Almeida et al., 2021). In addition to the high
demand for ATP, studies have shown that imidacloprid
can accumulate in muscle tissues (Frew et al., 2018).
In natural conditions, the change in cholinesterase
activity and constant muscle contraction lead the
fish to fatigue, making them vulnerable to predation
and, therefore, with less behavioral ability. As for the
group exposed to glyphosate, this xenobiotic binds
to membrane receptors, which activate messengers
that promote the release of Ca++ by the endoplasmic
reticulum, thus altering mitochondrial activity
(Cavalli et al., 2013). Carrée et al. (2021) showed that
rainbow trout exposed to glyphosate had significant
increase in the LDH, and this result may indicate a
need to produce more energy via anabolism rather
than aerobic metabolism. Finally, the analysis of the
energy metabolism and antioxidant system of fish
exposed to sublethal doses of pesticides alone or in
synergism showed a reduction in antioxidant capacity
and alteration in ATP synthesis, thus promoting
greater vulnerability of the animal to predation
events and decrease of survival potential. In this
way, we emphasize the importance of pesticide risk
analysis, environmental impact assessment, and,
mainly, an inspection of the misuse of such products.
Keywords: guppy; xenobiotics; iseticide; herbicide.
Acknowledgments: CNPq and FAPEMIG.

Ethnic-racial disparity in exogenous
intoxications in Brazil
Moraes, Niely Galeão da Rosa; Silva Júnior, Flavio Manoel Rodrigues
Introduction: Racism is a structuring system capable
of promoting avoidable and unjust disparities
between social groups, based on ethnicity or race.
In Brazil, ethnic-racial inequalities exist in virtually
all fields, obstructing access to goods, services and
opportunities, including health services. Studies
in other countries report ethnic-racial disparity
in drug poisoning and drugs of abuse, but studies
evaluating other toxic agents (such as pesticides)
are lacking. Objective: The present study aimed to
examine the relationship between race/ethnicity
and general exogenous intoxications, intoxication
by medicaments, by pesticides and by drugs of
abuse in different Brazilian states. Methodology:
An ecological study was carried out based on the
data available in the Notifiable Diseases Information
System (SINAN) and population data from the IBGE, for
the year 2017. Data on the notification of exogenous
intoxications in general and by toxic agents were
collected: medicaments, pesticides (for agricultural
use, domestic use, public health, rodenticides and
veterinary products) and drugs of abuse, in all
Brazilian states. Data were divided into: white and
non-white people (black, brown and indigenous), in
addition to ignored skin color. The percentages of
notifications with unknown skin color were recorded
and the ratio between the percentage of intoxications
of whites and non-whites and the population
percentage of each of the two groups was calculated.
Values above 1 for the intoxication/population ratio
among non-whites indicated racial-ethnic disparity.
Results: A high percentage of unknown race/skin
color was found in Amazonas, Federal District and
in states in the Northeast and Southeast regions.
Regarding the ethnic-racial disparity measured by the
170
Universidade Federal do Rio Grande - FURG.
ratio between the percentage of general intoxications
and population data among non-whites, there was
greater disparity in the states of Pernambuco (ratio
equal to 1.5), followed by Ceará, Rio Grande do Norte
and Paraíba (all with ratio equal to 1.4). In addition,
the type of intoxication that presented the highest
data was related to drugs of abuse, with a disparity
of 1.6 in the Federal District and Paraíba. In general,
in Brazil, racial disparities due to medications and
pesticides are almost non-existent. Conclusion and
Discussion: Racial inequality is a consequence of a
political and historical process, varying according
to the construction of society. This whole process
made racism persist and structure itself at different
levels. This condition affects blacks, brown people
and indigenous in a critical way and makes them
more likely to suffer from health problems, including
exogenous intoxications. This inequality imposes
worse living conditions, lower wages, low education
and less access to knowledge, increasing vulnerability
to intoxication scenarios. Among the toxic agents
investigated, drugs of abuse have the most expressive
rates of ethnic-racial disparity. In the country, non-
white families are known to be more vulnerable and
with people more subject to drug abuse. Regarding
the spatial distribution of ethnic-racial disparity,
the states of the North and Northeast regions stood
out with the highest rates. In these regions, there is
a higher percentage of non-white population when
compared to the states of the South and Southeast.
Finally, it should be noted that this type of study lacks
completely accurate data due to underreporting and
poor completion of notification forms. This scenario is
partially revealed in the study by the high percentage
of unreported skin color in several states.
 171

Evaluating the levels of toxic elements and

toxicity testing in water samples of Paraopeba
River after the Brumadinho dam rupture
Devóz, Paula Pícoli; Rocha, Cecília Cristina de Souza; Barbosa Jr, Fernando

University of Sao Paulo – Faculty of Pharmaceutical Science

of Ribeirao Preto, Ribeirão Preto - SP, Brazil.

Background/Introduction: Mining is one of the most
traditional economic activities carried out in Brazil.
The state of Minas Gerais (MG) extracts more than 160
million tons of iron ore per year. Vale is the world’s
largest company producer of iron minerals, with 133
dams in the country (90 of tailings) whereby 80%
are in Minas Gerais. On January 25, 2019, a rupture
in one of the ore tailings dams (B1), belonging to
Córrego do Feijão Mine in Brumadinho - MG, resulted
in 270 deaths. The mud reached the company’s areas,
neighboring communities, and a drainage channel of
the Ferro-Carvão stream, affecting the Paraopeba
River, responsible for water supply to several Minas
State municipalities. Objective: This study aimed
to evaluate the concentration of toxic elements in
several water samples collected from the Paraopeba
River in Brumadinho – MG a few days after the Dam
failure. Moreover, a toxicity test was performed
on root cells of Allium cepa in order to evaluate
water quality. Methods: Altogether, 136 samples
from seventeen areas were collected in the present
study. pH, temperature, electrical conductivity,
and dissolved oxygen were measured in loco using
a meter containing multiparameter probes. The
determination of chemical elements in the samples
was carried out using an inductively coupled plasma
mass spectrometer (ICP-MS). Results: All parameters
evaluated in loco showed a significant variation
according to the location of selected collection points.
Concentrations up to 1,000 times the maximum value
allowed by the CONAMA Resolution nº 357 were found
in some collection points for the toxic elements: Iron
(Fe), arsenic (As), nickel (Ni), aluminum (Al), and lead
(Pb). Discussion/Conclusion: The present study
provides evidence that it is essential to monitor
the water quality indices of the Paraopeba River to
avoid human and animal exposure to toxic elements
and impacts on the environment. It is also crucial
to promote conditions to guarantee contamination
reduction and the environmental recovery of the
affected area. Acknowledgments: CAPES, FAPESP,
CNPq.
 172

Evaluation of the effect of a nutrient solution

on the phytoremediation potential of a blue dye
by the aquatic macrophyte Salvinia auriculata
Schmitt, Maria Laura Videiro1; Carriço, Murilo Ricardo Sigal1; Nogueira, Caroline Lacerda1; Rodrigues,
Marina Diaz2; Molina, Higor Severo2; Denardin, Elton Luis Gasparotto2; Roehrs, Rafael3
1
 Student of the Postgraduate Program in Biochemistry, Federal University of
Pampa, Campus Uruguaiana; 2 Pharmacy Student, Federal University of Pampa,
Campus Uruguaiana; 3 Professor, Federal University of Pampa.

Background/Introduction: Dye compounds have been
used for many years in various industrial segments,
mainly in the field of textile products. Their excessive
use has been causing more and more damage to the
environment, such as preventing the passage of solar
radiation affecting living beings that inhabit aquatic
ecosystems. One option to reduce the impact of these
pollutants is phytoremediation, which consists of
a process that uses plants to purify contaminated
environments, preventing the pollutant from
dispersing further. Some phytoremediation plants are
already known, the species Salvinia Auriculata is one
of them. In agriculture, it is common to use nutrient
solutions to help the environment reproduction.
Objective: The objective of this study is to prove the
effect of a nutrient solution on the phytoremediation
potential of the macrophyte Salvinia Auriculata.
Methods: For this, a calibration curve of the dye was
performed in the Spectra Max M5 equipment, at the
wavelength of the blue dye, 570 nm, the calibration
curve was made with 6 different concentrations,
all in mg/l: 50, 100, 150, 250, 350 and 400. With the
absorbance reading at these concentrations, we
obtained the equation of the straight line y = 0.0005x
+ 0.0031 with r2 of 0.998. Experiment treatments
were performed with a solution of water and dye
at a concentration of 350 mg/l. A nutrient solution
adapted from Hoagland-Arnon was prepared for 100
mL, in the composition (weighed on an analytical
balance): calcium nitrate (4.609 g), potassium nitrate
(1.998 g), potassium phosphate (1.324 g), magnesium
sulfate (2.719 g), sodium chloride (0.788 g), boric acid
(0.116 g), manganese chloride (0.136 g), molybdic acid
(0.130 g) and Iron EDTA (1.850 g). The experiment was
divided into two groups and placed in a transparent
plastic container: group A, containing 100 ml of water-
dye solution and plant and group B, containing 100
ml of water-dye solution, plant and 1 ml of nutrient
solution. Each group was done in quadruplicate.
Collections were performed at 0, 2, 4, 8, 16, 24, 48,
72, 96 and 120 hours and the samples were stored
in the refrigerator until the absorbance reading was
taken. Results: At hour 4, the dye concentration in
the solution had dropped to approximately 79% in
both groups; from then on, it was noted that the
concentration of group B began to decline faster
than that of group A, reaching 26% at hour 48, while
group A, at this same hour, had a dye concentration
rate of 41%. In the last collection of the experiment,
group A presented 35% of the initial concentration
of dye, while group B, which contained the nutrient
solution, presented only 19% of initial concentration.
Discussion/Conclusion: It is therefore concluded that
the best remediation strategy for dye is to combine
phytoremediation techniques by the macrophyte
Salvinia Auriculata with the use of nutrient solutions,
such as Hoagland-Arnon. Acknowledgments: Capes,
CNPq, Federal Universitty of Pampa.
 173

New approaches to periphytic diatoms

as bioindicators of contamination by
iron ore tailings from the collapse of the
Fundão dam in the Doce River (Brazil)
Souza, Luiz Claudio Cindra1; Reis, Luciane Ayres Castro2; Rangel,
Lara Luiza Pimenta3; Barroso, Gilberto Fonseca4
1
 Federal University of Espírito Santo; 2 Federal University of Espírito Santo; 3 Federal
University of Espírito Santo; 4 Federal University of Espírito Santo.

Introduction: The contamination of aquatic
ecosystems by mining activities can cause numerous
human and ecosystem health. Periphytic diatoms have
been used to monitor environmental disturbances
associated with anthropogenic activities. Currently,
environmental monitoring tools require complex
and laborious metrics for ecological assessment
and management. Diatoms are increasingly being
evaluated using new non-traditional metrics of
low-cost labor and primary taxonomic expertise.
These new non-taxonomic approaches complement
traditional metrics, making environmental
assessments more sensitive, especially in cases of
metal contamination. Objective: The present study
assesses the response and resilience of periphytic
diatoms’ cell size (cellular biovolume) to exposure
to iron ore tailings. Methodology: The bioassay was
conducted in isolated recirculating systems with
replicates of control and treatments. The experiment
was conducted in experimental rivers (ERs) colonized
by periphyton from the lower Doce Doce River Valley
(ES, Brazil). The periphytic community of the ERs was
exposed to iron ore tailings from the Fundão dam,
with initial turbidity of 1,200 NTU and a progressive
reduction to less than 10 NTU. Changes in diatom cell
size were evaluated after 3 and 7 days of exposure
to mining tailings, corresponding to short and long-
term exposure. Results: There was a reduction in the
average size of algae in both experiments. Compared
to the control experiment, the initial phase of the
treatment showed an increase in cell size variability
and a sharp reduction after a short period of time.
Initially, exposure to tailings caused, on average, an
increase in the proportion of thicker cells and later,
a more accentuated reduction in the proportion
of narrower algae. Discussion and Conclusion:
The subsequent reduction in variability in cell size
modifies the composition of the diatom community,
leading to the decline in species richness. These short-
term responses to the disturbance can be related
to the increasing mortality rates of sensitive algae
to environmental stress. Combining these metrics
reveals a high potential for evaluating management
options in fluvial ecosystems. Combining the main
elements of sensitivity of periphytic diatoms to
environmental disturbances with the capacity to
control key environmental variables in ERs bioassays
allows for simulating ecological impacts and
providing an early warning system for environmental
management.

Periphytic community in experimental rivers
as a tool for the assessment of anthropogenic
stressors in fluvial ecosystems
Reis, Luciane Ayres Castro; Almeida, Stefano Zorzal; Barroso, Gilberto Fonseca
Introduction: The health of aquatic ecosystems is
one of the main concerns of this century. However,
developing and refining tools to assess contamination
and changes in aquatic ecosystems is a challenge.
For cause-effect assessments of contaminants and
biological communities, especially in highly dynamic
ecosystems, such as a river, enclosed mesocosms can
become the only reliable way to conduct investigations.
Experimental approaches are also valuable for assessing
environmental disasters associated with tailings spills,
providing essential guidelines to prevent or, at least,
mitigate environmental impacts. As a result, the use of
bioassays has advanced rapidly since 90’s, with many
well-documented applications. For running waters,
Experimental Rivers (ERs) stand out for providing a
better understanding of the factors of interest through
controlling key variables, offering certain realism, and
integrating ecological responses of different biological
groups. The community level has been considered the
most effective for ecotoxicological responses. In this
sense, the use of periphyton stands out as a micro-
ecosystem with critical ecological functions, among
which it is the primary production in fluvial ecosystems.
Combined with the short life cycle, easy development
of bioassays, sensitivity response for environmental
contaminants, and requiring little space, the approach of
the Periphytic Bioindication in ERs (PBERs) is promising.
Nevertheless, the main obstacle is the lack of procedural
review and methodological protocols that promote its
effectiveness. Objective: To systematize knowledge of
ecotoxicological responses of periphyton in ERs and to
propose key guidelines for PBERs. Methodology: The
study was carried out in two stages: 1) a systematic
literature review using an indexed database, Clarivate
Web of ScienceTM, with pre-selected keywords
associated with bioassays of PBERs, such as types of
resources used (i.e., water, periphyton, sediment, light),
and the community, population, and ecophysiological
parameters analyzed; 2) organize a database and
select key guidelines for the use of PBERs. Results and
174
Universidade Federal do Espírito Santo - UFES.
Discussion: The survey yielded 116 scientific papers
addressing ecotoxicological bioassays with PBERs.
Although various experimental designs and metrics
analyzed the capacity to simulate a certain degree of
realism, even in indoor experiments, the main advantage
is the realism simulation and control. Realism is the
experiment setup mimicking the structure and dynamics
of fluvial ecosystems. On the other hand, control refers
to the ability to relate cause and effect, to manipulate,
replicate, and repeat experiments with reliability. In
general, ERs are primarily developed outdoors, limiting
control variables. However, in PBERs, most (58%) of the
bioassays are indoors, which despite the complexity of
the periphytic community, the dimensions of ERs required
for its development are small, and the simulation of the
main variables limiting the periphyton development are
relatively simple. As for the experimental design, 74%
of the PBREs case studies were designed with water
recirculation, with sufficient renewal to maintain the
nutrients in the water (22%), or adding culture media
(30%). In general, 110 anthropogenic stressors were
investigated in the ecotoxicological assessment, with
pesticides and fungicides being the most common (33%),
followed by metals (18%). Most experiments (84%)
were planned with treatment replicates with different
contaminant concentrations as independent systems,
although multiple stressors approach has also been
investigated. The exposure of periphytic communities
(over 7 days of colonization) ranged from 16 to 30 days.
Very few studies (6%) investigated the resilience after
the disturbance. In this review, 117 different periphyton
metrics were found, grouped in terms of functional,
structural, molecular, and physiological processes of
the entire community structure of its biological groups,
algae, protozoa, fungi, bacteria. Microalgae was the
most studied group (85%). Conclusions: The PBERs
bioassays review indicated a very diverse approach for
ecotoxicological assessments. Guidelines and protocols
for bioassays are needed to provide more concise and
standardized results for environmental regulation.
 175

Urinary lead levels and factors associated with

their increase in schoolchildren living in a coal
mining area in the extreme south of Brazil
Brum, Rodrigo de Lima1,2; Baisch, Ana Luíza Muccillo1,2; Silva Júnior, Flavio Manoel Rodrigues1,2

 Laboratório de Ensaios Farmacológicos e Toxicológicos, Instituto de Ciências Biológicas,

1

Universidade Federal do Rio Grande, Brazil. 2 Programa de Pós-graduação em Ciências da

Saúde, Faculdade de Medicina, Universidade Federal do Rio Grande, Rio Grande, Brazil.

Background: Mining areas can result in a series of
damages to the environment and also to human
health. People who work in these areas, as well
as the population that lives close to these places,
are subject to pollution from these activities. Coal
mining and processing can trigger pollution by
different potentially harmful elements to the exposed
population. Among these elements, lead (Pb) stands
out due to its high toxicity, especially for children.
Objective: Therefore, the aim of this study was to
evaluate urinary Pb levels in schoolchildren living
near a coal mining region. Methods: A cross-sectional
study was conducted with 92 school-age children
residing in 7 municipalities in southern Brazil. The
municipalities evaluated in this study were Aceguá,
Bagé, Herval, Hulha Negra, Pedras Altas, Pinheiro
Machado and Candiota, with two coal mines and
two coal-fired thermoelectric plants located in the
latter. Demographic and socioeconomic data were
collected using a semi-structured questionnaire.
For the determination of urinary Pb levels, atomic
absorption spectrophotometry in a graphite furnace
(AAS-GF) was used and they were normalized from
the creatinine concentration. A spatialization map
was prepared to identify the sites that contained
the samples with the highest urinary Pb levels. To
assess the factors associated with urinary Pb levels
was used Poisson regression with a robust estimator
and based on the model theoretical. Results: Urinary
Pb levels in the study population ranged between
~0 and 21.6 µg.g-1-creatinine and presented 50% and
95% percentiles of 3.50 and 11.13 µg.g-1-creatinine,
respectively. The highest urinary Pb levels were
found in samples from children living in Candiota
and in areas closer to potentially polluting sources.
In the Poisson regression, it was observed that some
conditions such as mothers with less schooling, non-
white, living in Candiota, fathers with occupational
exposure and underweight children were associated
with high urinary Pb levels (p < 0.01). Discussion:
Urinary Pb levels identified in Candiota are above
the reference values available from the Centers for
Disease Control and Prevention for the unexposed
population. While in our study the 95% percentile was
11.13 µg.g-1-creatinine, the CDC value for the population
in the same age group was 1.14 µg.g-1-creatinine. Our
spatialization map showed that the samples with the
highest urinary Pb levels are located in Candiota, as
can be seen in other studies that evaluate the exposure
of metals in mining areas. In addition, studies carried
out in this region have already shown that Pb levels
in soil samples are low/moderate, but increase as
the samples approach the pollution sources and it
was also seen that the Pb concentration in surface
water samples was above limits recommended by
Brazilian legislation. In the literature, it has already
been seen that the concentration of Pb in the urine of
children can reach 3.95 µg.g-1-creatinine in areas with
recognized pollution from the activities of coal-fired
thermoelectric plants. In addition, as in our study,
it has already been observed that socioeconomic
factors may be associated with high levels of Pb,
such as age, parents who smoke, parents’ education,
drinking water, family income and housing conditions.
Conclusion: Therefore, we conclude that coal mining
areas can have a great impact on high urinary Pb
levels in children living close to these areas and that
these levels can also be associated with mother’s
educational level and skin color, father’s occupational
exposure, and child’s weight. Acknowledgments:
We thank the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior - Brasil (CAPES) for funding
our research.
 176

Water quality assessment of the Rio

do Peixe: a preliminary study
Gemelli, Bianca Aparecida Martins1; Summy, Maria Julia1; Cesaro, Humberto Luis1;
Alves, Rômulo Couto1; Saraiva, Illyushin Zaak1; Remor, Aline Pertille2; Baú, Morgana2;
Lima, Daina3; Almeida, Eduardo Alves4; Müller, Gabrielle do Amaral e Silva1
1
Instituto Federal Catarinense - IFC; 2 Universidade do Oeste de Santa Catarina-UNOESC;
3
 Universidade Federal de Santa Catarina – UFSC; 4 Universidade Regional de Blumenau- FURB.

Introduction: Manganese (Mn) is an essential metal
used for metallurgical industries purpose. Despite
its importance, Mn is considered a potential source
of contamination, which includes damage to human
health, and compromise of aquatic ecosystem. In
this context, Luzerna is the capital of metalworking
in middle west of Santa Catarina State - Brazil in
which 35.3% of workers have employment in the
metalworking sector. Although, there is a lack of
information of occupational or environmental metal
exposure in the region. Objectives: Then, the purpose
of this work was to evaluate the environmental
impacts of metals and water quality from the main
river of the city, named Rio do Peixe, which is used for
water supply to several cities around. Methodology:
To determine water quality physical-chemical and
bacteriological parameters, water samples were
analysed in duplicate from two sites of the river (non-
contaminated site and contaminated site) using in
situ instrumentals. Results: Water parameters in
reference site showed as fallows: pH = 7.67 ± 0.04;
temperature (ºC) = 27.0 ± 0,14; dissolved oxygen
(mg/l) = 7.85 ± 0.07; electrical conductivity (μS/
cm) = 106.5 ± 10,61 and refractive index = 0.0 ± 0.0.
In exposure site the values were: pH = 7.36 ± 0.02;
temperature (ºC) = 26.9 ± 0.01; dissolved oxygen
(mg/l) = 7.90 ± 0.28; electrical conductivity (μS/cm)
= 103 ± 4.24 and refractive index=0.0 ± 0.0. Total and
fecal coliform bacteria were found in both sites. Metal
analysis are being conducted by atomic absorption
spectrometry. Conclusion: Although our results are
still preliminary and only include duplicate samples
collected, it highlight the importance of monitoring
the environmental, since the results found were so far
similar between two sites of the river. In addition, in
order to analyse the environmental impact of metals
and occupational exposure, we are evaluating the
concentration of manganese metal as a biomarker of
occupational metalworking in the same region, using
nails and hair from 60 participants (30 controls and
30 exposure group) as sample. These future results
will make it possible to implement appropriate
prevention and control measures to avoid the harmful
consequences arising from the bioaccumulation of
metals, mainly, the compromise of the respiratory
and neurological system resulting from exposure
to manganese. Acknowledgments: This research
was made possible in part by a grant from Federal
Catarinense Institute– IFC/Luzerna, FURB, UNOESC
and in part by CAPES scholarship.
 06 
EXPERIMENTAL
TOXICOLOGY
 178

Acute toxicity of 2,4-dichlorophenoxyacetic acid

in the early life of the zebrafish (Danio rerio)
Viriato, Cristina1,2; Andrade, Ítalo B.L.2,3; Peixoto, Paloma V.L.2,3; Pereira, Lílian C.2,4

 São Paulo State University (Unesp), Institute of Biosciences, Botucatu; 2 Center for Evaluation
1

of Environmental Impact on Human Health (TOXICAM); 3 São Paulo State University (Unesp),
Medical School, Botucatu; 4 São Paulo State University (Unesp), School of Agriculture, Botucatu.

Introduction: The 2,4-dichlorophenoxyacetic acid
(2,4-D) is one of the components of the herbicide
2,4-D (DMA® 806) considered one of the most used
herbicides in the world due to its low cost and good
performance. This pesticide is an eye irritant and has
been classified as extremely toxic to humans. In 2018,
the International Agency for Research on Cancer (IARC)
classified this herbicide as possibly carcinogenic to
humans. Also, it has been classified as dangerous to
the environment and slightly toxic to fish and aquatic
invertebrates. However, in 2019, 2,4-D was classified
as a low toxic product (class IV) by Global Harmonized
System (GHS). Objective: In this context, the
objective of this study was to elucidate the potential
teratogenic and understand the toxic effects induced
by the active ingredient, 2,4-dichlorophenoxyacetic
acid, using embryonic stage of zebrafish (Danio rerio)
as a model organism. Methods: Embryo’s evaluation
followed Fish Embryo Toxicity (FET) Test n. 236 (OECD,
2013). After the fish reproduction, 140 fertilized eggs
in the blastula stage were collected and placed in
24-well culture plates. Assays were performed in
triplicate, and each replicate consisted of 24 embryos
for the negative control group (ISO-7346 standard
water), 20 embryos for the positive control group (4
mg/L 3,4-dichloroaniline) and 20 embryos for each
concentration of 2,4-dichlorophenoxyacetic acid: 0.1;
0.4; 1; 10; 100 and 200 µM. These concentrations are
realistic since that have been already found in the
environment. The culture plates were incubated at 26
± 1 °C, in cycles of 14 hours light and 10 hours dark and
the development of zebrafish embryos and larvae were
evaluated at 8, 24, 48, 72, 96, 120 and 144 hpf (hours
post fertilization) through of a stereomicroscope
(Leica M205FA). Mortality, yolk sac edema, swim
bladder inflation defects, pericardial edema and
caudal fin deformation were analyzed. The values
were submitted to the analysis of variance test (One-
way ANOVA) and a posteriori Dunnet’s test. Results:
After 144 hpf of exposure, the negative and positive
controls resulted in compliance with the test (mortality
≤ 10% and mortality ≥ 30%, respectively). Regarding to
mortality, there were significant (p ≤ 0.05) only in the
concentrations 100 and 200 µM (144 hpf), with 100%
embryo mortality. There were not differences about
Yolk Sac Edema, Pericardial Edema, Non-Inflated
Swim Bladder and Caudal Fin Deformity, between the
treatments, resulting in a maximum value of 20%
affected embryos in each deformity classification.
Discussion and Conclusion: We can conclude that
the active ingredient 2,4-dichlorophenoxyacetic acid,
one of the components of the herbicide 2,4-D (DMA®
806) can cause more toxic effects than teratogenic
effects in zebrafish embryos. Acknowledgments:
Acknowledgments to Coordination for the
Improvement of Higher Education Personnel - (CAPES)
and TOXICAM.
 179

Alterations in the activity of antioxidant

enzymes and NA+K+-ATPase in lead poisoning
in the cerebral cortex and hippocampus of rats
Ferrazza-Heitich, Magda1; Lima, Daniela Delwing1,2; Borgmann, Gabriela1; Plautz, Katherine1
1
 Programa de Pós Graduação em Saúde e Meio Ambiente; 2 Departamento
de Medicina - UNIVILLE, Joinville, SC, Brasil.

Introduction: Lead (Pb) poisoning is a public health
problem, as it presents high toxicity to the body,
acting on several biochemical targets, and the central
nervous system is especially sensitive to damage
caused by this metal. Some of the most important
sources of environmental lead contamination include
mining, smelting and, in some countries, the use
of leaded paint, and leaded fuels. Oxidative stress
(EO) is considered a possible molecular mechanism
involved in Pb neurotoxicity. Objectives: Considering
the vulnerability of brain structures to Pb, this study
evaluate the effects of chronic Pb administration
(16, 64 and 128 mg/kg) on the activity of antioxidant
enzymes and on Na+K+-ATPase activity (128 mg/kg)
in rat cerebral cortex and hippocampus. Methods:
60-day-old male Wistar rats received the following
treatment via gavage for 35 days, separated in four
groups: Group I: Saline Solution; group II: Pb 16 mg/
Kg; group III: 64 mg/Kg and group IV: 128 mg/Kg. Rats
were sacrificed by decapitation, without anesthesia,
the brain was removed and the hippocampus and
cerebral cortex were homogenized in 10 volumes
of appropriate buffer. The following antioxidant
enzymes were measured: catalase (CAT), glutathione
peroxidase (GSH-Px) and superoxide dismutase (SOD),
and the activity of Na+K+ATPase, data were analyzed
by one-way ANOVA followed by Duncan’s test when
the F test was significant (p<0.05). Results: Chronic
administration of lead acetate (128mg/kg) decreased
SOD activity in the cerebral cortex [p<0.05]; and, at
doses of 64mg/Kg and 128mg/Kg, decreased SOD
activity in the hippocampus [p<0.001] of rats. With
regard to CAT activity, chronic administration of lead
acetate (128mg/Kg), in the cerebral cortex [p<0.01]
increased this enzyme’s activity, as compared
to the control group. Regarding GSH-Px activity,
chronic administration of lead acetate decreased
this enzyme’s activity at a dose of 128mg/kg in the
cerebral cortex [p<0.01]. However, CAT and GSH-Px
activities in the hippocampus were not altered by
chronic administration of Pb. With regard to Na+K+-
ATPase activity, chronic administration of lead acetate
(128mg/Kg) decreased its activity in the hippocampus.
Discussion: The brain structures chosen for this
study derive from the importance of their functions.
Hippocampus is fundamental to process emotions
and information associated with memory. The
cerebral cortex has an overlap of functions related to
several processes such as motor behavior, receiving
information, coordinating complex movements,
multisensory information processing and language
comprehension. Results indicated that Pb causes
significant alterations in the antioxidant enzymes,
thus demonstrating an oxidative stress. Alterations
were different in the analyzed cerebral regions, which
suggest that the oxidative potential is different in
these regions in relation to the susceptibility to Pb.
Conclusion: Pb alters the activity of antioxidant
enzymes, causing oxidative stress, and compromises
the activity of Na+K+-ATPase, important for brain
homeostasis, contributing to brain dysfunction caused
by chronic exposure to Pb. Keywords: Lead exposure,
oxidative stress, cerebrum, antioxidant enzymes
 180

Carvacrol and its anti-inflammatory effect under

cigarette smoke-induced acute lung injury
Moura, Maria Joana Nogueira1,2; Silva, Aline Gabrielle Gomes1,2, Santos, Caio Cesar Araújo1; Pereira,
Artemia Kelly Holanda1; Felix, Renata Gleysiane de Sousa1; Lima, Crystiane Calado3, Golçalves, A.P.3;
Melo, Paolo Oliveira4; Silva, Gerlane Modesto1; Feitosa, Emanuel Kenedy1; Borges, Cibele dos Santos1
1
 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of
Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-
Arid Region - UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular
Biology (PMBqBM), State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande
do Norte, Brazil; 3 Higher Institute of Biomedical Sciences, State University of Ceará (UECE),
Fortaleza, Brazil; 4 Heart Institute, University of São Paulo (USP), São Paulo, Brazil.

Introduction: Carvacrol is a phenolic monoterpenoid
found in essential oils of oregano, thyme, pepper, wild
bergamot and other plants, possessing a wide range
of useful bioactivities for clinical applications such
as antimicrobial, antioxidant, anti-inflammatory and
anticancer activities. Due to its anti-inflammatory
action, it is possible to make use of its functions in
acute lung injury (ALI) induced by cigarette smoke,
which causes a condition characterized by tissue
damage and an increase in inflammatory markers.
Objective: Investigate the pharmacological activity
of carvacrol (CAR) in the acute lung injury induced
by cigarette smoke in mice. Methods: Mice C57BL/6,
male, (25-28g) were divided into two groups: control
and cigarette smoke (CS). The CS group were exposed
to 12 cigarettes per day for 5 days. The control group
was exposed to sham smoking. The CS group was
treated with CAR (1, 3 or 10 mg/mL) or vehicle by
inhalation (15 min/daily) for 5 days. After 5 days
bronchoalveolar lavage (BAL), trachea and lungs
were collected for inflammatory profile, functional
and morphological analysis. Significant difference
when p<0.05. Results and discussion: The number of
inflammatory cells increased in the BAL of the CS group
when compared to control one (0.02 ± 0.01 vs 0.34 ±
0.03; p<0.001). The treatment with CAR 1, 3 and 10 mg/
mL reduced this number (0.23 ± 0.02, p=0.02; 0.18 ±
0.01, p=0.002 and 0.010 ± 0.01, p=0.001, respectively).
The treatment with CAR 10 mg/mL reduced the airway
hyperresponsiveness in tracheal segments in the
electro and pharmachomecanical coupling (p<0.05).
The pulmonary parenchyma analysis showed an
increase of the epithelium damage (76.3 ± 2.26 vs
112 ± 3.62 control group; p<0.001), lung inflammation
(3.5 ± 0.17 vs 1.9 ± 0.21 control group; p<0.001) and
bronchoconstriction index (4.66 ± 0.2 vs 2.99 ± 0.18
control group; p<0.05). CAR recovery epithelium
damage with 1 mg/mL (91.9 ± 2.12 p<0.01) and 3 mg/
mL (102.3 ± 2.51 p<0.0001). The lung inflammation
was reduced by all the doses (1 mg/mL 2.25 ± 0.09; 3
mg/mL 2.40 ± 0.11 e 10 mg/mL 2.59 ± 0.08; p<0.001).
The bronchoconstriction index was reduced in 1 mg/
mL (3.79 ± 0.17; p<0.05) and 10 mg/mL (3.85 ± 0.16;
p<0.05). Alveolar septal volume density (VvSept) and
mean linear intercept (Lm) did not show any difference.
Conclusion: Carvacrol has anti-inflammatory and
bronchodilator activity without change in the lung
parenchyma in the lung injury induced by cigarette
smoke. Acknowledgments: UFERSA and UERN
 181

Chronic exposure to methylphenidate

during juvenile period alters
zebrafish behavior in adulthood
Nardi, Jessica1; Freddo, Natália1; Biazus, Inara Carbonera2; Oliveira, Ana Paula2; Soares, Suelen
Mendonça3; Fortuna, Milena3; Pompermeier, Aline1; Siqueira, Lisiane1; Cole, Amanda Carolina3;
Tamagno, Wagner3; Amaral, Francieli Ubirajara India1; Costa, Vitoria Cadore4; Mozzato,
Mateus Timbola4; Barcellos, Leonardo José Gil1,3; Grando, Luciana Grazziotin Rossato1
1
 Programa de Pós-graduação em Bioexperimentação, Universidade de Passo Fundo,
Passo Fundo, Rio Grande do Sul, Brazil; 2 Curso de Farmácia, Universidade de Passo Fundo,
Passo Fundo, Rio Grande do Sul, Brazil; 3 Programa de Pós-Graduação em Farmacologia,
Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil; 4 Curso de
Medicina Veterinária, Universidade de Passo Fundo, Passo Fundo, Rio Grande do Sul, Brazil.

Introduction: Methylphenidate is the main treatment
for Attention Deficit and Hyperactivity Disorder (ADHD)
in children. It is believed that there is an overdiagnosis
and consequently over treatment of this problem and
many children end up using methylphenidate without
real need. The consequences of this exposure are
still unknown, thus, in this context, the zebrafish is a
suitable animal model for studying neurobehavioral
changes related to exposure to drugs in a translational
perspective. Objective: we aimed to evaluate
whether the chronic exposure to methylphenidate
during juvenile period leads to behavioral changes
regarding the response to a new environment in
zebrafish during adulthood. Methods: we exposed
juvenile zebrafish (n= 12) to methylphenidate 2
mg/L from the post-natal day (PND) 30 to PND 60.
The treatment was added in the tank water twice a
week. A non-exposed group (control, n=12) was kept
in the same maintenance conditions for comparisons.
The Novel Tank test (NTT) was performed on during
adulthood, on PND 120. Results: animals exposed
to methylphenidate presented behavioral changes
regarding the exploration of the novel environment,
spending less time on the bottom of the tank and
more time on the top zones compared to the controls.
Discussion/Conclusion: The behavioral pattern
observed here might indicate that animals presented
decreased anxiety-like behavior. On a translational
perspective, we can suggest that the decreased
anxiety due to exposure to methylphenidate during
childhood and adolescence might affect normal
behavior during adulthood. Acknowledgements:
Universidade de Passo Fundo for the financial support
and Laboratório de Fisiologia de Peixes for providing
the structure, materials and help during the study
development.
 182

Chronic plus binge ethanol feeding

synergistically induces abdominal aorta
dysfunction and gut dysbiosis in mice
Silva, Carla Brigagão Pacheco1; Rodrigues, Vanessa Fernandes1; Costa, Bruno Ruiz
Brandão2; Sartori, Daniela Carlos1; De Martinis, Bruno Spinosa3; Tostes, Rita C.1
1
 Ribeirão Preto Medical School; 2 School of Pharmaceutical Sciences of
Ribeirão Preto; 3 Faculty of Philosophy, Sciences and Letters of Ribeirão
Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.

Introduction: Excessive ethanol consumption
is a risk factor for cardiovascular disease (CVD),
one of the leading causes of death and disability
worldwide. However, the mechanisms underlying
ethanol effects on the cardiovascular system are
not fully understood. Considering that alterations
of gut microbiota composition or gut microbiota-
dependent metabolites are key to the development
and progression of diseases, including CVD. The
hypothesis of the present study is that excessive
ethanol consumption alters the gut microbiota
composition/function, leading to gut dysbiosis. In
addition, whether ethanol-induced gut dysbiosis
plays role in ethanol-induced vascular dysfunction is
not comprehended. Objective: The goal of this study is
to determine whether the gut microbiota plays a role
on vascular dysfunction induced by excessive ethanol
consumption. Methods: Male C57BL/6 mice were
treated according to the modified National Institute on
Alcohol Abuse and Alcoholism (NIAAA) model. Animals
were randomized into control or ethanol groups. All
mice from the ethanol groups were submitted to
one-week-adaptation period with 5% (v/v) ethanol.
Then, mice either received 1) 10% (v/v) ethanol in their
drinking water for 10 day and binge feeding on day 11
with 5 g/kg body ethanol solution (T1) or 2) 10% (v/v)
ethanol for 20 days combined with feeding on days 11
and 21 with higher doses of alcohol. Animals from the
control groups received water. Ethanol levels were
measured by gas chromatography. Abdominal aortic
tissues were used to perform functional experiments.
Analysis of the gut microbiota was performed by
high-throughput sequencing of the 16S rRNA gene
in fecal pellets. Results: Similar ethanol levels were
observed after the short (T1) and long (T2) treatments
(T1 = 298.3±15.4 mg/dL, n=8; T2= 303.9±13.1, n=5).
Conversely, changes in abdominal aorta reactivity
were observed only after chronic ethanol feeding for
10 days plus a single binge dose of ethanol. In this
treatment (T1), changes in reactivity were observed
in abdominal aortic rings with intact endothelium
and without perivascular adipose tissue (PVAT).
Reduced maximal contraction to phenylephrine was
observed in ethanol-treated mice versus the control
group (Emax: Control= 181.1±4.9, n=7; Ethanol=
128.8±6.3, n=8). Chronic plus binge ethanol feeding
led to the loss of the anticontractile effect of PVAT
(Emax – PVAT (-): Control= 181.1±4.9, n=7; Ethanol=
128.8±6.3, n=8; Emax – PVAT (+): Control= 148.5±4.4,
n=8; Ethanol= 116.2±6.2, n=10). No alterations were
observed in acetylcholine- or sodium nitroprusside-
induced relaxation. These results were accompanied
by increased abundances of Firmicutes and
Proteobacteria phyla. Conclusion: Ethanol-induced
alterations in vascular reactivity was not related to
ethanol levels. Negative effects of ethanol in the PVAT
contribute to vascular dysfunction. Gut dysbiosis may
be associated with vascular dysfunction induced by
chronic plus binge ethanol feeding, but this remains to
be tested. Keywords: Ethanol; Vascular dysfunction;
Abdominal aorta; Gut dysbiosis. Financial support:
FAPESP.
 183

Commercial 2,4-D and its isolated compounds,

2,4-dichlorophenoxyacetic acid and 2,4-
D dimethylamine, induces changes in
mitochondrial function parameters
Polido, Lucas Roberto Ferreira1,2; Rizzi, Joyce Santana1,2; Pereira, Lílian Cristina2,3

 São Paulo State University (Unesp), Medical School, Botucatu; 2 Center for
1

Evaluation of Environmental Impact on Human Health (TOXICAM), Botucatu;

3
 São Paulo State University (Unesp), School of Agriculture, Botucatu.

Background: The use of pesticides has increased, due
to their significant role in the economic development
of Brazilian agriculture. Pesticides are used on a
large scale, as they guarantee crop productivity while
preserving the species of interest. However, its use is
also related to negative impacts on the environment
and on human health, leading to ecological and
toxicological issues when incorrectly used. Analyses
on different organisms and exposure conditions are
conducted to assess the hazard and risk that these
compounds may induce. Some of the mechanisms
by which xenobiotics can exert their cytotoxicity are
mitochondrial pathways, as it is an organelle vital
to the maintenance of several biological processes,
such as energy balance and cell death. The study
of isolated mitochondria can provide information
about toxicity mechanisms induced by chemical
compounds. Thus, this study aims to investigate the
toxicity mechanisms by which 2,4-D, one of the most
widely used herbicides in Brazilian agriculture, may
be harmful to the liver mitochondria. For this purpose,
the redox state of mitochondria isolated from the
liver of young Wistar rats, was evaluated after in
vitro exposure to 2,4-D and its isolated components,
2,4-dichlorophenoxyacetic acid (C8H6Cl2O3) and 2,4-
D dimethylamine (C10H13Cl2NO3). Even though 2,4-D
is one of the most studied chemicals, little is known
about the toxicity mechanism of this herbicide, and
its cytotoxic action remains unclear. Objective: To
identify the toxic mechanism of 2,4-D and its isolated
components in hepatic mitochondria by in vitro
exposure. Methods: The exposure of mitochondria
isolated from the liver of Wistar rats to 2,4-D
was carried out for ten minutes, in four different
conditions: [1] the commercial formula (DMA 806 BR®);
[2] acid component (2,4-dichlorophenoxyacetic acid);
[3] amine salt component (2,4-D dimethylamine);
and [4] mixture of the acid and salt components,
emulating their commercial concentrations, thus free
from excipients and other components found in DMA
806 BR. Concentrations ranging from 0.1 to 200 µM
were used, as these comprises the concentrations
found in the environment. So far, the production
and accumulation of Reactive Oxygen and Nitrogen
Species (RONS) using the H2DCFDA probe and the
redox state of pyridine nucleotides based on their
auto-fluorescence has been assessed. The results
were evaluated by analysis of variance (ANOVA)
followed by the Dunnett test, assuming a significant
difference when p ≤ 0.05. Results: The assays showed
that, at the concentrations evaluated, the generation
of RONS was not detected in any of the four different
conditions. The highest concentrations (200 µM) of
2,4-D dimethylamine and 2,4-dichlorophenoxyacetic
acid evaluated were able to oxidize the mitochondrial
NAD(P)H, as well as the 10 µM concentration from
the latter and the 100 µM concentration from the
proportional mixture. Discussion: The oxidation of
NAD(P)H may suggest a possible imbalanced redox
state-related mechanism of action, which will be
further investigated through other analyses such as
the evaluation of antioxidant enzymes, since 2,4-
D and its isolated compounds do not induce RONS
production or accumulation. Financial Support:
TOXICAM
 184

Could Eugenol promote the reduction of the

inflammatory process in acute injuries to the
lung of rats caused by cigarette smoke?
Silva, Aline Gabrielle Gomes1,2; Moura, Maria Joana Nogueira1,2; Santos, Caio Cesar Araújo1; Pereira,
Artemia Kelly Holanda1; Felix, Renata Gleysiane de Sousa1; Araujo, Bruno Vinicios Silva1; Barbosa, Maria
Clara de Oliveira1; Melo, Paolo Oliveira3; Feitosa, Emanuel Kenedy1; Borges, Cibele dos Santos1
1
 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of
Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-
Arid Region - UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular
Biology (PMBqBM), State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande
do Norte, Brazil. Heart Institute; 3 University of São Paulo (USP), São Paulo, Brazil.

Introduction: Cigarette smoke can induce the lung
acute injury with inflammatory cells and lung
parenchyma damage. Eugenol (EUG) is a component
of clove oil, with anti-inflammatory and antioxidant
activities. Objective: To evaluate the pharmacological
activity of the EUG in cigarette smoke-induced acute
lung injury. Methods: C57BL/6 mice, male were
exposed to 12 cigarettes per day for 5 days (CS group).
The control group was exposed to sham smoking.
The CS group was treated with EUG (100 mg/mL) or
vehicle by inhalation (15 min/daily) for 5 days. The
anti-inflammatory markers and lung morphology
were evaluated. Results and Discussion: The
leucocytes number from mice BAL increased 3x on
the CS group compared to control and the treatment
with EUG (100 mg/mL) reduced 46% when compared
to the CS group (p<0.05). The number of macrophages
(142%) and neutrophils (3x) increased in the CS
group. EUG (100mg/mL) reduced in 60% (p<0.001) and
63% (p<0.001), respectively. The lung inflammation
score showed the increase of 100% of inflammation
in the CS group (p<0.001). The treatment with EUG
100 mg/mL reduced this score by 30% (p<0.01). The
bronchoconstriction index was increased in the CS
group (65%; p<0.01) and reduced in 29% (p<0.05) by
treatment with EUG 100 mg/mL. Conclusion: Eugenol
has an anti-inflammatory effect to attenuates the
inflammatory response induced by cigarette smoke.
Acknowledgments: UFERSA and UERN
 185

Cytotoxicity assessment of potential

antitumor 4-methyl coumarins
Göethel, Gabriela1; Souza, João Pedro Silveira1; Neves, Gustavo Machado1; Kagami,
Luciano Porto1; Peruzzi, Caroline Portela2; Cattani, Shanda2; Garcia, Solange Cristina2;
Battastini, Ana Maria Oliveira3; Figueiró, Fabrício3; Eifler-Lima, Vera Lucia1
1
 Laboratório de Síntese Orgânica Medicinal (LaSOM), Pharmaceutical Sciences Graduate
Program, College of Pharmacy, Federal University of Rio Grande do Sul, Avenida Ipiranga,
2752, Porto Alegre – RS, Brazil. 2 Laboratório de Toxicologia (LATOX), Pharmaceutical Sciences
Graduate Program, College of Pharmacy, Federal University of Rio Grande do Sul, Rua São
Luís, 150, Porto Alegre – RS, Brazil. 3 Laboratório de Bioquímica (Laboratório 22), Graduate
Program in Biological Sciences: Biochemistry, Institute of Basic Health Sciences, Federal
University of Rio Grande do Sul, Rua Ramiro Barcelos, 2600, Porto Alegre – RS, Brazil.

Introduction: lung cancer is one of the biggest causes
of death in the world, accounting for approximately
2.1 million new cases per year. In Brazil, it is the fourth
most common type of cancer among men and the
fifth among women. The use of platinum derivatives
associated with other chemotherapeutics is still
the main choice for the treatment of lung cancer,
however the use of these drugs causes serious
adverse effects and can present several resistance
mechanisms. Objective: To identify new antitumor
prototypes by virtual screening of coumarins from
the LaSOM library and the in vitro assays. Methods:
five coumarins were selected by the rank-by-rank
method and their physicochemical and toxicological
properties in silico were analyzed. From these results,
three coumarins (LaSOM 235, LaSOM 229 and LaSOM
234) were selected to continue the in vitro studies. In
vitro cell viability, oxidative stress and mitochondrial
membrane potential assays were performed using the
human lung cancer strain A549. Results: the results
showed that LaSOM 235 presented a decrease in cell
viability at concentrations of 30, 40 and 50 µM when
compared to the control group (100% of cell viability),
revealing to be the most promising substance among
the three evaluated in vitro. Additionally, in order to
investigate the action mechanism of coumarins the
inhibition of the CD73 enzyme (ecto-5’-nucleotidase)
was investigated. Ecto-5’-NT has been associated with
some pathological conditions, such as myocardial
ischemia and cancer. The enzymatic activity was
tested at concentrations of 50, 75, 100 and 200 µM
and no enzymatic inhibition was evidenced at these
concentrations. Discussion/Conclusion: the in vitro
evaluation of coumarins LaSOM 229, LaSOM 234 and
LaSOM 235 against the cell line derived from human
lung cancer A549 showed that with the results
obtained so far, LaSOM 235 is the most promising
hit, with chances of becoming a potential candidate
for an antitumor drug prototype. More tests should
be performed to corroborate the results obtained.
Acknowledgments: PNPD/CAPES
 186

Cytotoxicity evaluation in human hepatic cells

HepG2 induced by emerging contaminants
Decametilcyclopentasiloxane and Triclopyr
Kohori, Natália Akemi1,2; Teodoro, João Soeiro3; Palmeira, Carlos Manuel Marques3; Pereira,, Lilian Cristina1,4
1
 Botucatu Medical School, Department of Pathology, São Paulo State University (UNESP);
2
 Center for Environmental Assessment on Human Health (TOXICAM), Botucatu, São Paulo,
Brazil; 3 Department of Life Sciences, Faculty of Sciences and Technology, University of
Coimbra, Coimbra, Portugal; 4 Department of Bioprocesses and Biotechnology, Faculty
of Agronomic Sciences of Botucatu, São Paulo State University, Botucatu, SP.

Background/Introduction: Humans are exposed to density determination, based on the measurement

a multitude chemicals: pesticides, pharmaceuticals
and industrial chemicals, among other substances
whether through production, consumption, presence
in the environment or incorrect disposal. The
bioaccumulation and biomagnification of their
residues may increase to inadequate exposures and
lead to high incidence of human diseases. Experimental
studies have the capacity to predict the toxicity of
these emerging contaminants and are valuable tools
for understanding biological mechanisms potentially
involved in the hazard definition and consequently,
help in risk assessment to human health. Currently,
the use of so-called alternative methods to animal
use for assessing the toxicity of chemicals is an
international trend in response to the 3R’s (Reduce,
Refine and Replace) policy that restricts the use
of animals in research. Among them are included
cell cultures applied to toxicology. Objective: This
study characterizes the in vitro toxicity to herbicide
Triclopyr and the personal care product (PCPs) used
in cosmetics, Decamethylcyclopentasiloxane (D5),
in an attempt to elucidate their mechanism based
on mitochondrial dysfunction and cellular injury
using HepG2 cells. Methods: As a first step HepG2
cell lines were cultivated and plated to 96 or 24-
well plates, incubated for 24 hours, then exposed to
Triclopyr and D5 at different concentrations between
0.1 to 50 µM for 24 and 48 hours for MTT and SRB,
respectively. Cell viability analysis was performed
using the using MTT [3-(4,5-Dimethylthiazol-2-
yl)-2,5-DiphenyltetrazoliumBromide] test. The
Sulforhodamine B (SRB) assay was used for cell
of cellular protein content. Furthermore, the
Agilent Seahorse Cell Mito Stress Test kit was
performed to assessing mitochondrial function,
using concentrations of 0.5 to500 µM. This test
provided multiple mitochondrial parameters like
basal respiration, ATP-linked respiration, maximal
and reserve capacities. Results: There were no
significant results for MTT assay. However, outcomes
of SRB demonstrated that Triclopyr and D5 affect
cell proliferation, with statistical difference between
the negative control and the tested concentrations
of 0.1, to 50 µM. Effects induced by mitochondrial
multiparameter using a high-capacity respirometer
after exposure of HepG2 cells showed that both
compounds have the ability to induce mitochondrial
damage at intermediate concentrations of 10 and 100
µM, inhibiting basal and spare respiratory capacity.
Discussion/Conclusion: The present results indicate
that both emergenting contaminants are mitotoxic.
A decreasing at mitochondrial respiration and ATP
production was observed. In the SRB assay there was
a decrease in cell proliferation. Although no effect on
the ability to metabolize the MTT dye was observed,
there was an alteration on cell density. Nonetheless,
mitochondrial damage can have different
consequences for the cell that will be investigated
in more detail. Acknowledgments: This work was
financially supported by grants from the Coordination
for the Improvement of Higher Education Personnel
(CAPES). Process number: 88887.600147/2021-00 and
Fapesp (2018/00229-1 e 2020/11128-1).
 187

Different diets and physical exercises:

assessment of the impact on the female
reproductive system after 90 days
Moura, Maria Joana Nogueira1,2; Silva, Aline Gabrielle Gomes1,2; Santos, Caio Cesar
Araújo1; Gomes, Francisca Tayná da Silva3; Pereira, Artemia Kelly Holanda1; Felix, Renata
Gleysiane de Sousa1; Fonseca, Ivana Alice Teixeira3; Borges, Cibele dos Santos1
1
 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of
Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-
Arid Region - UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular
Biology (PMBqBM); 3 Multicenter Graduate Program in Physiological Sciences (PPGMCF),
State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande do Norte, Brazil.

Introduction: Obesity is considered a global public
health problem and diets rich in fats and sugars
are increasingly being consumed. Associated with
the intake of high fat-sugar foods is a sedentary
lifestyle, the number 1 aggravating factor of weight
gain problems. In addition to the problems intrinsic
to obesity, others are also directly related, such
as reduced fertility. It is also known that obesity
directly impairs reproduction, altering hormone
levels and gamete production. Objective: The present
study aimed to evaluate and compare the possible
beneficial or deleterious effects of physical training
associated with diets rich in cashew nut fat or lard in
reproductive systhem of female adult rats. Methods:
Adult female Wistar rats were divided (n=8/group)
into standard chow group (control, 10% lipid and 20%
protein content) without physical exercise (sedentary)
- SDCNT; standard chow with physical exercise
group - EXCNT; cashew nut group (diet enriched with
vegetable fat from cashew nut, 40% lipid and 20%
protein content) sedentary - SDCSC; cashew nut group
with exercise - EXCSC; lard group (diet enriched with
fat of animal origin based on lard, 40% lipid and 20%
protein content) sedentary - SDBHP and lard group
with physical exercise - EXBHP. During 90 days, the
food was offered ad libitum, and consumption was
monitored weekly. The physical exercise groups were
submitted to 30 minutes of daily physical exercise,
following the running protocol on the treadmill (8m/
min for 5 minutes, 12m/min for 20 minutes and 8m/
min for the last 5 minutes, during the experimental
protocol). In the last 15 days, the estrous cycle of
the rats was monitored and after this period, in the
first estrus, the rats were euthanized for collection,
weighing and fixation of the ovaries and uterus,
followed by further histopathological analysis.
Ovaries and uterus were fixed in formaldehyde,
processed and stained with eosin-hematoxylin for
histopathological (qualitative) analysis. The study was
approved by the Ethics Committee of UERN 007/19
and Ethics Committee of UFERSA (PIA10006-2021).
Results and Discussion: Feed consumption showed a
significant difference (p<0.05) between groups SDCSC
(14.23+0.83), EXCSC (16.54+0.60), SDBHP (13.69+0.47),
EXBHP (16.56+0.58) when compared to both EXCNT
(22.15+0.41) and SDCNT (19.19+0.68). The final body
weight of the rats revealed a significant increase
(P<0.5) in the weight of the exercise groups when
compared to the sedentary groups, except the SDCSC
group compared to the EXCNT group. The uterine
weight of the SDCSC group was also significantly lower
compared to the EXBHP group. On the other hand, the
weight of the ovaries did not change (p>0.05). There
was no alterations on ovary histology (p>0.05). All
groups presented well-defined gonads, characterized
by a cortex with the presence of ovarian follicles in
various degrees of development, mainly corpora
lutea (70%), since the rats were euthanized in estrus.
Uterine histology revealed in the SDCSC an increased
presence of leukocyte infiltrates compared to the
SDCNT. In the exercise group, both EXCNT and EXCSC
showed an increase in the presence of leukocyte
infiltrates in the uterine endometrium (100% of the
animals evaluated). In the group EXBHP, the presence
of leukocytes was observed, but at a lower frequency
when compared to the other groups. As for cyclicity,
it was observed that there was a lower percentage
(p<0.05) of proestrus in the EXCSC group (7.80+2.07)
in relation to the SDCNT group (19.94+4.03), but not
directly impacted the size and number of estrous
cycles when compared between groups (p>0.05).
Conclusion: Therefore, it can be concluded, based
on this experimental model, that both diets, rich in
vegetable or animal fat, do not have a direct impact
on reproductive systhem of female adult rats, at least
not on gonadal morphology. Acknowledgments: PIBIC
(CNPq) and PIVIC UFERSA Scholarship.
 188

Discreet alterations on sperm quality

promoted by Aripiprazole
Moura, Maria Joana Nogueira1,2; Felix, Renata Gleysiane de Sousa1; Pereira, Artemia
Kelly Holanda1; Angelo, Ana Beatriz Silva1; Santos, Caio Cesar Araújo1; Silva, Aline
Gabrielle Gomes1,2; Silva, Ana Lucelha dos Santos1; Freire, Livia Horrana Forte1; Dantas,
Joao Artur Diogenes1; Silva, Mateus Limerio Carlos1; Borges, Cibele dos Santos1
1
 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of Bioscience,
Biological and Health Sciences Center - CCBS, Federal University of the Semi-Arid Region -
UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular Biology (PMBqBM),
State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande do Norte, Brazil.

Introduction: Numerous antidepressants and
antipsychotics drugs have been continuously
approved and released for sale that have not yet
had all their effects evaluated, both in the short and
long term. These drugs are aimed at different types
of treatment of mental disorders. Among these drugs
is Aripiprazole. Aripiprazole has high affinity for
serotonin, dopamine and noradrenaline receptors
for the treatment of mental disorders, mainly
schizophrenia and depression. It is known that the
5-HT system can directly influence the reproductive
function of vertebrates, due to its communication
with the sex steroid system. This interaction
between the systems occurs through estrogen
and progesterone receptors located in central
serotonergic neurons, demonstrating an important
signaling pathway, in which it can modulate several
neural processes, including thyroid secretion and
sexual behavior. However, there are few reports of
the effects of second-generation antidepressants
and antipsychotics on the reproductive system and
sexual behavior of male rats, mainly with regard to
aripiprazole. Objective: The present study aimed to
evaluate the possible effects of exposure to different
doses of aripiprazole on the male reproductive
system, with emphasis on sperm quality and sexual
behavior. Methods: Adult male Wistar rats were
divided (n=12/group) into control group (CTRL, treated
with vehicle solution - 66% saline and 33% DMSO);
group treated with 3.0mg/kg of aripiprazole diluted
in vehicle (EXP1) and group treated with 6.0mg/kg
of aripiprazole diluted in vehicle (EXP2). The animals
were treated during 28 days. After the end of the
treatment, a set of animals (n=7/group) were weighted
and euthanized by overdose of anesthetic xylazine
and ketamine. Blood samples were collected to
hormonal levels. Vital organs (kidney, adrenal, heart,
liver, brain, pituitary and thyroid) were collected and
weighed. The reproductive organs (testis, epididymis,
ventral prostate, full and empty seminal vesicle)
were also collected and weighed. The right testis and
epididymis were fixed in modified Davidson’s solution
(MDF) for further histopathological processing. The
left epididymis was removed and the sperm sample
was used to determine the sperm motility and count.
The remained animals (n=5/group) were used to
perform sexual behavior. The study was approved by
the Ethics Committee of UFERSA 02/2020 (Protocol
number 23091.014948/2019-20). Statistical analysis:
ANOVA and Tukey test for parametric data and
Kruskal Wallis and Dunn’s test for non-parametric
data (P<0.05). Results and Discussion: No alterations
were observed in the final body weight of the rats
exposed to different doses of aripiprazole, as well as
no alterations in the absolute weights vital organs,
when compared to the CTRL group. However, when
compared the reproductive organ weights there was
a significant increased in the full seminal vesicle from
EXP2 group when compared to CTRL group (p<0.05).
The evaluation of the sperm motility showed a
significant reduction on mobile sperm with progressive
moviment from EXP1 compared to CTRL group (57.7%
x 37.7%; p<0.05). The sexual behavior showed the
same results, once the number of mounts before the
ejaculation was significantly decreased in EXP1 group
when compared to the CTRL group (16.0 + 3.2 x 6.0 + 1.5;
p<0.05). Conclusion: Therefore, it can be concluded at
this moment, based on this experimental model, that
the exposure to aripiprazole during adulthood does
not cause systemic intoxication effects. However,
although the differences were occured only in some
variable, it can cause impairment on male sperm
quality and/or libido. Acknowledgments: PIBIC-CNPq
UFERSA Scholarship and UFERSA/PROPPG (grant
number 23091.014593/2019-02).
 189

Diuron herbicide-induced toxicity

on Caenorhabditis elegans
Lima, Thania Rios Rossi1,2; Martins Jr., Airton Cunha3; Pereira, Lílian Cristina2,4; Aschner, Michael3
1
 Botucatu Medical School, Department of Pathology, São Paulo State University
(UNESP); 2 Center for Evaluation of Environmental Impact on Human Health (TOXICAM),
Botucatu, SP, Brazil; 3 Department of Molecular Pharmacology, Albert Einstein College of
Medicine, Bronx, NY, USA; 4 Faculty of Agronomy Sciences, Department of Bioprocesses
and Biotechnology, São Paulo State University (UNESP), Botucatu, SP, Brazil.

Background: Studies have indicated that the diuron
herbicide induces toxicity in different tissues,
especially in the urinary bladder of Wistar rats,
where at high doses it may lead to the development
of tumors. However, events involved in its intimate
toxic mechanism remain unclear. In this context,
Caenorhabditis elegans (C. elegans) has been widely
used as an experimental model to understand adverse
events related to chemical exposures. Objective:
This study aimed to identify the toxic mechanism of
diuron and its metabolites, 3,4-dichloroaniline (DCA)
and 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU)
in C. elegans. Methods: Wild-type N2 worms in L1
larval stage were acutely exposed (1 hour) to the
test chemicals in a six-concentration range of 0.5
to 500 μM and analyzed after 24 and 48 hours of
incubation for % lethality. Then, exposures were
carried out using the highest concentration (500 μM)
to evaluate % survival, reactive oxygen and nitrogen
species (RONS) , glutathione (GSH) and ATP levels,
as well as behavior. The QU1:izEx1 (Pllg-1::GFP) and
BY200 (Pdat-1::GFP) strains were used to determine
autophagy and dopaminergic neurodegeneration,
respectively. Results were evaluated by analysis of
variance (ANOVA) followed by Dunnet’s or Tukey’s
post hoc test, and significant differences were set at
p < 0.05. Results: Increased % lethality was found for
all chemicals at high concentrations, with statistically
significant difference for 500 μM DCA. Significant
difference was also observed in the % survival for
DCA. No changes in RONS production were observed
for diuron and its metabolites. Nevertheless, GSH
levels were significantly increased upon DCA
treatment. Moreover, ATP levels were decreased
for all test chemicals. There were no alterations
in the autophagy process. Under the conditions of
the present study, dopaminergic neurotoxicity was
observed for all tested chemicals, but only diuron
was induced alterations in the moving average and
smoothed speed (μm/s) of the worms. Discussion/
Conclusion: In this study, increased GSH levels acted
as a compensatory mechanism against the generation
of RONS by diuron and its metabolites. In turn, exposure
to diuron and metabolites was sufficient to impair
ATP levels, indicative of an alteration in oxidative
phosphorylation that takes place in mitochondria.
Such conditions may trigger autophagy as an adaptive
survival mechanism, but this was not observed in C.
elegans; in fact, necrosis and apoptosis have been
shown as the main types of cell death associated
with the test chemicals in previous studies. Although
dopaminergic neurodegeneration typically triggers
motor impairment, only diuron elicited this outcome;
alteration in the specific parameters evaluated may
occur at different time point for the metabolites and
was not accessed in the present study. Altogether,
the results suggest that the diuron metabolites are
more toxic than the parent compound, with DCA in
particular appearing to play an important role in the
toxicity observed on C. elegans. Acknowledgments:
Breno Pannia Espósito (Institute of Chemistry,
University of São Paulo) for technical assistance.
São Paulo Research Foundation (FAPESP) [Grant No.
2017/25402-5] and Coordination for the Improvement
of Higher Education Personnel (CAPES) [CAPES-PrInt
Grant No. 88887.467311/2019-00].
 190

Effect of inorganic selenium on triple-

negative breast cancer cell lines
Costa, Nayara de Souza1,2; Lima, Luiza Siqueira1,2; Oliveira, Franciele Aparecida Mendes1,2;
Galiciolli, Maria Eduarda de Andrade1,2; Oliveira, Cláudia Sirlene1,2; Irioda, Ana Carolina1,2
1
 Programa de Pós-graduação Strictu Sensu em Biotecnologia Aplicada à Saúde
da Criança e do adolescente, Instituto de Pesquisa Pelé Pequeno Príncipe,
Curitiba, PR, Brazil; 2 Faculdades Pequeno Príncipe, Curitiba, PR, Brazil.

Breast cancer causes a great number of deaths
worldwide, mainly in women. One of the most
aggressive breast cancer subtypes is triple-negative
breast cancer (TNBC), which represents about 20%
of the diagnosticated breast cancers. TNBC does
not express estrogen receptors (ER), progesterone
receptors (PR), and human epidermal growth factor 2
(HER2). In this way, hormone therapy is not an option,
and TNBC usually is treated with chemotherapy.
Unfortunately, chemotherapy treatment causes
several side effects to patients. Selenium (Se) is
an essential micronutrient, which has antioxidant
and anti-inflammatory effects. On the other hand,
some studies have been demonstrating that some
Se-containing compounds have pro-oxidant effects.
Thus, the aim of this work was to evaluate the
antiproliferative effects of sodium selenite in breast
cell lines. Two TNBC cell lines [BT-549 (primary tumor)
and MDAMB-231 (metastatic tumor)] and a non-tumoral
breast cell line (MCF-10A) were exposed to 1, 10, 50, and
100 µM sodium selenite for 48 hours. The following
assays were carried out (1) 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) test;
(2) identification of apoptotic and necrotic cells test;
(3) colony-forming unit test; and (4) cell migration.
For all tests, at least three independent experiments
were performed. Data were statistically analyzed
using Prisma Graphpad software, version 5.0, using
the Kruskal-Wallis test followed by Dunn’s test and
presented as median ± interquartile range. Effects
were considered significant when p<0.05. Kruskal-
Wallis test revealed an effect of exposure to sodium
selenite on the cell viability on the three cell lines
evaluated. In fact, MCF10-A and BT-549 cell lines
exposed to 50 and 100 µM sodium selenite had a
statistically significant decrease in cell viability, as
well as MDAMB-231 cells exposed to 100 µM sodium
selenite also had a statistically significant decrease
in cell viability. Kruskal-Wallis test revealed an effect
of sodium selenite on the percentage of apoptotic
and necrotic cells. In fact, the exposure to 50 and 100
µM sodium selenite caused a statistically significant
increase in the percentage of late apoptotic and/or
necrotic MCF-10A cells. However, only the exposure
to 100 µM sodium selenite caused a statistically
significant increase in the percentage of late
apoptotic and/or necrotic MDAMB-231 and BT-549
cell lines. Sodium selenite exposure totally inhibits
colony growth at concentrations of 10, 50, and 100 µM
in the three cell lines evaluated. Exposure to sodium
selenite (1 µM) caused a statistically significant
increase in the migration of MCF-10A and MDAMB-231
cells when compared to control (unexposed cells).
On the other hand, caused a statistically significant
decrease in cell migration of the BT-549 cell line.
Sodium selenite showed promising antiproliferative
results in different breast cell lines. In conclusion, it
was observed that sodium selenite has the potential
to be tested in pre-clinical studies of animal breast
cancer models. Keywords: Selenium, selenium
inorganic, breast cancer, triple-negative breast
cancer. Acknowledgments: We greatly appreciate the
Pequeno Príncipe Complex by scholarship.

Evaluation of irritability of Myrcia
pubipetala miq. by HET-CAM methodology
Perini, Camila Maria; Kohler, Cristofer José Weege; Wagner, Eduardo José; Machado, Isabel Daufenback
The HET-CAM method can be used to determine
the irritation potential of certain substances on
corioalantoic membrane in chicken eggs, and in
addition, it can determine anti-inflammatory activity
of this substances. The test is based on the exposure
of membrane to irritating agents with posterior
observation about vascular events triggered. HET-
CAM is considered a test in vivo that replaces the
use of animals, being a test considered useful and
well established to determine the potential of
pharmaceutical products. It should be emphasized
that ocular toxicity assessment of cosmetic products
is done alternatively to use of animals, since it is
prohibited. The species Myrcia pubipetala, commonly
known as Goiabão, is a tree registered on states
of Santa Catarina and Rio Grande do Sul, occurring
in the Atlantic Forest and Dense Anthropophilous
Forest. The present study aimed to evaluate the
irritability of Myrcia pubipetala extract by the HET-
CAM methodology. The hydroalcoholic extract (with
70% of ethanol) from the leaves of Myrcia pubipetala
was kindly ceded by Professor PhD Micheli A. Debiasi.
The fertilized chicken embryos were pre-incubated to
38º Celsius with 60% of humidity for 8 days. After a
hole highlighted with pencil was sanitized and drilled
gently over the overhead bag with tweezers to not
damage the eggshell, and the vascular zone was
easily identified in corioalantoic membrane (CAM).
Two drops of Saline solution 0,9% have been added
to dampen the inner membrane of the casing adjacent
to the CAM. After clamping and raised by ophthalmic
191
Universidade Regional de Blumenau – FURB.
tweezers, the membrane and CAM were separated
effortlessly, and in sequence, a space with 1x1cm on
membrane will be seccionated to expose vascular
zone. It was applied 300 µL of NaCl 0.9 % (negative
control), NaOH 1M (positive control) and extract of
Myrcia pubipetala (3, 30 and 300 µg/mL) directly on
vascular zone. After the compounds application, the
eggs have been observed for 300 seconds. During this
period, bleeding, coagulation, and vascular lysis it
was observed, and the experiment was performed in
triplicate. For the positive control group for irritation
was applied 300 µL of NaOH at 1 M, inducing a positive
response to irritation with the appearance of vascular
lysis, bleeding and coagulation, with an average score
of 20, categorized as severe irritation. In the negative
control group, the eggs were treated with NaCl 0.9 %,
with a negative response to irritation, with a score
of 0. Myrcia pubipetala test, 300 µL of plant extract
were applied at 300, 30 and 3 µg/mL and no irritative
effects were observed at all doses (p<0,001 vs
negative control). Thus, the extract was categorized
as non-irritating, with viability for dermal topical use.
Although the HET-CAM method is an established and
reliable, this test remains the most viable alternative
method to the use of animals, and inexpensive for
the purpose of controlling topical and ophthalmic
irritation of substances. We can suggest that the
extract of Myrcia pubipetala met the hypotheses
raised by the research group, feasible for topical
dermal use, because it does not cause irritation.

Evaluation of metabolic activity of
HepG2 cells co-exposed to micro or
nanoplastics and bisphenol A or S
Rocha, Cecília Cristina de Souza; Devoz, Paula Pícoli; Antunes, Lusania Maria Greggi; Barbosa Jr, Fernando
Introduction: Plastics are used wordwild due to
their excellent mechanical properties, versatility and
low cost. After their release into the environment,
plastics are degraded and fragmented into smaller
particles, at micro (<5 mm) and nano (<100 nm) scale,
which increases their toxic potential. Micro (MPs) and
nanoplastics (NPs) can be found in the air, sediment,
water and terrestrial and aquatic organisms.
Bisphenol A (BPA) is a industrial product used in the
plastic production and is known as an endocrine
disruptor. Bisphenol S (BPS) is an analogue BPA and
it is used as an alternative to this contaminant. BPS
has metabolism and mechanisms of action similar
to BPA, which represents a potencial human health
risk. Food and water contaminated with bisphenol
adsorbed in MPs and NPs are the major sources of
human exposure to these particles and contaminants.
The consequences for human health are still unknown
and there is no internationally accepted definition
for ingestion and exposure to these MPs and NPs. In
addition, few studies have shown the toxicity of MPs
and NPs in liver cells, in vitro and in vivo. Objective: To
evaluate whether micro and nanoplastics associated
to BPA or BPS can alter the metabolic activity of liver
cells. Methods: Commercial spherical micro and
nanoplastics (Thermo Fisher) were used. Liver cells
(HepG2) were co-exposed to BPA or BPS (100 µM) and
to two different sizes of MP (0.2 or 0.5 µm) at three
different concentrations (10, 100 and 300 µg.mL-1) for
24 hours. For the 72 hours experiments, HepG2 cells
were co-exposed to BPA or BPS (100 µM) and to two
different sizes of NP (0.02 or 0.1 µm) or one size of
MP (0.5 µm) at two different concentrations (5 and
10 µg.mL-1). Methylmethanesulfonate (600 µM) was
192
University of Sao Paulo – School of Pharmaceutical Sciences
of Ribeirao Preto, Ribeirão Preto - SP, Brazil.
used as a positive control. HepG2 metabolic activity
was measured by resazurin (spectrofluorimeter –
540/590 nm). Results: At the 24 hours experiments,
co-exposure to BPA or BPS and 0.2 µm MP (10 µg.mL-1)
decreased the cellular metabolic activity compared
to the group treated with MP only. The same results
occurred in the group BPA + MP (100 and 300 µg.mL-1).
No change in the cellular metabolic activity was
observed when the cells were exposed to BPA or BPS
only. Cellular metabolic activity decreased when the
HepG2 cells was co-exposed to BPA or BPS and 0,5
µm MP (10 µg.mL-1) when compared to the control
group and the MP group. In addition, BPA + MP group
decreased the HepG2 metabolic activity compared
to the BPA group. At the 72 hours experiments, co-
exposure to BPA or BPS and 0.02 µm NP (5 and 10
µg.mL-1) decreased the cellular metabolic activity
compared to the control and NP groups. Futhermore,
BPS + NP group decresead the HepG2 metabolic
activity in comparison to the BPS group. When co-
exposed to the 0,1 µm NP or 0.5 µm MP at both
concentrations (5 and 10 µg.mL-1) and BPA or BPS, the
cellular metabolic activity decreased in comparison to
the control group and the group treated with plastic
only, but not in comparison to the bisphenol group.
Discussion/Conclusion: At 24 hours, the 0.5 µm MP
co-exposed to the BPA is able to intensify the reduction
of HepG2 viability, demonstrating that the decrease in
cellular metabolic activity of HepG2 depends on the
concentration and size of the plastic. After 72 hours
exposure, the plastic size influences the co-exposure
response to MPs or NPs and bisphenol, but not the
concentration. Acknowledgments: This work was
supported by CAPES, FAPESP and CNPq.
 193

Evaluation of the female wistar rats reproductive

system submitted to different diet formulations
and activity patterns during 180 days
Dantas, Joao Artur Diogenes1; Silva, Aline Gabrielle Gomes1,2; Moura, Maria Joana Nogueira1,2;
Santos, Caio Cesar Araújo1; Gomes, Francisca Tayná da Silva3; Pereira, Artemia Kelly Holanda1;
Felix, Renata Gleysiane de Sousa1; Fonseca, Ivana Alice Teixeira3; Borges, Cibele dos Santos1
1
 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of
Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-
Arid Region - UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular
Biology (PMBqBM); 3 Multicenter Graduate Program in Physiological Sciences (PPGMCF),
State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande do Norte, Brazil.

Introduction: Incorrect nutrition with the absence of
physical activity associated with other factors can
contribute to the development of obesity. In rats,
specifically, the weight gain induced by high-fat diets,
represents an experimental model that has been
widely used to study the physiological and molecular
mechanisms of obesity development, as these animals
share similar gene expression patterns with humans.
In addition, it is known that the accumulation of fat
can directly harm reproduction, promoting hormonal
problems as well as fertility. Objective: To investigate
the possible impacts of different ketogenic diets with
or without physical activity during a period of 180
days on reproductive morphological parameters of
adult wistar rats. Methods: Adult female Wistar rats
were divided (n=8/group) into standard chow group
(control, 10% lipid and 20% protein content) without
physical exercise (sedentary) - SDCNT; standard chow
with physical exercise group - EXCNT; cashew nut
group (diet enriched with vegetable fat from cashew
nut, 40% lipid and 20% protein content) sedentary -
SDCSC; cashew nut group with exercise - EXCSC; lard
group (diet enriched with fat of animal origin based
on lard, 40% lipid and 20% protein content) sedentary
- SDBHP and lard group with physical exercise -
EXBHP. The physical exercise groups were submitted
to 30 minutes of daily physical exercise, following
the running protocol on the treadmill (8m/min for
5 minutes, 12m/min for 20 minutes and 8m/min for
the last 5 minutes, during the experimental protocol).
The treatment occured during 180days and in the last
15 days, the estrous cycle of the rats was monitored.
After that, in the first estrus, the rats were euthanized
for collection, weighing and fixation of the ovaries and
uterus, followed by further histopathological analysis.
Ovaries and uterus were fixed in formaldehyde,
processed and stained with eosin-hematoxylin for
histopathological (qualitative) analysis. Results and
Discussion: There were no statistical differences
observed in the body weight of the animals and also in
the uterine and ovarian absolute and relative weights
(p>0.05). In addition, no alterations were observed in
the number of estrous cycles and in the duration of
these cycles between the groups (p>0.05). However,
when each phase was analyzed individually, a higher
frequency of the diestrus phase was seen in the EXCSC
group (54.81+6.41) when compared to the SDBHP group
(25.96 + 8.09). In the histopathological evaluation of
the ovaries, no significant changes were identified. All
groups had well-defined and characteristic gonads.
Histopathological analyzes of the uterus showed that
both, in the sedentary and in the groups submitted
to exercises, the presence of leukocyte infiltrates in
at least half of the animals of all groups, including
the group treated with standard chow. On the other
hand, only in the group exposed to lard, both in the
sedentary group and in the group submitted to
exercise, alterations were observed in the glandular
epithelium in at least 20% of the animals. Conclusion:
Therefore, based on the present experimental model,
there was no deleterious effects caused by the
different types of high-fat diets and physical activity
in the on the female reproductive system. Which, on
the one hand, opens new perspectives on the intake
of different types of fat on health and fertility studies.
Acknowledgments: PIBIC (CNPq) and PIVIC UFERSA
Scholarship.
 194

Evaluation of the oil toxicity of

Astrocaryum vulgare Mart. through renal
and hepatic indicators of Wistar rats
Reginato, Fernanda Ziegler; Guex, Camille Gaube; Figueredo, Kássia Caroline;
Graiczik, James Ramires Penteado; Cassanego, Gabriela Buzatti; Pappis,
Lauren; Heck, Amanda Szymansky; Bauermann, Liliane de Freitas

Departamento de Fisiologia e Farmacologia, Centro de Ciências

da Saúde, Universidade Federal de Santa Maria.

Background/Introduction: Worldwide, the use
of natural products of plant origin grows both as
nutraceuticals and in drug therapy. Not only in
developing countries, but also in developed ones,
there is a greater acceptance of medicinal plants in
health treatment. However, many of these products
are used without studies that determine safe doses
and absence of harmful effects. Objective: We intend
to evaluate the safety associated with the use of
tucumã (Astrocaryum vulgare Mart.) fruit oil, based
on its effects on renal and hepatic parameters of
rats. Methods: Virgin tucumã pulp oil (TO), purchased
commercially, was applied daily, cutaneously, for 28
days, to three groups of five Wistar rats each (n=5),
in increasing doses of 250, 500 and 1000 mg/kg . The
control group (n=5) received saline solution. The test
followed the OECD 410 protocol and was approved by
CEUA-UFSM (8956120620). At the end of the 28 days of
treatment, the rats were euthanized. For euthanasia,
anesthesia was induced by ketamine in combination
with xylazine hydrochloride at a dose of 100 mg/kg
ketamine + 10 mg/kg xylazine (intraperitoneally).
When the absence of caudal and limb reflexes was
detected, blood was collected by cardiac puncture.
The blood was centrifuged and the serum used to
measure AST, ALT, blood urea nitrogen (BUN) and
creatinine using a semi-automatic biochemical
analyzer (Bioplus: Bio -2000). In addition, liver and
kidney were weighed and their relative weights were
determined. To analyze if the data presented a normal
distribution, the Kolmogorov–Smirnov test was used.
Results that showed a Gaussian distribution were
analyzed by parametric test one-way ANOVA followed
by Tukey’s test. Kruskal-Wallis and Dunn’s were used
as statistic tests when the requirements to perform
a parametric test were not satisfied. Difference was
considered significant when p < 0.05. Results: The
measurement of the absolute and relative weights
of liver and kidney showed no significant difference
between the different treatment groups. Likewise, the
dosage of renal markers BUN and creatinine remained
similar between the groups and within the values​​
considered normal for the species. The determination
of the activity of liver enzymes ALT and AST was
also within the reference values, however there was
a reduction in the activity of AST in the group that
received the highest dose of TO in relation to the
control group. Discussion/Conclusion: End-of-study
organ weights are relevant markers in medicinal plant
toxicity studies. Furthermore, liver and kidney are
organs very sensitive to xenobiotics and their markers
reflect the effect of the compound on the health of the
animal. Since our results were all within the normal
range and there was no difference between the
different groups, it is suggested that TO was not toxic
at the doses and conditions used. Keywords: Tucumã,
Astrocaryum vulgare, medicinal plants, topic, skin
toxicity. Acknowledgments: UFSM.
 195

evaluation of toxicity of extracts from Myrcia

tijucensis in Artemia salina leach model
Kohler, Cristofer José Weege; Perini, Camila Maria; Alberton, Michele Debiasi

Universidade Regional de Blumenau, Blumenau – SC, Brazil.

The species Myrcia tijucensis is a tree of Myrtaceae
family, widely distribute and abundant in areas of
anthropophilic forest and sandbank of Atlantic Forest.
Some trees of Myrtaceae family, like M. tijucensis, are
commonly know as “Guamirim” or “Ingabaú”. Chemical
and toxicological studies about this species is scarce.
The objetive of this study was assess the toxicity of
crude hydroalcoholic (EBH), dichloromethane (DCM)
and ethyl acetate (EBAE) extracts of M. tijucensis using
Artemia salina toxicity model according Meyer et al.
(1982). M. tijucensis leaves was collected in Blumenau
(SC), on August 2018. Leaves were separated from
the branches and ground in knife mill. The dry and
grounded material was extracted by maceration
during 7 days in solvents of different polarities. The
extracts were filtered and evaporated until dryness
in vacuum rotary evaporator. Artemia Salina larvae
grown in saline solution 38 g L-1 during 72 hours with
light and constant aeration at room temperature. The
extracts were diluted in saline solution (38 g L-1) with
Tween 2% at concentrations 1000, 500, 250 and 120
ppm. About 10 Artemia salina larvae were tranferred to
each well of a 24 wells microdilution plate containing
extracts and saline solution in the concentration cited.
The count of live and died animals was performed
after 24 hours of incubation. Tests were conducted
in triplicate using K2Cr2O7 solutions (10 to 50 ppm) as
positive control. After counting of larvae, the results
were analised by probitos statistical method. Results
show that in all concentrations tested, the extracts
of M. tijucensis presented a toxic profile (LD50 < 1000
ppm). In the lowest concentration tested (120ppm),
mortality results it were 72,50 ± 9,01, 78,10 ± 19,18 and
98,99 ± 1,74% for the extracts EBH, EBAE and DCM,
respectively. The results suggest toxicity of tested
species, opening up the possibility of further studies
in the area. So, future trials will be carried out using
other methodologies and in minor concentrations in
order to check the LD50 of these extracts.
 196

Exposure of adult female rats to different

doses of the antipsychotic Aripiprazole:
a look at the reproductive system
Silva, Aline Gabrielle Gomes1,2; Moura, Maria Joana Nogueira1,2; Pereira, Artemia Kelly
Holanda1; Felix, Renata Gleysiane de Sousa1; Angelo, Ana Beatriz Silva1; Santos, Caio
Cesar Araújo1; Silva, Ana Lucelha dos Santos1; Freire, Livia Horrana Forte1; Dantas, Joao
Artur Diogenes1; Silva, Mateus Limerio Carlos1; Borges, Cibele dos Santos1
1
 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of Bioscience,
Biological and Health Sciences Center - CCBS, Federal University of the Semi-Arid Region -
UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular Biology (PMBqBM),
State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande do Norte, Brazil.

Introduction: Numerous antidepressants and euthanized by saturation of anesthetic xylazine and

antipsychotics drugs have been continuously
approved and released for sale that have not yet
had all their effects evaluated, both in the short and
long term. These drugs are aimed at different types
of treatment of mental disorders. Among these drugs
is Aripiprazole. Aripiprazole has high affinity for
serotonin, dopamine and noradrenaline receptors
for the treatment of mental disorders, mainly
schizophrenia and depression. It is known that the
5-HT system can directly influence the reproductive
function of vertebrates, due to its communication
with the sex steroid system. This interaction
between the systems occurs through estrogen
and progesterone receptors located in central
serotonergic neurons, demonstrating an important
signaling pathway, in which it can modulate several
neural processes, including thyroid secretion and
sexual behavior. However, there are few reports of
the effects of second-generation antidepressants
and antipsychotics on the reproductive system and
fertility of females, mainly with regard to aripiprazole.
Objective: The present study aimed to evaluate the
possible impacts of exposure to different doses of
aripiprazole on the female reproductive system, with
emphasis on estrous cyclicity. Methods: Adult female
Wistar rats were divided (n=11/group) into control
group (CTRL, treated with vehicle solution - 66%
saline and 33% DMSO); group treated with 0.3mg/kg
of aripiprazole diluted in vehicle (EXP1); group treated
with 3.0mg/kg of aripiprazole diluted in vehicle (EXP2)
and group treated with 6.0mg/kg of aripiprazole
diluted in vehicle (EXP3). The animals were treated
during 15 days and the estrous cycle of the rats was
monitored. After the end of the treatment, in the first
estrus, six female rats per group were weighted and
ketamine. Blood samples were collected to hormonal
levels. Vital organs (kidney, adrenal, heart, liver, brain,
pituitary and thyroid) were collected and weighed.
The ovaries and uterus were collected, weighed
and fixed in modified Davidson’s solution (MDF) for
further histopathological processing. The study was
approved by the Ethics Committee of UFERSA 02/2020
(Protocol number 23091.014949/2019-90). Statistical
analysis: ANOVA and Tukey test for parametric data
and Kruskal Wallis and Dunn’s test for non-parametric
data (P<0.05). Results and Discussion: No alterations
were observed in the final body weight of the rats
exposed to different doses of aripiprazole, as well as
no alterations in the absolute and relative weights
of reproductive and vital organs, when compared to
the CTRL group. However, when compared between
the experimental groups, the relative weight of the
pituitary was significantly increased (p<0.05) in
the EXP1 group (0.07+0.01mg/g) in relation to the
EXP2 group (0.04+0.01mg/g). The evaluation of the
estrous cycle showed a significant decrease in the
number of estrus in the EXP3 group (2.4+0.5) when
compared to the CTRL group (5.2+0.7). On the other
hand, when performing sexual behavior, the only
group that showed a significant reduction in relation
to the CTRL group was the EXP1 group (62% x 34%;
p<0.05). Conclusion: Therefore, it can be concluded at
this moment, based on this experimental model, that
the exposure to aripiprazole during adulthood does
not cause systemic intoxication effects. However,
although subtle, it can cause impairment on female
cyclicity and/or libido. Acknowledgments: PIVIC
UFERSA Scholarship and UFERSA/PROPPG (grant
number 23091.014593/2019-02).
 197

Imidacloprid-based commercial pesticide

causes behavioral and biochemical
impairments in Wistar rats
Tonietto, Bruna Ducatti1,2; Laurentino, Ana Olívia Martins3; Costa-Valle, Marina Tuerlinckx4; Cestonaro,
Larissa Vivan1,2; Antunes, Bibiana Pereira1; Manfio, Cleofas Sates3; Santos, Nícolas Guimarães1;
Dallegrave, Eliane4; Garcia, Solange Cristina1,2; Leal, Mirna Bainy3; Arbo, Marcelo Dutra1,2
1
 Laboratório de Toxicologia (LATOX), Departamento de Análises, Faculdade de Farmácia - Anexo I,
Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre – RS. 2 Programa de Pós-Graduação
em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre - RS.
3
 Laboratório de Farmacologia e Toxicologia Neurocomportamental, Departamento de Farmacologia,
Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul (UFRGS), Porto
Alegre – RS. 4 Laboratório de Pesquisa em Toxicologia (LAPETOX), Departamento de Farmacociências,
Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre – RS.

Introduction: Imidacloprid (IMI) is a neonicotinoid MB fraction, butyrylcholinesterase, total proteins,

insecticide employed worldwide for crop protection.
IMI’s mode of action occurs through the agonism
of postsynaptic nicotinic acetylcholine receptors
(nAChRs), with high specificity for insect nAChRs
although there are reports of mammals’ toxicity.
Objective: Studies on IMI’s neurotoxicity are not
conclusive; therefore, the aim of this study was to
evaluate the behavioral and biochemical effects of
an IMI based commercial pesticide on rats. Methods:
Adult male Wistar rats received 1.5, 5, and 15 mg/kg of
an IMI suspension via the oral route for 45 consecutive
days. Behavioral toxicity was evaluated using rota-
rod for motor coordination, spontaneous locomotor
activity, and novel object recognition and OX maze
tests for memory and learning. At the end of the
treatment, the animals were euthanized, and blood was
collected for biochemical measures (urea, creatinine,
transaminases, γ-glutamil-transpeptidase, lactate
dehydrogenase, alkaline phosphatase, creatine kinase
C reactive protein, and glucose). The experiments
were approved by the University Ethics Committee
(number 37572). Results: No alterations were
observed in motor coordination and short- and long-
term memory tests. However, IMI-based insecticide
caused an increase in rearing and time spent at the
periphery in the locomotor activity test and a decrease
in time spent to finish the OX maze task (p<0.05;
ANOVA/Bonferroni). In blood, it was observed only a
significant increase in serum butyrylcholinesterase
activity (p<0.001; ANOVA/Bonferroni). Conclusion:
Subchronic administration of an IMI-based-pesticide
caused behavioral and systemic impairments in rats.
The behavioral altrations could point to an anxiogenic
effect. The increase in serum BuChE activity is
possibly a way to metabolize the acetylcholine excess.
Keywords: imidacloprid, acetylcholine, locomotor
activity, OX maze, butyrylcholinesterase.
 198

Immediate toxicological impairment of

Lisdexamfetamine Dimesylate in male rats
treated during juvenile period and periuberty
Stein, Julia1; Jorge, Bárbara Campos1; Reis, Ana Carolina Casali1; Nagaoka, Lívia Trippe1;
Manoel, Beatriz de Matos1; Valente, Letícia Cardoso1; Arena, Arielle Cristina1,2
1
 Department of Structural and Functional Biology, Institute of Biosciences of
Botucatu, UNESP – Univ. Estadual Paulista - Botucatu, São Paulo State, Brazil.
2
 Toxicological Assistance Center (CEATOX), Institute of Biosciences of Botucatu,
UNESP – Univ. Estadual Paulista - Botucatu, São Paulo State, Brazil.

Background: Attention deficit hyperactivity disorder
is one of the most diagnosed neuropsychiatric
disorders in school-age children and adolescents.
The pharmacological treatment is carried out
with psychostimulants, being Lisdexamfetamine
Dimesylate (LDX), a prodrug of dextroamphetamine
(D-AMF), one of the most used. The safety profile of
this drug is similar to other psychostimulants, But
it is still conflicting and needs to be better studied,
mainly when the target population belongs to a
critical period of development, such as peripuberty.
Considering this fact and the benefits of its use,
studies regarding aspects of toxicological evaluation
and drug safety in this period are still scarce.
Objective: the aim of this study was to evaluate the
toxicological impacts immediately after exposure to
LDX during the juvenile period to peripuberty in male
rats. Methods: Male Wistar rats (23 days old) were
divided into 4 groups: Control (deionized water); and
three different doses of LDX: 5.2; 8.6 and 12.1 mg/
kg/day. They were treated for 31 consecutive days,
orally (gavage). During the treatment, clinical signs
of toxicity, body weight, water and food consumption
and play behavior were evaluated. On postnatal day
(PND) 54, the animals were killed for organ weight,
hematological and biochemical analysis. Results: We
observed a reduction in the daily food consumption in
all experimental groups compared to the control, as
well as in the daily water consumption in the group
exposed to 8.6 and 12.1 mg/kg of LDX. Also, during
treatment, the animals in the highest dose group
had a reduction in body weight since the 15th day of
treatment (PND 37) until the end. On play behavior
evaluation, the animals of the highest dose group
presented a reduction in the percentage of social
behaviors. We observed an increase in the relative
weight of the liver (12.1 mg/kg), the spleen (12.1 mg/kg)
and the seminal gland (8.6 and 12.1 mg/kg), as well as
a reduction in the erythrocyte count and total protein
levels in these groups and reduction of albumin level
in the lowest dose group. Discussion/Conclusion:
The data regarding body weight and food intake are
supported by previous finds in the literature, being
able to corroborates with the information regarding
this growth delay in children. The altered organ weight,
hematological and biochemical parameters point
to systemic toxicity of this substance. Thus, we can
conclude that the continuous use of LDX during this
period can lead to significant toxicological changes
in the parameters and organs evaluated, negatively
impacting the health of the individual during its
exposure. Histopathological analyses of liver and
kidney are underway to support our hypothesis of LDX
toxicity. Acknowledgments: FAPESP (2021/04119‑9)
and Capes (88887.602916/2021-00). Ethics
committee: 3148130421
 199

Investigation of the lipid-lowering

and antiatherogenic effects of Plinia
cauliflora bark extract in an experimental
model of atherosclerosis
Dalmagro, Mariana1; Donadel, Guilherme2; Pinc, Mariana Moraes3; Berta, João Antonio4; Borba,
Maria Eduarda4; Gasparotto Junior, Arquimedes5; Jacomassi, Eliza6; Zardeto, Giuliana6; Hoscheid,
Jaqueline6; Ceranto, Daniela de Cassia Faglioni Boleta6; Lourenço, Emerson Luiz Botelho7
1
 Student Master in biotechnology applied to agriculture UNIPAR; 2 PhD student in
animal science at UNIPAR; 3 PhD student in biotechnology at UNIPAR; 4 Undergraduate
Student Veterinary Medicine UNIPAR; 5 Professor at the University of Grande Dourados
–UFGD; 6 Professor at UNIPAR; 7 Unipar Research and Graduate Coordinator.

Introduction: Atherosclerosis is the main cause
of morbidity and mortality, making it worrying
for humans and causing an incessant search for
experimental models to better characterize it and
for therapeutic alternatives to prevent or reduce its
evolution and severity. Because of this, studies with
supplements and medicinal plants have become very
promising. Among the plants that have potential for
the prevention and treatment of atherosclerosis, we
have Plinia cauliflora, which according to literature
data is effective as a vasodilator and vascular oxidative
stress reduction agent. Objective: Thus, the objective
of the present work was to evaluate the lipid-lowering
and antiatherogenic effect of P. cauliflora bark extract
in an animal model. Materials and Methods: With
the approval of the UNIPAR ethics committee, male
New Zealand rabbits with approximately 2 kg of live
weight were used, which were divided into 6 groups
with 6 animals per group, being the negative control,
control positive, doses of 10, 30 and 100 mg/kg of
the extract and simvastatin 2.5 mg/kg group, thus
totaling 36 animals. The negative control group was
fed a normal commercial diet and the other groups
were fed a high-cholesterol diet (CKD) for 60 days.
Thirty days after the beginning of the diet with CKD,
treatment was started orally with P. cauliflora extract
at doses of 10, 30 and 100 mg/kg and with simvastatin
2.5 mg/kg, once a day, for 30 days. At the end of 60
days, the animals were euthanized and blood was
collected for analysis of biochemical parameters and
indices of cholesterol and serum lipids, along with
histopathological analysis of the metabolizing and
cardiovascular organs (arteries, heart, liver, spleen
and kidneys). Results and Discussion: The results
showed that oral administration of P. cauliflora
extract significantly reduced cholesterol and
triglyceride levels in animals fed CKD when compared
to the positive control group and simvastatin. In
addition, when we looked at the lesions in the aorta,
abdominal and iliac arteries, the reduction occurred
in all treatments. Regarding the relative weights of
the organs, no significant differences were observed
in relation to the treated groups. The animals treated
with CKD did not show increases in liver enzymes
(ALT and AST) when compared to the negative control
group. However, an increase in these enzymes (ALT
and AST) was observed in the group treated with a
dose of 100 mg/kg of the extract, which may indicate
the beginning of liver injury, however, these can also
be found in other metabolizing organs. Conclusion:
In view of the results found, it can be concluded that
all doses of Plinia cauliflora bark extract analyzed in
the study were effective in reducing cholesterol and
triglycerides, thus, it has the potential to be a new
supplement to be used directly in the treatment and
prevention of atherosclerotic disease. Keywords:
Atherosclerosis, Cholesterol-rich diet, Animal Model,
Plinia Cauliflora
 200

Levamisole, a cocaine adulterant, promotes

acute and subchronic toxic effects in wistar rats
Laurentino, Ana Olívia Martins1,2; Salomón, Janaína1; Sebben, Viviane3; Tonietto, Bruna Ducatti4; Cestonaro,
Larissa Vivan4; Dallegrave, Eliane5; Garcia, Solange4; Arbo, Marcelo Dutra4; Leal, Mirna Bainy1
1
 Programa de Pós-graduação em Ciências Biológicas: Farmacologia e Terapêutica da
Universidade Federal do Rio Grande do Sul; 2 Curso de Psicologia da Universidade do Sul de
Santa Catarina; 3 Centro de Informação Toxicológica do RS, 4 Programa de Pós-graduação em
Ciências Farmacêuticas da Universidade Federal do Rio Grande do Sul; 5 Programa de Pós-
Graduação em Patologia da Universidade Federal de Ciências da Saúde de Porto Alegre.

Introduction: Levamisole (LVS) is an anthelmintic that
was withdrawn from the USA market due to its clinical
complications, but it still can be found in Brazilian
market. It is a cheap white powder that acts as a
nicotinic agonist in the worms. Studies shown that LVS
participates in the release of some neurotransmitters
and it is metabolized in some active metabolites, such
as aminorex, which has amphetamine-like effects.
However, since 2002, the use of LVS as a cocaine
adulterant increased worldwide and many case
reports associate diseases, such as agranulocytosis,
systemic vasculitis, neutropenia, and renal injury, with
LVS-adulterated cocaine use. Nevertheless, there are
no consistent data about toxicity of LVS. Objective:
The aim of this study was to evaluate acute and
subchronic toxicity of LVS in Wistar rats. Methods:
This study was approved by CEUA/UFRGS (protocol
nº: 34357). Male adult Wistar received saline (control
group) or LVS at the doses of 12 mg/kg, 24 mg/kg
and 36 mg/kg in the acute toxicity assay (n=5/group)
(adapted from protocol OECD 420); and at 3 mg/kg, 6
mg/kg and 12 mg/kg in the subchronic toxicity assay
(n=10/group) (adapted from protocol OECD 407). All
the administrations occurred by intraperitoneal route.
Doses were calculated through the relative doses
usually ingested by users considering recent studies
and then, it was extrapolated for the treatments.
Toxicity was investigated through behavioral (rotarod,
spontaneous locomotor activity and novel object
recognition test), renal, hematological, biochemical,
and histopathological parameters. Results: In
the acute toxicity assay we observed behavioral
alterations in all LVS groups (p < 0.05), decreased
GSH levels and urinary alterations in LVS 24 mg/kg
group (p < 0.05), and death (p < 0.05) and macroscopic
alterations in LVS 36 mg/kg group. Moreover, in the
subchronic toxicity assay we observed behavioral
alterations in the rotarod test in LVS 6 and 12 mg/
kg groups (p ≤ 0.001 and p < 0.0001), decreased
recognition index in short and long-term memory
tests in LVS 6 mg/kg group (p < 0.05), increased
liver relative weight in LVS 6 and 12 mg/kg groups
(p < 0.0001), increased ALP activity in LVS 6 mg/kg
group (p < 0.05), increased erythrocytes GSH levels
in LVS 12 mg/kg group (p < 0.01), and decreased total
thiols levels in the liver of all LVS groups (p ≤ 0.0001).
Besides, it was also observed some hematological,
urinary, and macroscopic alterations in all LVS groups
(p < 0.05). Conclusion: LVS promoted toxic effects and
its use as a cocaine adulterant is unsafe. We hope
that our results contribute to the clinical practice
by helping to correctly diagnose the unusual clinical
manifestations found in cocaine users and avoiding
more critical complications. Acknowledgments:
CREAL-UFRGS, CNPq, CAPES, FAPERGS (Process nº
33416.465.34568.26032018), and PROPG/UFRGS for
the support.
 201

Maternal exposure to a phthalate

combination associated with prostate
lesions and cancer in F1 and F2 offspring
Aquino, Ariana Musa1; Costa, Luiz Guilherme Alonso1; Santos, Sérgio Alexandre Alcantara1; Magosso,
Natália1; Souza, Patrick Vieira1; Rocha, Vanessa Aguiar1; Oliveira, Marcos Antônio Fernandes1; Barbisan,
Luis Fernando1; Justulin Junior, Luís Antonio1; Flaws, Jodi A.2; Scarano, Wellerson Rodrigo1
1
 Department of Structural and Functional Biology, Institute of Biosciences, São Paulo
State University (UNESP), Botucatu, São Paulo, Brazil; 2 Department of Comparative
Biosciences; University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

Phthalates represent a class of molecules detected
in different concentrations in breast milk, in the urine
of pregnant women, and in the maternal-fetal tissue.
Previous studies have shown that perinatal exposure
to isolated phthalates increased susceptibility to
prostatic perturbations. Thus, the study seeks to
investigate proteomic profile and identify the most
important pathway altered on ventral prostate (VP)
of rats exposed to a mixture of phthalates in the
perinatal period (F1), as well as their descendants (F2).
For this, Sprague-Dawley pregnant rats were exposed
to different concentrations of phthalates mixture (C:
control; vehicle; T1:20µg/kg/day and T2:200mg/kg/
day). The pregnant females received treatment by
oral route from gestational day (GD) 10 to postnatal
day (PND) 21. Exposed and unexposed animals were
mated to obtain the F2 generation. Male rats were
euthanized on PND22 and PND120 (F1) and PND22
(F2). At the end of the treatment, VPs from all groups
were collected for proteomic analysis. The data were
analyzed by string platform, to create a PPI network
and enrichment analysis; PiNet platform (to show
interaction with kinases) and WebGestalk platform
were performed to evaluate the pathway enrichment.
The results showed a great number of downregulated
proteins among treatments and generations. Among
them, Rabs, Histone and, Chaperones clusters were
altered in both treated groups and generations.
Fourteen downregulated proteins interacted with
one or more Kinase (39), that are involved in VEGF,
Wnt and GnRG signaling, and Aldosterone synthesis
and secretion. Furthermore, there were five proteins
(Ywhae; Ywhag; Ywhaz; Hsp90ab1 and Hsp90b1) which
have enriched for the Pi3k-Akt signaling pathway, the
most deregulated pathway in prostate cancer and
other prostatic disorders. In conclusion, the exposure
of fetuses and newborns to this mixture, containing
the most abundant phthalates found in the urine of
pregnant women, alter proteins and cell pathways
related to prostate homeostasis, increasing the
susceptibility for developing prostatic diseases in F1
and F2 generations. Ethical approval: 1040/CEUA.
Funding support and Acknowledgments: FAPESP:
(2017/08306-2; 2018/50002-3; 2018/09510-5 and
2019/13823-1) and CAPES.

Methylmercury toxicity induces structural
and cellular damage in cardiac ventricles
Krüger, Nathália Ronconi Zilli; Pinheiro, Jacqueline; Simioni, Carmen; Nazari, Evelise Maria
Organisms exposed to methylmercury (MeHg) usually
present morphological, metabolic and behavioral
damage, and the central nervous system is an
important target of this metal. However, the effect of
MeHg on heart development remains poorly explored.
The heart is the first functional organ in vertebrate
embryos, modeling itself from a single-chamber
tubular structure to a four-cavity organ. Thus, the
aim of this study was to investigate the effects of
a single dose of MeHg on heart morphology and on
cardiomyocytes compartments in cardiac ventricles,
using Gallus domesticus embryos as a model. Fertilized
eggs were incubated at 37.5ºC (± 0.5) and 65% humidity.
At 33 hours of incubation (i.e., E1.5, corresponding
to the developing heart with a single chamber),
embryos were exposed in ovo to 0.1 µg MeHg/50 µL
saline solution, being analyzed at 10th embryonic day
(E10, corresponding to the developing heart with four
chambers) (n = 45 embryos). Control embryos were
exposed to 50 µL of saline solution at E1.5 and analyzed
at E10 (n = 45 embryos) (Ethics Committee of the
Federal University of Santa Catarina, CEUA - protocol
5843231018). For morphological analysis, the heart
was submitted to light microscopy and transmission
electron microscopy techniques. Initially, a significant
reduction in heartbeats was observed after exposure
to MeHg (76 ± 6 heartbeats/min) compared to the
control (104 ± 8 heartbeats/min; p < 0.01). About
60% of embryos exposed to MeHg showed changes
in the tissue organization of the heart. Therefore,
changes in the morphology of most embryos exposed
to MeHg were observed, such as differences in the
202
Laboratório de Reprodução e Desenvolvimento Animal /
Universidade Federal de Santa Catarina (LRDA/UFSC).
organization of the walls of the cardiac ventricles and
in the appearance and consistency of the trabecular
muscle fibers. Moreover, after exposure to MeHg,
a reduction in the thickness of the left ventricular
wall was observed (61.78 μm; ± 17.86) compared to
the control (154.83 μm ± 25.90; p < 0.05), while for
the right ventricle no differences were observed.
Additionally, MeHg induced in 100% of exposed
embryos, the appearance of ultrastructural changes
in the ventricular trabeculae, as well as subcellular
changes in mitochondria (dilatations in mitochondria
ridges), increase in perinuclear space and increase
in the number of vesicles (electrontransparent,
electrondense and autophagic). These results
demonstrate that exposure to MeHg was able to
promote important cellular changes in the walls of the
heart chambers. Morphological changes, such as the
observed reduction in trabecular thickness, have been
shown to compromise the structure of trabecular
cardiomyocytes. These alliterations may be closely
related to the reduction in heart rate. Furthermore,
the interference of MeHg in the heartbeat of embryos
is a determining factor for the development of the
embryo. The morphological evaluation performed in
this study can be used as an indication of patterns
for visualization such as biomarkers. We would
like to thank the Fundação de Amparo à Pesquisa
e Inovação do Estado de Santa Catarina (FAPESC),
the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES), the Laboratório Multiusuário
de Estudos em Biologia (LAMEB) and the Laboratório
Central de Microscopia Eletrônica (LCME).
 203

Mitochondrial injury induced by Diuron

and its metabolites in the rat liver
Seloto Danielle Gabriel1,2; Lima Thania Rossi Rios Rossi1,2; Camargo
João Lauro Viana1,2; Pereira Lilian Cristina2,3.
1
 São Paulo State University (Unesp), Medical School, Botucatu; 2 Center for Evaluation
of Environmental Impact on Human Health (TOXICAM), Botucatu, São Paulo, Brazil;
3
 São Paulo State University (Unesp), School of Agriculture, Botucatu.

Background: The use of pesticides is of concern
because of the possible toxic effects on human health.
Among herbicides, Diuron, a compound derived from
urea, is the twelfth best-selling herbicide in Brazil,
despite its classification by the North American
Environmental Protection Agency (USEPA) as “probable
carcinogen to the human species”. Successive studies
on the potential carcinogenic mode of action (MoA) of
Diuron in the urothelium of rats have been published
by our research group TOXICAM in journals with
selective editorial policy abroad. However, it remains
unknown what is the mechanism of cytotoxic action
of this herbicide, and which of its forms is responsible
for cytotoxicity, whether the parental Diuron or any
of its metabolites. Objective: Considering that the
initial biotransformation of Diuron occurs in the
liver, the present study aims to assess the in vitro
damage induced in mitochondria isolated from the
liver after exposure to Diuron and its metabolites,
DCA and DCPMU. Methods and Results: Hepatic
mitochondrial isolation was performed by differential
centrifugation and the mitochondrial membrane
potential was analyzed using safranine-ο as a probe;
a significant decrease in the membrane potential was
observed at highest concentration. Mitochondrial
Respiration was monitored polarographically in
an oxygraph equipped with a Clark electrode using
succinate and glutamate and malate as oxidizable
substrates. Mitochondrial swelling was assessed
by the decrease in the apparent absorbance of the
suspension of mitochondria resulting from a decrease
in the turbidity of the suspension. Since mitochondrial
swelling was observed after exposure to DCA 500
µM, the effect of substances against mitochondrial
swelling modulators was evaluated: Cyclosporin A
(3 μM), Ruthenium Red (0.5 μM and 3 μM) , N-Ethyl-
Maleimide (NEM) (15 μM) and N-acetyl-cysteine ​​(NAC)
(12 μM). Discussion/Conclusion: Our findings suggest
that Diuron and its metabolites can be classified
as mitotoxicants, inducing uncoupling of oxidative
phosphorylation, which was evidenced by the
dissipation of the mitochondrial membrane potential
and the consumption of basal oxygen. In addition,
DCA appears to play an important role in the chemical
toxicity, since the metabolite caused mitochondrial
swelling at 500 µM, a morphofunctional indicator
of severe organelle damage. Acknowledgments:
Supported by São Paulo Research Foundation
(FAPESP) [Grant No. 2017/25402-5] and National
Council for Scientific and Technological Development
(CNPq) [Grant No. 133611/2019-1].
 204

Paternal Origins of Health and Disease: BaP

exposure via paternal germ cells causes
negative reproductive consequences
in female offspring (F2) in rats
Jorge, Bárbara Campos1; Stein, Julia1; Reis, Ana Carolina Casali1; Nogueira, Jéssica
Bueno1; Paschoalini, Beatriz Rizzo1; Moreira, Suyane da Silva1; Manoel, Beatriz de
Matos1; Kassuya, Cândida Aparecida Leite2; Arena, Arielle Cristina1,3
1
 Department of Structural and Functional Biology. PPG Pharmacology and Biotechnology,
Institute of Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São Paulo
State, Brazil; 2 College of Health Science, Federal University of Grande Dourados, Dourados,
Mato Grosso do Sul, Brazil; 3 Center of Toxicological Assistance (CEATOX), Institute of
Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São Paulo, Brazil.

Background: Benzo(a)pyrene (BaP) is a ubiquitous
chemical and has recently been classified as an
endocrine disruptor. In our research lab it has
already been shown to have harmful effects on the
reproduction of males exposed to BaP, but whether
these changes persisted until F2 generation in their
female offspring was still unknown. Objective:
to evaluate the reproductive repercussions of F2
generation in female offspring rats after exposure to
BaP via paternal germ cells. Methods: Pubertal males
were allocated to two experimental groups: control
(vehicle) and BaP-exposed (0.1 μg/kg) during post-
natal day (PND) 23 to 53 by gavage; and in adulthood
they were mated with unexposed females to generate
F1. The males of the F1 generation, in adulthood, were
mated with unexposed females to generate F2. In
this study, we evaluated the sexual development of
the female rats, as well as estrous cyclicity, fertility
potential, sexual behavior, hormone dosage, and
the histological structure of the ovaries and uterus.
All data obtained in this analysis were submitted to
statistical tests. Results: The anogenital distance
(AGD) were unchanged in all measurements, but
the females of the BaP-group showed a decrease
in weight on PND1 and 13. There was a precocious
vaginal opening and first estrus in the BaP-group.
The frequency of lordosis in female sexual behavior
was not significantly altered, but the score of
lordosis was decreased in the BaP-group. In addition,
the fertility of the F2 female offspring decreased
when compared to control, as well as an increase in
placental weight and pre-implantation loss rate. The
percentage fetuses large for gestational age was
increased in the BaP-group. Estradiol, progesterone,
FSH and LH levels remained similar between the
experimental groups. In the follicular composition of
the ovarian on PND 90, the percentage of atrophic
follicles increased and the corpora lutea had an
abrupt decrease in the BaP-group. The morphometric
measurements of the uterus showed an increase in
myometrial thickness and luminal area in the BaP-
group. Discussion/Conclusion: We can conclude
that F2 generation showed impairment reproductive
parameters, with negative transgenerational impacts
in this experimental models, probably due to changes
in the germ cells of the generation directly exposed
to benzo(a)pyrene by epigenetic mechanisms
(next analyses). Ethics committee, nº 1148/2019.
Acknowledgment: CNPq – 140826/2019-0; FAPESP -
2019/03264-5; 2019/03284-6.
 205

Pathological findings arising from

continuous exposure to trace concentrations
of organochlorine DDT residues in
multigeneration of wistar rats
Guimarães, Ana Tereza Bittencourt1; Silva, Fernanda Coleraus1; Quiozini, Nathaly de Matos1; Simon,
Jaqueline1; Pavlak, Jaíne Luana1; Azevedo, Camilla de Marchi Sanches1; Rangel, Ana Lúcia Carrinho Ayroza2
1
 Department of Biosciences and Health, Universidade Estadual do Oeste do Paraná, UNIOESTE, Brazil.
2
 Department of Oral Pathology, Universidade Estadual do Oeste do Paraná, UNIOESTE, Brazil.

Introduction: Dichlorodiphenyldichloroethylene described qualitatively. Results: We identified the

(DDE) and dichlorodiphenyldichloroethane
(DDD) are decomposition products of
dichlorodiphenyltrichloroethane (DDT). These
compounds are equivalent in the aspects of
environmental contamination and toxic effects.
Despite extensive knowledge about these substances,
investigations are needed on the synergism of
DDD and DDE at trace concentrations, simulating
environmental conditions of exposure to multiple
generations. Objectives: Describe the pathological
findings of multiple generations of Wistar rats
exposed to trace concentrations of organochlorine
residues of DDT. Methodology: The study (CEUA/
Unioeste protocol n. 21-20) was carried out with two
generations (F1 and F2) of Wistar rats, exposed to trace
concentrations of organochlorine DDT residues (DDD
and DDE), since the period intrauterine until the end
of life. These offspring were obtained from parental
generations (10th week), not consanguineous. The
groups were defined from day 0 of pregnancy as:
Control, DDD (0.0147 µM of DDD), DDE (0.0056 µM
of DDE) and DDD and DDE (association of the two
substances). The placement of substances was carried
out via water ad libitum, continuously, throughout
the life of the multigenerations. At the end of the 5th
and 15th week of life of the F1 and F2 generations,
males (n=6-8 per group) and females (n=6-8 per
group) were euthanized with specific anesthetics,
followed by exsanguination and submitted to autopsy
for identification of morphological changes in
internal organs (heart, kidneys, liver, pancreas, brain,
adipose tissue and organs of the male and female
reproductive system). Cases of urinary bladder
stones and their frequencies were compared between
groups, in each generation, using the Chi-Square
test for k proportions, followed by Marascuilo test.
Histopathological studies were performed according
to the identification of macroscopic findings and
presence of pathological morphological changes in
the renal and hepatic systems. In a male from the
DDD group (5th week, F1) the presence of changes in
appearance, loss of turgidity and asymmetry in size
and weight of the right kidney (1.76x1.2 cm; 0.842 g)
in relation to the left kidney (1.48 x0.89 cm; 0.530 g).
In the 15th week of both generations, urinary bladder
stones were found, which more frequent in the DDE
group (F1 = 57%; F2 = 33%) when compared to the
other groups, with significant differences between
this group and Control, especially in F1 generation
(p=0.048). Regarding the hepatic system, in the 15th
week of F2 generation, the presence of liver nodules
was identified in a male from the DDE group and a
female from the DDD/DDE group. The nodules were
similar, both sessile, measuring approximately 343
mm3, with a translucent surface and serous-looking
liquid content. Through histopathological analysis, a
cavity surrounded by dense fibrous connective tissue
was observed, associated with intense angiogenesis
and chronic inflammation, predominantly plasmacytic,
with Russell bodies and eosinophils. Additionally,
the presence of xanthomatous macrophages was
observed bordering the cavity lumen, which was filled
with mucoid material, suggesting a possible evolving
hepatitis. The other analyzed organs showed no
morphological alterations. Discussion/Conclusion:
Studies show the residues of the organochlorine
DDT, mainly with regard to DDE, as inducers of
nephrotoxic and hepatotoxic effects. However, the
mechanisms and toxic effects generated by long-
term exposure to low concentrations still generate
gaps, especially with regard to the chronicity of the
effects in multiple generations. Thus, although these
findings are preliminary, these described pathologies
suggest possible relationships with exposure to trace
concentrations of DDT residues. Acknowledgments:
Cardiff University, GCRF, CNPq.
 206

Production and in vitro toxicological

studies of natural dyes
Yli-Öyrä, Johanna1; Herrala, Mikko1; Albuquerque, Anjaina Fernandes2; Farias, Natália Oliveira2; Morales,
Daniel Alexandre2; Räisänen, Riikka3; Freeman, Harold S.4; Umbuzeiro, Gisela de Aragão2,4; Rysä, Jaana1
1
 School of Pharmacy, University of Eastern Finland, Kuopio, Finland; 2 School of Technology, University
of Campinas, Limeira, Brazil; 3 Helsinki Institute of Sustainability Science, Craft Studies, University of
Helsinki, Helsinki, Finland; 4 Wilson College of Textiles, North Carolina State University, Raleigh, USA.

Background: Massive amounts of synthetic dyes
and pigments are used around the world. In textile
dyeing, the consumption of fresh water contributes
to shortages and contamination of freshwater bodies.
Also, the dye synthesis is problematic when it requires
the use of toxic chemicals such as heavy metals.
Additionally, certain workers can become sensitized
to these compounds, and the incidence of some
cancers is higher in highly exposed workers. Due to
these problems, a new take on the natural colorants
is emerging. The goal is to develop biodegradable,
renewable, and sustainable alternatives to synthetic
dyes. There is a long tradition of harvesting colorants
from natural sources. Some of these include fungal
anthraquinones and bisorbicillinols, and indigo from
plants. More recently, some of the color-coding
genes have been identified. They could be inserted
to a host, enabling colorant production in large-scale
tanks, reducing the need for farming land. In addition,
waste material could be deployed following circular
economy principles. Several colorants can also be
used in waterless dyeing media, supercritical CO2,
reducing the need for freshwater resources. One dye
source of interest is Cortinarius sanguineus fungus,
a producer of several Sc-CO2 soluble anthraquinone
dyes. Objective: Despite their commercial potential,
almost nothing is known about the toxic properties
of the anthraquinones produced by C. sanguineus.
Our aim was to study their toxicity in vitro to assess
their acceptability for widescale use. Methods: First,
dermocybin and dermorubin were extracted from
bloodred webcap by multiple liquid-liquid partition
and their purity was verified by HPLC-DAD-MS and
NMR. Their genotoxic potential was studied in five
strains of Salmonella typhimurium in a miniaturized
Ames test protocol with and without S9 fraction.
Thereafter, the dyes were studied in human HepG2
hepatocytes and human THP-1 monocytes. The cells
were exposed to six dermocybin and dermorubin
concentrations of 0.035-7 and 0.03-10 µg/ml,
respectively. Following the exposure, cell viability was
studied with MTT and lactate dehydrogenase (LDH)
assays, cytotoxicity with propidium iodide–digitonin
(PI-DI) assay, and oxidative stress endpoints with
H2DCFDA (DCF), dihydroethidium (DHE), and MitoSOX
assays. In addition, their allergic potential was tested
with KeratinoSens, a reporter gene assay kit which
follows OECD in vitro skin sensitization guidelines.
Results: High-purity fungal dyes were obtained.
Neither caused genotoxic effects. Dermocybin caused
slight cytotoxicity in HepG2 cells in the LDH assay
at the two highest concentrations, and reduced the
HepG2 cell viability in MTT test to 76.7% at the highest
concentration of 7 µg/ml. Dermocybin also caused
mitochondrial superoxide production in both cell lines
at the two highest concentrations, 3.5 and 7 µg/ml.
Dermorubin results were similar: some cytotoxicity
was detected in LDH assay with both HepG2 and THP-
1 cells at the concentrations of 1-10 µg/ml. No effects
were seen in other viability tests. Dermorubin also
caused mitochondrial superoxide production in THP-1
cells at the 10 µg/ml concentration. In KeratinoSens
assay, neither dye caused the sensitization pathway
activation. Conclusions: As the interest in the natural
dyes increases, the need for toxicity studies arises.
Natural origin is not a safety guarantee. Our results
offer new information on the potential toxicity of
natural anthraquinone dyes. However, more studies
are needed on natural anthraquinone mixtures,
which is normally the form of the dyes when first
isolated from plants. Their ecotoxicological potential
should also be evaluated. Acknowledgments: This
research is a part of the BioColour (Bio-based Dyes
and Pigments for Colour Palette) Consortium funded
by the Strategic Research Council at the Academy of
Finland. In addition, this study was funded by FAPESP
and CAPES. A travel grant was awarded by the UEF
Water research community.
 207

Reproductive repercussions in adulthood

of male rats exposed to Lisdexamfetamine
Dimesylate on peripuberty
Stein, Julia1; Jorge, Bárbara Campos1; Reis, Ana Carolina Casali1; Nagaoka, Lívia Trippe1; Manoel,
Beatriz de Matos1; Valente, Letícia Cardoso1; Pupo, André Sampaio2; Arena, Arielle Cristina1,3
1
 Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, UNESP
– Univ. Estadual Paulista - Botucatu, São Paulo State, Brazil. 2 Department of Biophysics
and Pharmacology, Institute of Biosciences of Botucatu, UNESP – Univ. Estadual Paulista
- Botucatu, São Paulo State, Brazil. 3 Toxicological Assistance Center (CEATOX), Institute of
Biosciences of Botucatu, UNESP – Univ. Estadual Paulista - Botucatu, São Paulo State, Brazil.

Background: Lisdexamfetamine Dimesylate parameters. Results: No experimental group had

(LDX), is the newest psychostimulant drug with
prolonged action and the first and unique prodrug of
dextroamphetamine (D-AMF) used in the treatment
of attention deficit hyperactivity disorder, a condition
most associated with children and adolescents, with
a higher incidence in male sex. This treatment aims
to increased neurotransmission levels, like dopamine
and noradrenaline, improving the symptoms of this
disorder and bringing a lot of benefits. On the other
hand, this can compromise hormonal pathways,
affecting the release of hormones, including
those related to reproductive function, which can
negatively impact the reproductive system of this
individuals. Besides that, the gonads are susceptible
to direct influence of neurotransmitters derived
from local synaptic innervation or brought by the
circulation, so, altered levels may impact its normal
function. Objective: evaluated the impacts of LDX
in reproductive and health parameters in adulthood
of male rats exposed to this drug on peripuberty.
Methods: Male Wistar rats (23 days old) were divided
into control group (deionized water) and three groups
exposed to LDX in therapeutical doses (correct by
body surface of the rat): 5.2; 8.6 and 12.1 mg/kg/
day. The treatment occurred from post-natal day 23
(PND) to 53, by gavage. During the treatment, puberty
onset, an important reproductive parameter, were
evaluated. In adult life (PND 90), the animals were
evaluated in relation to male sexual behavior and
fertility parameters. On PND 120, the animals were
killed to blood and organ collection, to evaluation
of organ weight, sperm motility and hematological
the day of preputial separation altered compared to
control, but the weight of the animals in the highest
dose group were reduced in this day. The male sexual
behavior was not altered between the groups. In the
fertility test, also in the 12,1 mg/kg animals, we found
a reduced placental weight and increased percentage
of fetuses small for gestational age, as well as an
increased number of corpora lutea, implantations and
live fetuses. The experimental groups did not differ
among themselves in the organs weight. Besides that,
there were an increased in type C sperm (immobile)
in the animals exposed to 8,6 mg/kg of LDX and
hematological alterations (reduced neutrophiles and
increased lymphocyte percentage) in the lowest dose
group. Discussion/Conclusion: The reduced placenta
can be related to a higher incidence of fetuses small
for gestational weigh, and it can be related to paternal
influence since placental development depend on
both from maternal and paternal influence. However,
the others fertility alterations may be related to
maternal facts, independent of this treatment. The
altered motility may represent an important alteration
in sperm function which could compromise the
reproductive system, but further analyses are being
performed to confirm and support its finds. Thus, so
far, LDX was able to impact negatively some important
parameters related to reproductive function of the
animals, but other analyses should be conducted to
enrich these statements. Acknowledgments: FAPESP
(2021/04119-9) and Capes (88887.602916/2021-00).
Ethics committee: 3148130421
 208

Skeletal abnormalities caused by L-mimosine:

development toxicity study in rats
Almeida, Elaine Renata Motta1; Pereira, Edimar Cristiano2; Hueza, Isis Machado1,2
1
 Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade
de São Paulo, São Paulo, S.P., Brasil; 2 Instituto de Ciências Ambientais, Químicas e
Farmacêuticas, Universidade Federal de São Paulo (ICAQF-UNIFESP), Diadema, S.P., Brasil.

Leucena leucocephala is an invasive plant that has
attractive properties for use in folk medicine to treat
different conditions, such as diabetes, antioxidant,
gastric diseases and abortifacient. Its active
ingredient is L-mimosine, a metabolite that, although
much investigated for its antitumor properties, is
poorly studied during fetal development. Thus, we
aimed to evaluate the possible maternal-fetal toxicity
in Wistar rats treated with increasing oral doses of
L-mimosine during pregnancy. Thirty-four pregnant
rats were divided into 4 groups: control (n=8); and
3 experimental groups treated with 60 (n=9), 100
(n=8) and 140 (n=9) mg/kg of L-mimosine. which was
administered orally from the 6th to the 19th gestational
day (GD). The control group only received the vehicle
through the same route and period. Feed and water
were provided ad libitum. Every 3 days the rats’ weight
and feed consumption were measured. In the 20th GD,
an exploratory cesarean section was performed for
maternal-fetal assessment and blood and lymphoid
organs (thymus and spleen) were also collected.
During the experimental period, no statistically
significant changes were observed between groups
in feed and water consumption (P>0.05). Likewise,
the hematological and biochemical parameters were
within the reference range and without statistical
differences between the groups, with the exception
of the ALT enzyme, which was increased in the groups
that received 60 and 100 mg/kg of L-mimosine
(P<0.05). No statistical differences were observed
in relation to the relative weight of lymphoid organs
and spleen cellularity of these animals, showing
no evidence of immunotoxicity. In relation to
reproductive parameters, no changes were observed
between the groups. There was a decrease (P<0.05)
in weight gain (WG) of the 100 L-mimosine group
between GD15 and GD18, but without impacting the
total WG of these animals. Skeletal analysis showed
an increase in the number of affected fetuses only in
the higher dose group (140 mg/kg) and an increase
in delayed ossification of the skull and in sternal and
vertebral bodies variations in all treated groups, in
relation to control. Thus, with the results obtained in
this study of the administration of L-mimosine during
pregnancy, it can be seen that the doses used were
sufficient to cause skeletal variations, although they
were not sufficient to change the other parameters
evaluated here in a dose-dependent manner. These
results suggest a negative effect of L-mimosine
on the skeletal development of the animals, which
may be an indication of toxicity on the offspring.
Complementary visceral analysis studies will help to
understand these results. Keywords: immunotoxicity,
skeletal variations, phytotoxin
 209

Study of the toxicological effects of thimerosal

and aluminum hydroxide on Danio rerio’s kidney
Galiciolli, Maria Eduarda A.; Silva, Juliana F.; Guiloski, Izonete C.; Oliveira, Cláudia S.

Programa de Pós-graduação Strictu sensu Aplicada à Saúde da Criança e do Adolescente. Instituto de

Pesquisa Pelé Pequeno Príncipe, Curitiba, PR, Brazil; Faculdades Pequeno Príncipe, Curitiba, PR, Brazil.

Vaccination is considered one of the greatest
techniques in terms of disease control and eradication.
However, with the decrease in cases of vaccine-
preventable diseases, the population’s attention has
turned to the potential adverse effects caused by
vaccines, especially concerning preservatives and
metal-based adjuvants used in their composition
(thimerosal and aluminum hydroxide, respectively).
Based on this, this study evaluated the effects
during 24 h, 96 h and 21 days after the exposure to
thimerosal and aluminum hydroxide (isolated or in
mixture) in kidney of Danio rerio (zebrafish) through
biochemical biomarkers. The zebrafish were divided
into four groups (n=30 per group) and exposed to
the compounds intraperitoneally. The groups were:
control (saline), thimerosal (TMS - 7.5 mg/kg),
aluminum hydroxide (Al - 175.0 mg/kg) and mixture
(TMS+Al - 7.5 mg/kg of TMS + 175.0 mg/kg of Al).
Concentrations were chosen after conducting a pilot
study, and the highest concentration that did not cause
animal mortality within 96h was selected for this
study. The bioassay was divided into three periods:
24 h, 96 h, and 21 days. After this, the animals were
anesthetized with 0.001% benzocaine and euthanized
by spinal section. The kidney was removed for analysis
of the enzymes superoxide dismutase (SOD) and
glutathione S-transferase (GST); and, for the levels
of reduced glutathione (GSH) and metallothioneins
(MT). The results were statistically analyzed using
the Graph Pad Prism 5. For homogeneous data, one-
way ANOVA was performed, followed by Dunnett’s
test. The Grubbs test was performed to remove the
outliers. The results were considered statistically
significant when p<0.05. After 24 h of exposure, fish
exposed only to Al showed a significant increase in
renal SOD activity [F (3, 23) = 6.334; p = 0.0027] and
GST [F(3, 22) = 6.336; p = 0.0029] when compared to
the control group; and animals exposed only to TMS
showed a significant decrease in renal GSH levels [F(3,
22) = 4.954; p = 0.0089]. There was no change in MT
levels. Ninety six hours after the exposure, animals
exposed intraperitoneally to Al (Al and TMS+Al groups)
showed a significant decrease in SOD activity [F(3, 35)
= 14.15; p < 0.0001] and MT levels [F(3, 8) = 4.724; p =
0.0351] when compared to the control group. The GST
and GSH biomarkers were not altered. Finally, after
21 days of exposure, animals exposed to Al have a
statistically significant decrease in renal SOD activity
[F (3, 33) = 15.51; p<0.0001] and renal GSH levels [F (3
35) = 3114; p<0.0385] when compared to the control
group. Animals exposed to TMS + Al had a statistically
significant decrease of renal SOD activity [F (3, 33)
= 15.51; p<0.0001]. There was also a statistically
significant increase in GST renal activity [F (3, 34) =
3.702; p = 0.0209] in animals exposed to TMS. Renal MT
levels were not altered. Exposure to metals present in
the composition of vaccines, thimerosal and aluminum
hydroxide, caused biochemical changes in Danio rerio
kidney when evaluated alone or in a mixture, and
these alterations remain up even 21 days after the
exposure. The increase observed in the first hours may
indicate an adaptive response of the organism and
the inhibition observed in the 21 days probably occurs
because the compounds are bioaccumulated in the
kidney, due to the excretion process. We emphasize
the lack of scientific information on the subject and
the importance of further studies to assess possible
long-term effects since a better understanding of
these compounds will result in a better understanding
of the possible adverse effects after vaccination,
contributing to better solutions to the problem.
 210

Systemic toxicity and testicular damage

produced by exposure to benzo(a)pyrene in low-
dose from juvenile to peripuberty in male rats
Jorge, Bárbara Campos1; Reis, Ana Carolina Casali1; Stein, Julia1; Paschoalini,
Beatriz Rizzo1; Nogueira, Jéssica Bueno1; Arena, Arielle Cristina1,2

 Department of Structural and Functional Biology. PPG Pharmacology and Biotechnology,

1

Institute of Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São

Paulo State, Brazil; 2 Center of Toxicological Assistance (CEATOX), Institute of Biosciences
of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São Paulo, Brazil.

Background: Among the substances with the
potential for endocrine disruption, benzo(a)pyrene
(BaP) is a persistent organic pollutant diffused in the
environment in a ubiquitous way. This substance has
some well-established mechanisms at high doses;
however, our group has demonstrated that BaP can
directly impact the sperm quality of male rats at low
doses. Our study showed the potential endocrine
disruptor of BaP in a highly androgen dependent
phase: juvenile period and peripuberty. Objective:
Thus, this study aimed to evaluate if BaP in low-dose
causes immediate toxicity and testicular disorders.
Methods: For this, juvenile male Wistar rats (23
days, n = 8 animals/group) were allocated in control
group (corn oil + DMSO - vehicle) and BaP (vehicle +
0.1 µg/kg); and the exposure to BaP occurred for 31
consecutive days (gavage), until post-natal day (PND)
53. The clinical signs of toxicity, the body weight
progression and water and food of consumption of
these animals were evaluated. On PND 54, the rats
were killed for immediate toxicological and testicular
histological evaluation. The organs of these animals
were weighted and blood collection to hematological,
hormone and biochemical tests. All data obtained
in this analysis were submitted to statistical tests.
Results: During the treatment, the animals showed
no clinical signs of toxicity; however, the body
weight gain of BaP-treated group was reduced when
compared to control group although water and food
intake is not altered. On PND 54, the relative weight of
kidney and liver were decreased. The leukocyte counts
also decreased in treated group and the cholesterol
have biological increased. The tubules seminiferous
diameter and height was altered in treated group,
as well as Leydig cells atrophy. Testosterone was
decreased in BaP-group and FSH was increased.
Discussion/Conclusion: The data on immediate
systemic toxicity at low doses of BaP were similar
when compared to high doses already established in
the literature. Although indirect involvement due to
systemic toxicity cannot be ruled out, the decrease
in testosterone in the present study is key to support
the hypothesis that BaP can occupy the steroidogenic
acute regulatory protein (StAR) receptor, preventing
cholesterol from entering the mitochondria at the
proper physiological rate. This hormonal decrease
negatively affects the reproductive system and
testicular structure of the animal. Expression
of steroidogenic enzymes and StAR are being
performed to support our hypothesis. Approved by
ethics committee, nº 1148/2019. Acknowledgment:
CNPq – 140826/2019-0; FAPESP - 2019/03264-5;
2019/03284-6.
 211

Teratogenic and toxic effects induced

by the herbicide 2,4-D (DMA® 806) in
zebrafish (Danio rerio) embryos
Viriato, Cristina1,2; Andrade, Ítalo B.L.2,3; Peixoto, Paloma V.L.2,3; Pereira, Lílian C.2,4

 São Paulo State University (Unesp), Institute of Biosciences, Botucatu; 2 Center for Evaluation
1

of Environmental Impact on Human Health (TOXICAM); 3 São Paulo State University (Unesp),
Medical School, Botucatu; 4 São Paulo State University (Unesp), School of Agriculture, Botucatu.

2,4-D is one of the most used herbicides in agriculture,
despite its classification as “possibly carcinogenic
to humans”. Some of its components in commercial
formulations have high lipophilicity, resistance to
degradation and bioaccumulative potential, being
evaluated as persistent organic pollutant (POP),
probably at high levels in the environment (air, water
and sediments), biota and humans. In this context, the
objective of this study was to elucidate the potential
teratogenic of the herbicide 2,4-D (DMA® 806), using
embryonic stage of zebrafish (Danio rerio) as a model
organism. Embryo’s evaluation followed Fish Embryo
Toxicity (FET) Test n. 236 (OECD, 2013). After the fish
reproduction, 140 fertilized eggs in the blastula stage
were collected and placed in 24-well culture plates.
Assays were performed in triplicate, and each replicate
consisted of 24 embryos for the negative control
group (ISO-7346 standard water), 20 embryos for the
positive control group (4 mg/L 3,4-dichloroaniline)
and 20 embryos for each concentrations of 2,4-D
(DMA® 806): 0.1; 0.4; 1; 10; 100 and 200 µM. The culture
plates were incubated at 26 ± 1°C, in cycles of 14
hours light and 10 hours dark and the development of
zebrafish embryos and larvae were evaluated at 8, 24,
48, 72, 96, 120 and 144 hpf (hours post fertilization)
through of a stereomicroscope. Mortality, yolk sac
edema, swim bladder inflation defects, pericardial
edema and caudal fin deformation were identified. The
values ​​were submitted to the analysis of variance test
(One-way ANOVA) and a posteriori Dunnet’s test. After
144 h of exposure, the negative and positive controls
resulted in compliance with the test (mortality≤10%
and mortality≥30%, respectively). There was 15%
embryo mortality in the maximum concentration
treatment (200 µM) 144h, with no difference, as in all
other concentrations. Regarding to the deformities
Non-Inflated Swim Bladder, Caudal Fin Deformity,
Pericardial Edema and Yolk Sac Edema, there was not
differences in the lowest concentrations tested (0.1,
0.4 and 1 µM). Despite of this results, these treatments
had adverse effects. In all type of deformities
observed, there were significant differences (p<0.05)
in the treatments of concentrations 100 and 200 µM
in 100% of the embryos in these two concentrations.
The yolk sac edema deformation, in addition to the
differences obtained at concentrations of 100 and
200 µM, there was also significant (p<0.05) in the
treatment of 10 µM concentration in 50% of the
embryos. This last result evidences the susceptibility
of zebrafish to this pesticide, as it is a concentration
of the commercial formula 2,4-D (DMA® 806) that
approaches values ​​found in the environment, being
a substance with teratogenic potential, requiring
further studies that will be done in our laboratory
(TOXICAM). Acknowledgments to Coordination for the
Improvement of Higher Education Personnel - (CAPES)
and TOXICAM.
 212

The relationship between oxidative system

and sperm motility in wistar rats continuously
exposed to organochlorine DDT residues
Silva, Fernanda Coleraus1; Quiozini, Nathaly de Matos2; Simon, Jaqueline2; Schwengber,
Heloysa Talia3; Lesniewski, Isabela Ramos3; Azevedo, Camilla de Marchi Sanches1; Itinose,
Ana Maria4; Marek, Carla Brugin4; Guimarães, Ana Tereza Bittencourt1
1
 Department of Biosciences and Health, Universidade Estadual do Oeste do Paraná,
UNIOESTE, PR, Brazil; 2 Pharmacy, Universidade Estadual do Oeste do Paraná, UNIOESTE,
PR, Brazil; 3 Odontology, Universidade Paranaense, UNIPAR, PR, Brazil; 4 Department of
Toxicology, Universidade Estadual do Oeste do Paraná, UNIOESTE, PR, Brazil.

Introduction: Given the modest sperm antioxidant
defenses and knowing that excessive damage in
male reproductive organs can lead to infertility. All
aspects of sexual reproduction can be affected if
the balance of free radicals and antioxidant defense
goes awry. That way, seeing the organochlorine
dichlorodiphenyltrichloroethane (DDT) and its
residues, Dichlorodiphenyldichloroethylene (DDE)
and dichlorodiphenyldichloroethane (DDD), are
associated to metabolic alterations, causing free
radical productions and oxidative stress. The
present study becomes relevant to evaluate if the
spermatozoa motility was affected by oxidative
system changes induced by continuously exposed
to DDT residues. Objective: Evaluate the association
of oxidative system to sperm motility in Wistar rats
continuously exposed to DDT residues. Methodology:
The study (CEUA/Unioeste protocol n. 21-20) was
performed with one generation of Wistar rats,
exposed to trace concentrations of organochlorine
DDT residues (DDD and DDE), since the intrauterine
period until the end of life. The offspring was
obtained from parental generation (10th week), not
consanguineous. The groups were defined from day
0 of pregnancy as: Control, DDD (0.0147 µM of DDD),
DDE (0.0056 µM of DDE) and DDD/DDE (association
of the two substances). The placement of substances
was carried out via water ad libitum, continuously,
throughout the life. At the end of 5 and 15 weeks of
age, males (n=6-8 per group) were euthanized with
anesthetics, followed by collected of right testicle
and sperm samples through the deferens ducts.
The oxidative parameters, peroxidase glutathione
(GPx), glutathione-S-transferase (GST), reductase
glutathione (GR), superoxide dismutase (SOD), catalase
(CAT) and lipoperoxidation (LPX), were measured in
the testicle samples. The spermatozoa motility was
evaluated at the 15 weeks of age, differentiating into
motile and immotile spermatozoa. Data collected at 5
and 15 weeks of age were compared between groups
using one-way ANOVA followed by Tukey-HSD test.
Matrices of oxidative system data from 5th and 15th
weeks were evaluated using Principal Component
Analysis (PCA). The factor loadings resulting from
the first two dimensions of each data matrix were
later related to the number of immotile spermatozoa,
using linear models evaluated through Analysis of
Variance (α=0.05). Results: There were no significant
differences in the variables means among the groups
at 5 and 15 weeks of age (p>0.05). However, when
performing the integrative assessment (PCA) at
the 5th week of age, opposite linear relationships
between the number of immotile spermatozoa and
GST and GR activities were observed, and direct
linear relationships between the number of immotile
spermatozoa and SOD activity. The results indicate
that the changes in antioxidant function at 5 weeks
of age in animals exposed to DDD/DDE led to the
increased number of immotile spermatozoa observed
at 15 weeks of age. The integrative assessment at the
15th week of age showed the direct linear relationship
among the number of immotile spermatozoa and
SOD, CAT and GR activities. The results indicate that
an induction of antioxidant system at 15 weeks of
age, especially in the DDE group, led to the increased
number of immotile spermatozoa. Conclusion: The
testis is a site of intense cell division and relies heavily
of NADPH, hence it is susceptible to oxidative stress
and depends of antioxidant enzymes to repair the
damages that can affect the spermatozoa. In this way,
knowing that DDT can contribute to oxidative damage
in different tissues, the results showed like small
changes in oxidative system can affect the quality
of sperm, contributing to possible male infertility
in experimental animals exposed to DDT residues.
Acknowledgments: Cardiff University, GCRF, CNPq.
 213

The white blood cells behavior in

multigenerations of wistar rats exposed
to DDT organochlorine residues
Silva, Fernanda Coleraus1; Quiozini, Nathaly de Matos2; Simon, Jaqueline2; Azevedo, Camilla de Marchi
Sanches1; Vieira, Bruna Todeschini3; Marek, Carla Brugin4; Guimarães, Ana Tereza Bittencourt1
1
 Department of Biosciences and Health, Universidade Estadual do Oeste do Paraná,
UNIOESTE, PR, Brazil; 2 Pharmacy, Universidade Estadual do Oeste do Paraná,
UNIOESTE, PR, Brazil; 3 Veterinary laboratory, VitaLab, PR, Brazil; 4 Department of
toxicology, Universidade Estadual do Oeste do Paraná, UNIOESTE, PR, Brazil.

Introduction: The organochlorine dichlorodiphe-
nyltrichloroethane (DDT) and its residues, Dichlo-
rodiphenyldichloroethylene (DDE) and dichlorodi-
phenyldichloroethane (DDD), are persistent organic
pollutants (POPs) that are resistant to degradation
and can accumulate at high levels in the environment
and human body. Besides that, DDT and its residues
have been associated with several pathological con-
ditions including endocrine disruption and immuno-
logic dysfunction. Studies reveal that DDT has es-
trogen-like properties and can affect some immune
cells functions. That way, the present study becomes
relevant to evaluate the white blood cells behavior
in multigenerations of female Wistar rats exposed to
DDT organochlorine residues. Objectives: To evaluate
possible changes in the white blood cells of Wistar
rats exposed to trace concentrations of DDT residues.
Methodology: The study (CEUA/Unioeste protocol n.
21-20) was carried out with two generations (F1 and
F2) of female Wistar rats, exposed to trace concen-
trations of organochlorine DDT residues (DDD and
DDE), since the intrauterine period until the end of life.
These offspring were obtained from parental gener-
ations (10th week), not consanguineous. The groups
were defined from day 0 of pregnancy as: Control,
DDD (0.0147 µM of DDD), DDE (0.0056 µM of DDE) and
DDD/DDE (association of the two substances). The
placement of substances was carried out via water
ad libitum, continuously, throughout the life of the
multigenerations. At the end of the 5th and 15th week
of life of the F1 and F2 generations, females (n=6-8
per group) were euthanized with specific anesthetics,
followed by cardiac puncture to blood collect. White
blood cells, mononuclear cells and polymorphonu-
clear cells, were estimated using the hematological
analyzer Mindray bc 2800 vet. White blood cell fre-
quency data were evaluated using linear models, con-
sidering experimental groups and time of experimen-
tation as predictors. The time of experimantation was
representaded of ages (5th and 15th weeks of life) in
the respective generations (F1 and F2). The models
were evaluated for their significance through Anal-
ysis of Variance (α=0.05). Results: When evaluating
the data, it was possible to verify that during the de-
velopment of two generations of Wistar rats exposed
to the trace concentrations of DDD and DDE there was
a white blood cells productions modulation. The ani-
mals of the DDE and DDD/DDE group of the F1 gener-
ation, with 5 weeks of life, showed a polymorphonu-
clear cells reduction, especially neutrophils. However,
in the following ages and respective generations (F1-
15th week, F2-5th week and F2-15th week) there was
an increase in the polymorphonuclear cells frequen-
cy, indicating permanent primary defense activation
(r2=0.25; F3.105=4 .50; p=0.005). In relation to mon-
onuclear cells, the opposite behavior was observed,
especially in the DDE and DDD-DDE groups, with an
increase in the frequency of these cell types, especial-
ly by lymphocytes, among the animals with 5 weeks
of life of the F1 generation and subsequent decrease
in the following periods, indicating a possible loss of
antibody production cells capacity (r2=0.22; F3,105=2.24;
p=0.088). Discussion/Conclusion: Knowing that DDT
is classified as a xenoestrogen, these results are in
agreement with studies which suggest that polymor-
phonuclear cells alterations is partially caused by the
impact of estrogen-like effect on differentiation of
those cells, especially for the neutrophilic way. The
main way of biological effects induction of xenoestro-
gens in neutrophils is by their interaction with estro-
gen receptors, being able affect neutrophilus abilities,
like chemotaxis, adhesion and phagocytosis. As this
way, the results suggest an important modulation of
white blood cells caused by DDT residues, especial-
ly for the DDE. Being important to conduct an exten-
sive research and make a comprehensive assessment
of DDT and its residues effect on immunocompetent
cells. Acknowledgments: Cardiff University, GCRF,
CNPq.
 214

Toxicological effects of Color Index Disperse Red

1 textile azo dye in sperm of mice orally exposed
Tanamachi, Amanda Rodrigues1; Fernandes, Fábio Henrique1; Lima, Geovana Cristina Ribeiro1; Mariani, Noemia
Aparecida Partelli2; Silva, Alan Andrew dos Santos2; Silva, Erick José Ramo2; Salvadori, Daisy Maria Fávero1
1
 Medical School, UNESP - São Paulo State University, Botucatu, SP, Brazil; 2 Institute of
Biosciences of Botucatu, UNESP - São Paulo State University, Botucatu, SP, Brazil.

Reproductive health has received special attention as
the incidence rate of human infertility has increased.
Exposure to environmental xenobiotics constitutes a
serious risk, particularly for male infertility. Despite
some evidence, however, the molecular processes
underlying lower fertility remain unknown. The aim
of this study was to investigate toxicological effects
of Color Index Disperse Red 1 (DR1) in mice F0 and F1
sperms (sperm count, mitochondrial activity, vitality
and acrosome integrity). DR1 is a textile azo dye
commonly discharged into water bodies that can be
consumed by humans. Herein, mice were distributed in
6 groups, including: 1 - negative control: treated with
filtrated water; 2 - positive control: intraperitonealy
treated with 3 doses of n-etil-n-nitrosourea (ENU;
100mg/kg body weight); 3 - vehicule control: orally
treated with 0.5% DMSO; 4, 5 and 6 - orally exposed to
DR1 at doses of 5, 50 and 500 µg/kg/b.w., respectively.
Treatments lasted 14 days, and mating took place 35
days later. Animals were euthanized two days after
mating (F0) or at the age of eight weeks (F1). The
highest DR1 dose (500 g/kg/b.w.) reduced sperm count
in both F0 and F1 mice; mitochondrial activity damage
was observed in all DR1-treated groups of animals.
Changes in sperm vitality and acrosome integrity were
also observed in F1 mice. In conclusion, DR1 was able
to cause transgenerational damage, implying that
water contamination with these substances could
be hazardous to human health. Acknowledgments:
CNPq (141322/2020-9) and CNPq-INCT DATREM
(465571/2014-0).
 215

Toxicological safety studies of plant extract of

Eugenia uniflora L. at different safety doses
Donadel, Guilherme1; Dalmagro, Mariana2; Pinc, Mariana Moraes3; Berta, João
Antonio4; Borba, Maria Eduarda4; Gasparotto Junior, Arquimedes5; Zardeto,
Giuliana6; Hoscheid, Jaqueline6; Lourenço, Emerson Luiz Botelho7
1
 PhD student in animal science at UNIPAR; 2 student Master in biotechnology applied
to agriculture UNIPAR; 3 PhD student in biotechnology at UNIPAR; 4 Undergraduate
Student Veterinary Medicine UNIPAR; 5 Professor at the University of Grande Dourados
–UFGD; 6 Professor at UNIPAR; 7 Unipar Research and Graduate Coordinator.

Introduction: The use of medicinal plants has been a
therapeutic alternative for thousands of years, mainly
in Middle Eastern countries and Asia in the treatment
of health disorders, prevention of disease epidemics
and microbial control, with more than 235 species of
plants medicinal plants do not have a detailed study
of their beneficial health effects. Crude extracts of
medicinal plants are the most used form in research
both in vitro and in vivo studies, in addition to the use
of isolated bioactive compounds in order to evaluate
possible mechanisms of action. Among the medicinal
plants, Eugenia uniflora L. stands out, belonging to
the Myrtaceae family, whose origin is the Brazilian
Atlantic Forest. It is characterized by having great
economic exploitation potential for the Northeast
region, in which it is effective for the treatment
of various diseases, having anti-inflammatory,
antioxidant, analgesic, antihypertensive, antibacterial
and antifungal pharmacological activities, and its
action It takes place through the consumption of its
leaves and fruits, as these are where there is a source
of nutrients. Objective: The objective of this work is
to perform the administration and evaluation from a
vehicle (vaginal gel) of E. uniflora at concentrations
of 5%, 10% and 15%, in an animal model (female
Wistar rats). Materials and Methods: With the
approval of the UNIPAR ethics committee by protocol
38108/2020, 56 female animals were used, divided
into 8 groups of 7 animals per group, where they were
evaluated for the estrous cycle, On-site Tolerance test
of Administration, clinical signs such as pain, edema,
pruritus, irritation, secretion and sensitivity of the
vaginal gel and aqueous extract of E. uniflora, being
carried out macroscopic analyzes of the metabolizing
organs (liver, kidneys and spleen), histological
diagnoses of the vaginal cavity, uterus and ovaries,
blood count and biochemical analyses, estradiol
and progesterone dosage. Results and Discussion:
The results obtained after the statistical tests of
the PRISMA program using the one-way ANOVA test
followed by the post-bonferroni test, against the
parameters evaluated for estrous cycle, macroscopic
analysis and relative weight of liver, kidneys and
spleen, together with histology of the reproductive
system (ovaries, uterus, vaginal cavity) and local
tolerance test were not significant and there were no
changes in blood count, biochemical and hormonal
tests. Conclusion: Due to the arguments presented in
the study, it is concluded that, after carrying out the
tests in the vaginal region with the gel and extract
at concentrations of 5, 10, 15%, they did not show
significant differences in terms of hematological,
biochemical, hormones, estrous cycle and sensitivity
in the region of administration. However, the use of
Eugenia uniflora gel or extract for the vaginal region
does not pose a risk of toxicity in the application site
and may be suggested as a dermocosmetic. Keywords:
Medicinal Plants; Pitanga; Vaginal Gel; Toxicity.
 216

Transgenerational reproductive effects

(F2) in male offspring exposed to benzo(a)
pyrene by paternal germ cells in rats
Jorge, Bárbara Campos1; Reis, Ana Carolina Casali1; Stein, Julia1; Paschoalini, Beatriz Rizzo1; Nogueira,
Jéssica Bueno1; Moreira, Suyane da Silva1; Manoel, Beatriz de Matos1; Arena, Arielle Cristina1,2

 Department of Structural and Functional Biology. PPG Pharmacology and Biotechnology,

1

Institute of Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São

Paulo State, Brazil; 2 Center of Toxicological Assistance (CEATOX), Institute of Biosciences
of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São Paulo, Brazil.

Background: Benzo(a)pyrene (BaP) is a chemical
substance formed by the union of a molten benzene
into a pyrene produced by the incomplete burning of
organic compounds. Because it is ubiquitous, it can
cause adverse events and behave as an endocrine
disruptor at low doses; also targeting the reproductive
system. Puberty, a critical period of development,
is vulnerable to the effects of substances, leading
to effects throughout life or in next generations.
Transgenerational repercussions involving low-
dose BaP exposure and reproductive aspects is still
unexplored; and furthermore, we seek to fill in gaps
about the importance of paternal health in offspring.
Objective: to evaluate the transgenerational (F2)
reproductive repercussions in male rats offspring,
first generation non-exposed, which the paternal
generation (F1) was exposed to benzo(a)pyrene by
germ cells. Methods: For this, juvenile male Wistar
rats (F0, 23 days, n = 8 animals/group) were allocated
in control group (received corn oil + DMSO - vehicle)
and BaP-group (exposed to vehicle + 0.1 µg/kg/day)
for 31 consecutive days (gavage). In adult life, they
were mated with untreated females to obtain male
offspring (F1) and, later, the F2 generation, which
was evaluated in relation to their initial development
and reproductive parameters in male offspring (n = 8
litters/group). All data obtained in this analysis were
submitted to statistical tests. Results: There was a
reduction in absolute and relative anogenital distance
(AGD) in post-natal days (PND) 1, 13 and 22, and
decreased on the body weight (PND 1) in F2 generation
male offspring. The preputial separation was
advanced in BaP-group. In adulthood (PND 90), there
were no changes in male sexual behavior and total
ejaculations number; however, fertility potential was
reduced in BaP-group. There was a reduction in body
weight and an increase of reproductive organs weight.
Type A sperm percentage (progressive motile) was
decreased, and Type B/C were increased (immobile)
as well as in the epididymal transit time in BaP-
group. In the stereological analysis of the epididymal
caput, we found that the height of the epithelium was
decreased while the luminal area was dilated in the
BaP-group. Testosterone levels was reduced in BaP-
group, as well as Leydig cells number and volume.
Discussion/Conclusion: The results obtained so far
on the reproductive repercussions of the male F2
generation indicate that these animals showed losses
in both sexual development and fertility potential
when compared to the control group. This damage
was generated in the sperm of the F1 generation by
indirect exposure to BaP, probably due to changes in
its epigenetic pattern (next analyses) that modified
the functioning of the reproductive system of the F2
generation. These data also reinforce the importance
of paternal health in offspring development.
Molecular analyses will be performed to confirm
our mechanistic hypothesis. Ethics committee, nº
1148/2019. Acknowledgment: CNPq – 140826/2019-
0; FAPESP - 2019/03264-5; 2019/03284-6.

Rizzi, Joyce Santana1,2; Seloto, Danielle Gabriel1,2; Pereira, Lílian Cristina2,3
1
 São Paulo State University (Unesp), Medical School, Botucatu; 2 Center for
Evaluation of Environmental Impact on Human Health (TOXICAM), Botucatu;
3
 São Paulo State University (Unesp), School of Agriculture, Botucatu.
Background: The herbicide triclopyr (3,5,6-trichloro-
2-pyridinyloxyacetic acid, CAS 55335-06-3), is already
considered an environmental problem due to the high
concentrations found in the environment. This fact
occurred, probably, due to incorrect disposal, leaching
and dispersion aerial. This type of contamination can
pose risk to both environment and human health.
Studies have evaluated the metabolism, absorption,
excretion and active transport of triclopyr but there is
no clear information about its potential mode of action
(MoA), and its cytotoxic action remains unknown. In
this context, mitochondria have been used to assess
the toxicity of xenobiotics because it is a complex
and highly regulated organelle. It is essential for the
maintenance of various biological processes such
as cell survival and metabolic homeostasis and,
therefore, for the interaction of potentially toxic
substances in various points in this system can
result in mitochondrial dysfunction and consequently
cytotoxicity. Objective: Identify the toxic mechanism
of triclopyr in Rat liver mitochondria isolated from
young Wistar rats. Methods: The exposure of
isolated mitochondria with triclopyr was carried out
in vitro using different concentrations between 0.5
to 500 µM as it comprises concentrations found in
the environment, in biological samples of animals
and humans. The formation of reactive oxygen
species and nitrogen (RONS), redox state of pyridine
nucleotides, dissipation of membrane potential,
membrane lipoperoxidation, mitochondrial levels of
membrane protein sulfhydryl (P-SH) groups, reduced
glutathione (GSH) and oxidized glutathione (GSSG)
217
Triclopyr induces changes in
mitochondrial parameters
levels and mitochondrial respiration was assessed.
The results were evaluated by analysis of variance
(ANOVA) followed by the Dunnett test. Results: The
results showed that was no statistical significance
in ADP/O rate, RCR value and Oxygen consumption
rate in state 3 (V3) when compared to the negative
control. However, the Oxygen consumption rate in
state 4 (V4) was increased at highest concentration
(500 µM) demonstrating that triclopyr can has an
uncoupling characteristic. However, ERONs was not
detected. In addition, neither inducing the formation
of malondialdehyde (MDA), nor alterations in the
levels of P-SH in the mitochondrial membrane were
observed. Therefore, at 500 µM was able to oxidize
NAD(P)H but did not alter the redox homeostasis from
GSH/GSSG ratio. Discussion/Conclusion: All results
taken together suggests that it can act as an uncoupler
of oxidative phosphorylation, which associated with
dissipation of the membrane potential demonstrates
that triclopyr is able to induce in vitro alterations on
parameters of mitochondria, not involving damage
related to the redox state due to the normality of
the GSH/GSSG ratio, suggesting that the oxidation
of NAD(P)H may be related to other contexts besides
those analyzed so far. Triclopyr also does not have
the properties of formation of RONS, membrane
lipoperoxidation and preserves the thiol groups of
the mitochondrial membrane. Acknowledgments:
Coordination for the Improvement of Higher Education
Personnel (CAPES) [Grant No. 88887.481968/2020-
00] and São Paulo Research Foundation (FAPESP)
[Grant No. 18/00229-1].
 218

Use of zebrafish as animal model

for research for neurotoxicological
substances: a literature review
Silva, Luíza Madureira1; Pinheiro, Dávylla Rennia Saldanha2; Braga, Ádrya Lariela
Lima2; Holanda, Wiron Pimentel2; Honório Júnior, José Eduardo Ribeiro3
1
 Academic of Nursing in the University Center Christus, Laboratory of Neurociência Translacional-
Neurocit; 2 Academic of Biomedicina in the University Center Christus, Laboratory of Neurociência
Translacional-Neurocit; 3 Doctor in Biotechnology for the RENORBIO/UFC. Professor in the University
Center Christus, Coordinator of the Laboratory of Neurociência Translacional-Neurocit.

Introduction: Studies involving zebrafish have to different concentrations of Methiopropamine and

highlighted genetic, anatomical, physiological and
cellular similarities as well as mammals, allowing
the science to expand to tests on human research.
Some toxicological research, which elucidate disease
prevention, has recently adopted the use of zebrafish,
after proving that this model would be adequate. The
small teleost stands as an alternative and innovative
proposal for use in toxicological experiments and
tests, and may offer several benefits, such as rapid
development of the nervous system, low costs, easy
handling, high level of neural homology with humans
and a complex immune system with mammalian
characteristics. Objectives: To identify in the
literature the use of Zebrafish as an animal model
for toxicological tests, highlighting its advantages
and benefits. Materials and Methods: Descriptive
study of the narrative type of literature, with selected
articles published in the last five years. Data were
obtained through a search in the PUBMED and
MEDLINE databases, using the descriptors: Zebrafish,
Toxicology, Animal Model. The inclusion criteria for
this study were clinical trials and comparative studies,
in Portuguese and English. Those who did not meet
the inclusion criterion were excluded. Results: The
comparative study between mice and zebrafish was
carried out with the aim of evaluating the latter as a
high-throughput model for a first screening of New
Psychoactive Substances. Each model was exposed
APINAC, and to equivalent amounts for each species,
and resulted in similar behaviors. In another study,
PfrHEPA also had its toxicity evaluated in different
models; and their results corroborate each other,
confirming that drug screening in zebrafish can
become effective in toxicology, which may benefit
several fields of study in the future. Another study
done to observe neurotoxicity from acute exposure to
acrylamine, an alkene widely used in water treatment,
mineral processing and cosmetics, which showed very
satisfactory results, and how it was observed that
the nervous system and neurotransmitter systems
are similar to humans. The zebrafish is a widely used
model used in neuropharmacological research. It is
observed in another clinical study, in which a library
with 87 chemicals were used, as generally toxic or
neurotoxic for development, or unknown, and were
exposed in zebrafish and planarians; resulting in
similar behavioral trends, but the zebrafish presented
itself as a more sensitive indicator of bioactivity.
Conclusion: It is possible to conclude that the animal
model presented gives a positive response in the
experiments used, revealing its scope in the types of
neurotoxicological compounds that can be studied
from zebrafish. Its potential is evidenced for further
studies to be carried out, enabling a better assessment
of the capacity of this organism.
 219

Whole-mount skeletal staining in rabbits fetuses

for teratogenicity evaluation (OECD 414)
Matsui, Andresa; Alves, Paula Daniela Sabino de Freitas; Santana, Thatiane Nunes;
Vecina, Juliana Falcato; Bechtold, Bruna Assunção; Fava, Luis Paulo

Mérieux NutriSciences / Bioagri Laboratórios Ltda, Piracicaba-SP, Brazil.

Introduction/Objective: Exposure to chemical
substances during pregnancy can result in abnormal
development and congenital malformations of the
fetus. OECD 414 is a guideline for developmental
toxicity testing designed to provide general
information concerning the effects of prenatal
exposure on the pregnant test animal and on the
developing organism; this may include assessment
of maternal effects as well as death, structural
abnormalities, or altered growth in fetus. This OECD
mandates that all fetuses should be screened for
skeletal and soft tissue changes, whether the test
animal is a rodent or a non-rodent. Specifically for
the skeletal assessment, although required by the
guideline, there is no description of the methodology
for its performance. A methodology was developed
by Bioagri toxicology laboratory for the skeletal
assessment of rabbit fetuses. Techniques described
in the literature were adapted for the analysis of
whole-mount skeletal preparations. This technique
allowed detecting changes in skeletal patterns and
evaluating the skeletal maturation rhythm through
specific bone and cartilage staining. Methods: After
evisceration of rabbit fetuses, proceed with the
removal of the skin, eyes, adipose tissue and bubbles
from the body cavity. Fix the embryos in 95 % ethanol
alcohol (EtOH) overnight at room temperature.
Place the samples in acetone for three days at room
temperature. Stain for cartilage by submerging the
embryo in a vial containing at least enough Alcian
blue solution to cover it. Incubate the sample for 01 to
08 hours at room temperature. Wash fetuses in 70%
EtOH twice in a row. Incubate fetuses in 95% EtOH,
at room temperature, for 24 hours. Wash the fetuses
in deionized water. Submerge them in 1% potassium
hydroxide (KOH) solution, at room temperature for 01
to 04 hours. If soft tissue clearing has not occurred,
change the 1% KOH solution for another one and leave
the fetuses submerged for the same period. Remove
1% KOH solution by washing fetuses in deionized
water. Submerge the fetuses in a solution of Alizarin
Red S, at room temperature, for at least 04 hours. A
period longer than 24 hours should be avoided due
to over-coloring effects. Replace the Alizarin Red
S solution with a 50% liquid Glycerin:50% KOH 1%
solution, with enough quantity to cover all fetuses.
The fetuses should remain incubated in this solution,
at room temperature, until the excess of the reddish-
purple color is removed. When the fetuses reach a
gelatinous and transparent appearance, transfer them
to a container with 100% liquid glycerin. Results/
Discussion: Alcian blue and Alizarin red stain cartilage
and bone, respectively. As a cationic dye, Alcian blue
binds strongly to sulfated glycosaminoglycans and
glycoproteins, while Alizarin red, an anionic dye,
binds to cationic metals such as calcium. Fetuses
can have different size, which influenced the time
of immersion in each of the solutions of the double
staining process. Care must be taken to not have an
excess of blue or reddish coloration, as well as for
the time of immersion in the KOH solution; which can
irreversibly damage the fetus, especially the smallest
ones. As expected, it was possible to evaluate the
shapes and sizes of the rabbits’ fetuses skeletal
elements in their appropriate locations. Conclusion:
In view of the results, the use of whole-mount skeletal
staining is valid to the teratogenicity evaluation of
the rabbit’s fetuses, as long as each step is carefully
followed. Acknowledgments: Bioagri Laboratórios
Ltda (Merieux NutriSciences), Piracicaba-SP, Brazil;
Faculdade de Ciências Agrárias e Veterinárias/UNESP
Jaboticabal-SP, Brazil
 07 
FOOD SAFETY

Aluminum levels in fruits and in
1
 Curso de Pós-Graduação (lato sensu) em Tecnologia e Qualidade na Produção
de Alimentos da UNIFAL; 2 BioTox - Laboratório de Bioquímica e Toxicologia de
Alimentos, Departamento de Alimentos, Faculdade de Farmácia da UFMG.
Introduction: Aluminum occurs naturally in
the environment and may be in drinking water;
unprocessed foods such as fruits and vegetables;
food of animal origin and processed foods. Due to its
technological properties, it is widely used, e.g. in food
packaging, kitchen utensils and food additives. At high
levels aluminum can be toxic to human health. It is a
neurotoxic metal, especially in patients with renal
failure on dialysis, and it is related to several diseases,
such as osteomalacia and microcytic anemia. Joint
FAO/WHO Expert Committee on Food Additives (JECFA)
established provisional tolerable weekly intake (PTWI)
for aluminum of 2 mg/kg bw. The Brazilian Health
Surveillance Agency (ANVISA) specified as PTWI for
aluminum in food additives 1 to 7 mg/kg bw, but it did
not establish any maximal level for aluminum in food.
Taking this scenario into consideration, it is necessary
to study aluminum occurrence in foods, especially in
fruit and vegetables that are directed impacted by
water and soil contamination and also the influence of
materials in contact to food in aluminum occurrence
in processed food. Objective: The main objective
of this study was to evaluate the world situation of
aluminum contamination in fruits and fruit/vegetable
juices. Methods: The worldwide occurrence of
aluminum was evaluated in fruits and fruit/vegetable
juices from data available in GEMS/Food database.
The inclusion criteria were: i) all regions; ii) results
from edible parts. The exclusion criteria were i) dried
fruits; ii) concentrated juices; iii) results in dry basis;
and iv) samples with limit of detection (LOD) and
limit of quantification (LOQ) higher than the average
levels observed. The results of aluminum in foods
221
fruit and vegetable juices
Pinelli, Juliana Junqueira1; Custódio, Flávia Beatriz1,2
were expressed as mean, median, standard deviation
and 95th percentile. When results were not detected,
they were replaced by zero in lower bound treatment
(LB) and by LOD value in upper bound (UB). Results: It
was evaluated 443 results of aluminum in fruits with
53.5% of positive samples. LOD and LOQ ranged from
0.02-0.236 mg/kg and 0.1-0.5 mg/kg, respectively.
The mean level of aluminum at LB was 0.57 ± 0.85
mg/kg and at UB was 0.64 ± 0.81 mg/kg. Median and
95th percentile were respectively 0.24 mg/kg and
2.2 mg/kg. Fruit and vegetable juices had 48.08% of
positive samples (n=52) with mean and median levels
at LB of 0.51 ± 0.86 mg/kg and zero, respectively. At
UB mean and median levels were 0.58 ± 0.81 mg/kg
and 0.22 mg/kg, respectively. The 95th percentile
in LB and UB was 2.33 mg/kg. LOD and LOQ for fruit
and vegetable juices ranged from 0.05-0.236 mg/
kg and 0.218-0.5 mg/kg, respectively. Discussion/
Conclusion: The mean levels of aluminum found both
in fruits and their derived products and in fruit juices
were very similar, suggesting that the processing
used by food industry does not interfere in aluminum
concentration in final product. This is probably due to
the most industrial equipment is made of stainless
steel and not aluminum. However, in Brazil, which is
one of the largest aluminum producers, most homes
have aluminum utensils in their kitchens, which can
favor migration of this metal to the foods, increasing
aluminum exposure. Further studies evaluating
migration of aluminum from kitchen utensils to
food are necessary to understand its impact in the
accumulation of this metal in human body has on
public health. Acknowledges: CAPES
 222

Analysis of physical chemical parameters

and cyanogen residues in macaxeira flour
(Manihost esculenta Crantz), produced
in a municipality of Paraense
Araújo, Maria Eduarda Lima; Estumano, Saulo Braga; Oliveira, Sidney Julio Vieira; Santiago, Mayla
Andra de Andrade; Costa, Eliene dos Santos da Silva; Oliveira, Cláudia Simone Baltazar

Laboratório de Toxicologia, Centro Universitário Fibra, Belém-PA, Brasil.

Introduction: In the North Region, cassava flour
(Manihot esculenta) stands out for being a versatile
food and its production plays an important role in
the economy of the involved sectors, mainly for
the producing municipalities in the west of Pará.
Traditionally, flour is produced from cassava known
as “brava”, but a new flour allegedly produced from
cassava, a subspecies of cassava, has been sold in
natural food stores. The difference between the two
products is that the flour produced from cassava
can be consumed immediately after a short cooking
period, while cassava is indicated for consumption
only after prolonged preparation to reduce cyanogenic
compounds. Cassava is being a preferred product in
the market, as sellers point out to consumers that
cassava has a high content of hydrocyanic acid (HCN)
compared to cassava flour. Considering that there are
no quality standards for the manufacture of products
derived from cassava, only Normative Instruction nº
52 for table flour, it was proposed to verify the content
of cyanogenic compounds in commercialized cassava
flour. Objective: To evaluate the physical-chemical
parameters and free cyanogen residues of cassava
flour. Material and Methods: The study was carried
out with 3 different samples from each supplier
from the municipality of Bragança-PA. Analyzes were
performed in triplicate for moisture, pH, soluble
solids (ºBrix), foreign bodies, titratable acidity, based
on the methodology of the Instituto Adolfo Lutz, 1985.
For the determination of free cyanide it was based on
the study by Essers et al (1993), adapted. Results and
Discussion: In the verification of foreign bodies in all
the samples were found: ants, ash and insect wings,
which indicates that the manufacturing process
directly influences this contamination of foreign
matter, it is believed than by the lack of structure,
being the flour produced in open places, with dirt
floors, presence of domestic animals and wooden
utensils. In the analysis of soluble solids, the following
mean values ​​were obtained: 2.1; 1.6 and 4.3, where
only the A3 sample presented a discrepant value. In
the pH verification, the samples obtained an average
of 5.15 meq NaOH (0.1N) /100g, thus, according to the
regulation n° 52 of BRAZIL, 2011 is considered high
acidity for values ​​above 3.0 meq NaOH (0.1N) /100g.
In the analysis of titratable acidity, they presented
values ​​of 5.84%; 4.85%; 8.58%. The moisture levels
of the samples were between 4.5 and 9.0%, according
to the standards of normative nº 52 of BRAZIL 2011.
As for the analysis of free cyanide, the average
found was respectively 0.75; 0.96; 12.78 mg/HCN/kg.
Second, the WHO (World Health Organization) and FAD
(Organization of Food and Agriculture United Nations)
have set a limit of 10 mg HCN/kg. According to the
Pinto study (2020), in their physicochemical analyzes
of cassava flour, such as pH, titratable acidity and free
cyanide, they presented respectively: 4.95; x̄ ≥ 3%; 1.42
mg/HCN/kg. Thus, most samples of cassava flour had
a low content compared to cassava flour. Conclusion:
In view of the results obtained, cassava flour has a
low free cyanide content compared to cassava flour.
It is worth mentioning the presence of foreign bodies
in all samples, so it is pertinent to evaluate good
production practices. Therefore, quality control is
necessary in the production of flour, to provide food
security to consumers. Acknowledgments: I thank
the collaborators who participated in this study.
 223

Assessment of the incidence and

concentration of polycyclic aromatic
hydrocarbons in cocoa-derived products
Silva Júnior, Clovis Reis; Almeida, Gabriela de Oliveira; Moretti, Gabriela; Jager, Alessandra Vincenzi

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil.

Cocoa and its products are foods widely consumed
by the population in all age groups. For cocoa to be
transformed into the final product to be marketed, it
must go through several processing steps that can
have a great influence on the contaminants present in
the final products. Among the possible contaminants
are polycyclic aromatic hydrocarbons (PAHs), a group
of organic compounds consisting of two or more fused
aromatic rings, which are related to cellular mutation
processes and carcinogenicity. European legislation
sets the maximum limit of Benzo[a]pyrene (BaP) at 5.0
ng.g-1 fat, and 30.0 ng.g-1 fat for the sum of Benzo[a]
pyrene (BaP), Benzo[a]anthracene (BaA), Chrysene
(Chr), Benzo[b]fluoranthene (BbF) in cocoa beans and
derived products. This work aimed to determine the
incidence and concentrations of BaP and ΣPAH4 in
commercial chocolate samples consumed in Brazil.
Eighty-eight samples (n=88) of various chocolates
were purchased from local markets in the city of
Ribeirão Preto, Brazil. PAH concentrations were
determined in all collected samples using a liquid
chromatograph system equipped with a fluorescence
detector and a Supelcosil LC-PAH column, 250 x 4,6
mm, 5 μm particle size. Gradient elution with mobile
phase composed of water and acetonitrile was used at
1.0 mL/min. Around 0.7 gram of lyophilized sample was
extracted with n-hexane on ultrasound for 1 hour. The
extract was centrifuged, and the solvent evaporated
to near-dryness. The residue was dissolved in hexane
and eluted through a Strata SI-1 silica cartridge (5g,
20 mL). PAHs were eluted from the column with
n-hexane/dichloromethane (70:30, v/v). The eluate
was dried, dissolved in 200 μL of acetonitrile, and
injected into the liquid chromatography system. The
method evaluated presented quantification limits of
0.45 ng.g-1 BaA, 0.45 ng.g-1 Chr and BbF, and 0.45 ng.g-
1
BaP. Analytical curves were evaluated in terms of
linearity, following the necessary criteria for linear
regression and quantification of analytes using the
external calibration method. Recovery rates ranged
from 62 to 88%, and accuracy (n = 3) varied between
3 and 18%. The results of BaP obtained are described
as follows (n; mean ± standard deviation; range): milk
chocolate (n=18; 2.8 ± 2.2 ng.g-1; <LOQ – 7.1 ng.g-1 ),
bittersweet (n=22; 3.2 ± 1.7 ng.g-1; 0.40 – 7.0 ng.g-1),
bitter (n=10; 2.9 ± 1.9 ng.g-1; 1.0 – 5.0 ng.g-1), white (n=12;
3.8 ± 1.9 ng.g-1; <LOQ – 7.0 ng.g-1), milk chocolate diet
(n=13; 2.0 ± 2.4 ng.g-1; <LOQ – 8.4 ng.g-1); bittersweet
diet (n=4; 1.4 ± 0.7 ng.g-1; 0.79 – 5.5 ng.g-1), bitter diet
(n=4; 3.5 ± 4.6 ng.g-1; <LOQ – 10.0 ng.g-1), white diet
(n=5; 2.8 ± 1.0 ng.g-1; 1.5 – 3.8 ng.g-1). ΣPAH4 results are
described as follows (n; mean ± standard deviation;
range): milk chocolate (n=18; 36 ± 29 ng.g-1; 2.0 – 89
ng.g-1 ), bittersweet (n=22; 44 ± 23 ng.g-1; 7.0 – 85 ng.g-
1
), bitter (n=10; 28 ± 25 ng.g-1; 6.8 – 90 ng.g-1), white
(n=12; 43 ± 31 ng.g-1; 2.0 – 115 ng.g-1), milk chocolate
diet (n=13; 17 ± 17 ng.g-1; < LOQ – 60 ng.g-1); bittersweet
diet (n=4; 15 ± 9 ng.g-1; 5.5 – 26 ng.g-1), bitter diet (n=4;
27 ± 37 ng.g-1; <LOQ – 80 ng.g-1), white diet (n=5; 26
± 11 ng.g-1; 17 – 42 ng.g-1). Twelve out of 88 samples
(13%) had a BaP concentration greater than 5.0 ng.g-1
fat, while 44 out 88 samples (50%) presented ΣPAH4
values greater than 30 ng.g-1 fat. These preliminary
results show a high incidence of BaA, Chr, BbF, and BaP
in cocoa-derived products manufactured in Brazil. The
absence of PAH legislation in several food products
in Brazil can be revised by regulatory agencies in the
future.
 224

Caffeine content in pre-workout

supplements and energy drinks marketed
in Brazil: are the actual levels safe and
in accordance with the labels?
Costa, Bruno Ruiz Brandão1; El Haddad, Lohanna Pereira2; Freitas, Bruno
Toledo2; Marinho, Pablo Alves3; De Martinis, Bruno Spinosa2
1
 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São
Paulo, Ribeirão Preto, Brazil; 2 Faculdade de Filosofia, Ciências e Letras de Ribeirão
Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; 3 Instituto de Criminalística
da Polícia Civil do Estado de Minas Gerais, Belo Horizonte, Brazil.

Background: The stimulating and performance‐
enhancing properties of caffeine are often explored
in pre‐workout supplements (PWS) and energy
drinks (ED). However, despite the great popularity
of these products, previous studies have reported
incompatibilities between what is described in their
labels and their actual caffeine content. Objective:
This study aimed to apply simple and fast methods
to quantify caffeine in PWS and ED, and to analyze
commercial samples marketed in Brazil to estimate
the caffeine daily intake. Methods: The PWS sample
preparation consisted of weighting 50 mg of the
supplement and adding 5 mL of a lidocaine solution
prepared in isopropanol (internal standard – IS – at
25 μg/mL). Then, the extraction was performed by
ultrasonic bath (70°C for 10 minutes - conditions
that were previously optimized by central composite
rotatable design). After the extraction, the samples
were centrifuged (10 minutes at 2800 rpm), and then
100 μL was transferred for a vial containing 900 μL of
the IS. The ED sample preparation consisted of a simple
dilute and shoot: 20 μL of the ED was transferred for
a vial containing 980 μL of a 2,2′-Bipyridyl solution
prepared in isopropanol (IS for ED analysis – at 40
μg/mL). Both PWS and ED samples were analyzed
by gas-chromatography with nitrogen phosphorous
detector (GC-NPD). In addition, both methods were
previously validated following the ANVISA Guideline
on Analytical Method Validation. Fifty-two PWS
and 37 ED commercial samples marketed in Brazil
were analyzed. Results: The presence of caffeine
was declared in 40 of the 52 PWS samples (77%).
However, the GC-NPD analysis revealed that actually
45 products contained caffeine (87%). The actual
caffeine concentration ranged from 1.95 to 904
mg/g of supplement. From the 36 PWS labels that
in fact specified the caffeine amount, seven (19%)
presented more than 120% of the declared quantity,
whereas 15 (42%) contained less than 80% of the
labeled caffeine. Additionally, six products presented
undeclared caffeine. Considering the label stated
doses, five supplements exceeded the safe caffeine
daily intake (400 mg). Regarding the ED products,
caffeine was declared and detected in all samples.
The determined caffeine concentrations ranged from
11 to 68 mg/mL. Only one ED sample (2,7%) presented
more than 120% of the declared quantity, whereas
four (10.8%) contained less than 80% of the labeled
caffeine. No ED products exceeded the safe caffeine
daily intake. Discussion/Conclusion: More than 50%
of the PWS products presented inconsistencies in
caffeine content. These issues may affect consumers
in two ways: either the product contains less caffeine
than expected, so the consumer is paying for a
supplement that may not provide the desired effect;
or it contains more caffeine than expected, leading
to higher caffeine intake than the amount that is
considered safe, which may cause adverse effects.
The quantified caffeine content in ED samples was
much more consistent with the labels. Thus, the
production and/or quality control of these products
are more efficient comparing to PWS. Therefore, the
present findings showed that a stricter quality control
is still needed to ensure that PWS actually contain
what is declared. This is an issue that deserves
more attention from consumers, manufacturers,
and regulatory agencies. Acknowledgments: This
work was supported by Conselho Nacional de
Desenvolvimento Científico e Tecnologico (CNPQ) and
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES).
 225

Cytotoxic activity of green banana (Musa

sinensis) flour by Allium cepa test
Tadeu, Vitória Costa1; Roehrs, Rafael2; Farias, Fabiane Moreira1; Kieling, Ketelin Monique
Cavalheiro2; Nogueira, Caroline Lacerda1; Denardin, Elton Luis Gasparotto1
1
 Laboratório de Estudos Físico-Químicos e Produtos Naturais (LEFQPN); 2 Laboratório
de Análises Químicas Ambientais e Toxicológicas (LAQAT), Campus Uruguaiana,
Universidade Federal do Pampa (Unipampa), Uruguaiana – RS, Brasil.

Introduction: Banana is a world-renowned fruit,
cultivated in several countries with predominantly
tropical and subtropical climates. The fruit is known
for its nutritional value, being an excellent source
of vitamins and minerals. In addition to in natura
consumption, part of its production is transformed
into banana flour and biomass. This process takes
place through the use of green bananas. The use of
green bananas, a nutritional flour, minimizes future
losses that occur in the production chain, due to factors
such as distribution and storage. Green banana flour
is a source of carbohydrates, phenolic compounds,
vitamins and minerals. This food has been gaining
prominence in the functional food market for having
high concentrations of resistant starch that helps
in the weight loss process due to its composition.
Several tests are done to determine the composition
and physicochemical properties of this type of food,
however, tests that can determine its toxicity are
necessary to determine its safe and effective use
through a cytotoxic and mutagenic investigation due
to the increasing consumption of natural products.
The Allium cepa (also known as onion) test is a
simple and widely used test as a cytotoxicity and
genotoxicity of plants, aquatic systems and extracts.
In addition, an efficient test for the analysis of several
environmental substances. Objective: The goal of this
study was to evaluate the cytotoxicity of the green
banana flour (Musa sinensis) using Allium cepa test.
Methods: Hydroalcoholic extract (80:20 EtOH:H2O)
of green banana flour was obtained by extraction
in an orbital shaker (150 rpm, 24h, 35 ºC). After, the
material was dried out in a rotary evaporator. The
flask was weighed before and after the process
to obtain the corresponding mass and then, the
extract obtained was resuspended in a 50% ethanol
solution (Conc.=5.0 mg.mL-1) for posterior analysis.
For Allium cepa assay, roots were cultivated for 48h
(25ºC) using a cylindrical reservoir containing water.
After, they were divided in three groups: (a) negative
control (NC) (distilled water); (b) banana flour extract
(BFE) (0.5 mg.mL-1); (c) positive control (PC) (5%
glyphosate solution). Three Allium cepa bulbs size
were measured at the initial treatment and every 24h
during 120h (total exposure). Root growth rates were
analyzed statistically using one-way ANOVA /Tukey
test (p≤0.05). Results: Our results demonstrated
that both positive control (glyphosate solution)
and extract (BFE) reduced root growth significantly
when compared to NC. In relation to negative control
(NC) the growth of 18.5% and 3,5% for BFE and PC,
respectively. Discussion/Conclusion: According to
results obtained, Banana Flour Extract exhibits, for 5
mg.mL-1, an apparent toxicity, however, lower than the
positive control. Mitotic index and genotoxicity assay
will still be carried out, aiming at greater knowledge
about the toxicological effects of this food, as well
as tests using animal models. Acknowledgments:
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES).
 226

Design of Experiments as a tool for HS-

SPME-GC-MS method development
applied to qualitative analysis of volatile
alcohol congeners in seized whiskeys
Bigão, Vítor Luiz Caleffo Piva1; Costa, Bruno Ruiz Brandão1; Gomes, Nayna Cândida1; Marinho,
Pablo Alves2; Silva, Jonas Joaquim Mangabeira3; De Martinis, Bruno Spinosa3
1
 Faculdade de Ciências Farmacêuticas de Ribeirão Preto – USP. 2 Superintendência de
Polícia Técncio-Científica da Polícia Civil de Minas Gerais –SPTC/PCMG. 3 Faculdade de
Filosofia, Ciências e Letras de Ribeirão Preto – Departamento de Química – USP.

BACKGROUND: Counterfeiting alcoholic beverages
can lead to harmful effects on consumers’ health
due to the lack of quality control, which can originate
a formulation that contains alcohol congeners
(i.e., substances which are responsible for the
sensorial properties of the beverage) in improper
concentrations or even in hazardous levels. In this
sense, the monitoring of alcohol congeners can serve
as counterfeiting markers, aiding the elucidation of
possible frauds. Objective: To develop and optimize a
HS-SPME-GC-MS method for the qualitative analysis of
volatile alcohol congeners in seized whiskeys in cases
of fraud suspect. Methods: A pool of six authentic
whiskeys samples was used for the optimization of
the method. For the SPME fiber selection, two types
of stationary phase coating material were evaluated
(Carboxen/PDMS and PDMS). The pool of whiskeys
was analyzed in triplicate on the following conditions:
extraction temperature (40ºC), extraction time (40
min), and equilibration time (12.5 min). The responses
evaluated were the number of peaks and the total
chromatographic area. A Student’s t-test was applied
to assess any statistical significance between the
mean of the responses for the two treatment groups.
Next, a two-level-three-factor Full Factorial Design
(FFD 23) was conducted in triplicate to examine the
impact of the factors pH (pH 3-5) and salting-out (0-
1g of NaCl) on the responses. The data were evaluated
by ANOVA to verify if any of the four possible
combinations resulted in any statistically significant
differences in the responses. Finally, a Box-Benhken
Design (BBD) with 16 experiments was conducted
to model the response in function of the following
factors: extraction temperature (30-50ºC), extraction
time (20-60min.), and equilibration time (5-20min.).
The geometric mean of the total chromatographic
area was adopted as the response. The desirability
function of Derringer & Suich was conducted after
BBD optimization to find the theoretical optimum
condition for the HS-SPME extraction. For validation
of the model, experiments in triplicate on the
predicted optimum conditions were conducted and
the relative error was measured. Results: The fiber
type evaluation results revealed that Carboxen/PDMS
presented better responses than PDMS. The Student’s
t-test revealed a statistically significant difference
between the means of the total chromatographic area
for each type of fiber (p=0.041). Since Carboxen/PDMS
presented a higher total chromatographic area, we
choose it for further experiments. Regarding the pH
and salting-out effects, the ANOVA results indicated
no statistical difference between the means of each
of the four treatment combinations (p>0.05). Thus,
we decided to choose the treatment with no pH and
salt adjustment. The predicted optimal settings
for the SPME factors obtained through the BBD 23
and the desirability function were: 60 minutes for
extraction time, 5 minutes of equilibration time, and
50ºC for extraction temperature. The experimental
validation of the model presented a relative error
of -1.59%, which demonstrates the adequacy of the
model. Discussion/Conclusion: The HS-SPME-GC-MS
method was developed and successfully optimized
through the aid of the design of experiments, which
propitiated a reduction in the number of experiments
and standard experimental work required. The method
will be applied to 39 seized whiskeys provided by the
Superintendência da Polícia Civil Técnico-Científica de
Minas Gerais to verify materiality in possible causes
of fraud. Acknowledgments: This research was
supported by CAPES – Brazil (Financing Code 001). We
also thank the Superintendência de Polícia Técnico-
Científica de Minas Gerais.

Development of a new quantitative
field-testing assay for the analysis
of histamine in seafood samples
Background/Introduction: Scombrotoxin fish
poisoning or also known as histamine fish poisoning,
occurs after the consumption of dark-muscled fish
that has been not properly handled. Histamine is
produced from putrefied fish after the inadequate
chilling of raw fish enables allows bacterial conversion
of free L-Histidine. Histamine intoxications have been
reported to occur at concentrations lower than 1000
mg/kg, the United States Food and Drug Administration
uses a guidance level of 50 mg/kg for histamine in
fish, whereas in the European Union Critical levels
for histamine in fish and simple fish products are 100
mg/kg histamine. So far, the established analytical
methodology to quantitatively measure histamine
involves complex laboratory instrumentation such
as high-pressure liquid chromatography. However,
most fish processors do not have the expertise or
the infrastructure to deploy these technologies on
the field to make rapid decisions. In this research, the
validation of a simplified quantitative workflow for
the field testing of histamine in fish samples using
a new lateral flow immunochromatography assay
is discussed. Objectives: The objective of this study
was to develop a new quantitative and portable
methodology that enables fish processors to analyze
histamine in fish samples in 3 minutes. The use of
an aqueous extraction in combination with lateral
flow immunochromatography was evaluated and
developed to provide reproducible and high recoveries
of histamine in spiked fish samples and quality control
materials at critical levels, as well as the comparison
of these results with those derived by other analytical
methods. Methods: The following fresh and thawed
frozen raw Histamine-free fish samples were used for
the analysis: anchovy, sardine, tuna, cod, mackerel,
fish meal. These matrices were spiked at both 50
mg/kg and 100 mg/kg. The extraction involved a
227
Cabrices, Oscar G.
G-Flo Scientific Laboratory Testing & Consultants (USA).
homogenization step with distilled water. The extracts
were filtered subsequently diluted for analysis. 100
µL of the extracts were loaded into testing wells
and were analyzed using Symmetric Histamine
Lateral Flow immunochromatography kits (3-minute
incubation time), subsequent quantitative analysis
using the portable Lateral Logic S-Flow reader. Both
technologies were produced by Prognosis Biotech.
Results: The overall lateral flow method’s recovery
for histamine was above 90% and coefficient of
variation of all the fish matrices spiked and the quality
control materials were within acceptable range (%CV
< 15%). The extraction process using distilled water
in average took less than 5 minutes to complete, in
comparison to other methodologies that use long
acylation procedures (i.e., more than 20 min) and
require expertise to avoid errors in accurate histamine
measurements. Comparison to other analytical
approaches showed good correlation between
methods (R2> 0.9), and no significant difference in the
results between fresh and thawed frozen raw fish
samples was found. Conclusion/Discussion: A new
quantitative histamine assay was developed using
lateral flow immunochromatography technology.
Histamine was efficiently extracted (>%90 recovery)
using a simplistic aqueous extraction protocol. The
3-minute analysis time as an alternative to other
analytical techniques can help fish processors
to maintain product integrity on the field while
maintaining histamine quantification requirements
by regulatory agencies. This approach provides
portability and reduces the complexity presented
by other methodologies. Acknowledgments: The
author would like to acknowledge Research and
Development team at Prognosis Biotech for the
technical development and method implementation
of this assay.

Food Safety: regulatory and toxicological
aspects of food packaging
Introduction: In the Food Safety context, packaging
and materials that come in food contact must not:
endanger human health; change the composition of
the food and/or cause organoleptic deterioration.
Through migration, packaging can transfer
components into the food. Migrants are low
molecular weight substances, with mobility and
ability to be extracted and/or absorbed by food, which
may represent a health risk to the consumer. Food
packaging are classified as: Cellulosic; Elastomeric;
Metallic; Plastic; Regenerated Cellulose; and Glass.
Objective: Approach the legislative and toxicological
aspects of migrants in food packaging, emphasizing
Anvisa’s role in Food Safety. Methods: Bibliographic
review of articles, legislation and national regulations
that address the regulatory and toxicological
aspects of food packaging. Results and Discussion:
In Brazil, the National Health Surveillance Agency
(Anvisa) is responsible for regulating the use of
materials intended to come into contact with food.
Thus, it establishes the maximum tolerated limits of
contaminants relevant to human health through the
Collegiate Directorate Resolutions (RDC), organized
by type of packaging material. In this scope, the
RDC’s describe that every substance used in the
composition of a package must appear in the positive
lists, which present the list of substances proven safe
for the intended application, once the specifications,
restrictions and limits established are met. Until the
present moment, there is no negative list about this
subject, that is, with substances prohibited to be used
in food packaging materials. A substance is not listed
as positive when: use is unsafe; safety has never been
studied; there is not enough information to conclude
on safety; inclusion has not been requested. When
a component is not in the positive list, it cannot be
used in the application to which this list refers. Some
228
Almeida, Giulia Forni; Pinheiro, Fabriciano
Intertox Ltda. - São Paulo, SP.
RDC’s establish parameters for: total migration,
specific migration, and the composition of the
material. Therefore, because of packaging migrants,
the human health risk assessment is related to the
hazard of the component (toxicological aspects of
that migrating material) and the potential exposure
to this given component (amount that will migrate
from the packaging to the food). In the cellulosic
packaging issue, it is possible to highlight RDC’s No.
88/2016, No. 89/2016 (coction and hot filtration) and
No. 90/2016 (coction or oven heating). The positive
list present in RDC No. 88/2016, which approves
the technical regulation about cellulosic materials,
packaging and equipment intended to come into
contact with food and makes other provisions, has
numerous substances. Among these substances,
with the function as additive for raw materials and
authorized as accelerator for lignin and cellulose
separation, Anthraquinone (CAS No. 84-65-1), is
classified within the Carcinogenicity class - Category
1B (risk phrase: may cause cancer), by the European
Chemicals Agency (ECHA). RDC No. 88/2016, in terms
of risk assessment, establishes for Anthraquinone the
maximum limit of 0.10% by weight of lignocellulosic
material and the specific migration limit of 0.01mg
of Anthraquinone/kg of food. Conclusion: According
to what has been analyzed, it can be concluded that
Anvisa, through the parameters described in the RDC’s,
acts clearly stamping the toxicological concepts of
risk assessment concerning the composition and
possible migration of food packaging materials,
ensuring the adequate protection of human health in
Food Safety. As was also evident the importance of
toxicological studies to establish the conditions under
which foods can be ingested without causing harm.
Acknowledgments: To Intertox Ltda. and everyone
who participated in the development of this work.
 229

Ozone processing of peanut “milk”: degradation

of aflatoxins, reduction of allergenic
proteins and impact on lipid oxidation
Romero, Alessandra de Cássia1; Sartori, Alan Giovanini de Oliveira1; Caetano-Silva, Maria Elisa2;
Alencar, Severino Matias1,3; Calori-Domingues, Maria Antonia1; Augusto, Pedro Esteves Duarte1
1
 Department of Agri-food Industry, Food and Nutrition (LAN), “Luiz de Queiroz” College of
Agriculture (ESALQ), University of São Paulo (USP), Piracicaba, Brazil. 2 College of Agricultural,
Consumer & Environmental Sciences, University of Illinois at Urbana-Champaign, IL, USA. 3 Food
and Nutrition Research Center (NAPAN), University of São Paulo (USP), São Paulo, SP, Brazil.

The market for plant-based products has been
expanding over the last few years due to consumer’s
concerns about vegetarianism, healthier and/or
more sustainable lifestyle. Then, vegetable products
analogous to conventional animal-products has
been developed, such as the plant-based “milks”.
Peanut is appreciated both for its flavor and for
its high nutritional value, despite its allergenic
potential and mycotoxin contamination. Aflatoxins
are carcinogenic and immunosuppressive mycotoxins
whose presence in peanut grain and derivatives
is commonly reported in the literature. If present
in the peanut grain, aflatoxins can be carried into
the extract during processing, reaching the peanut
“milk” and compromising the product safety. In
the same way, allergenic proteins found in peanut
are resistance to process of milk production. Thus,
we studied the “peanut milk” ozonation process,
evaluating the kinetics of degradation of aflatoxins
AFB1, AFB2, AFG1, and AFG2, considering their effect on
lipid oxidation and the allergenicity of peanut proteins
and oxidative parameters. Aflatoxins degradation
was evaluated through ozonation of commercial
peanut milk samples for 0, 5, 10, 20, 30, 60, 90, 120,
150, 180 and 210 min (initial concentrations: AFB1 =
43 μg/L, AFB2 = 38 μg/L, AFG1 = 59 μg/L, and AFG2 =
30 μg/L). Oxidative parameters evaluated comprised
the fatty acid profile and the volatile compounds
formation. Ozonation effect on the allergenic proteins
from peanut milk was measured at times 0, 10, 30,
60, 120 min. Ozonation was efficient to degrade
AFB1 and AFG1 within 30 min, despite AFB2 and AFG2
were more resistant even after 210 min. A higher
ozone consumption occur between the first 30 min,
suggesting that the most accessible and reactive
points on the molecules from peanut milk were
exhausted after this time. The fatty acid profile shown
a reduction of oleic acid and an increasing of volatile
compounds whose signal the oxidative degradation.
Nonanal and hexanal are markers of lipid oxidation
and can be detected by the human nose at levels from
1-5 ppb. The oleic acid degradation after ozonation
produced nonanal and hexanal levels several times
above these threshold levels, even after only 10 min
of process. Thus, it suggests that the evaluation of
the improvement of the ozonation process should be
done in this initial range of time. In addition, additional
strategies to prevent lipid oxidation and improve the
ozonation efficiency could be useful. Ozonation was
suitable to degrade the most toxic aflatoxins, AFB1,
and AFG1 from peanut milk.In addition, the ozonation
of peanut milk has shown the potential to reduce
the concentration of the allergenic peanut proteins.
However, the process should be improved to find the
best compromise to the reduction of the undesirable
compounds and the quality maintenance of the
ozonated product. Acknowledgments: This work
was supported by the National Council for Scientific
and Technological Development (CNPq, Brazil) (CNPq,
Brazil) (funding the productivity grant of P.E.D. Augusto
306557/2017-7 and 310839/2020-3); Coordination
for the Improvement of Higher Education Personnel
(CAPES, Brazil) [funding the postdoctoral fellowship
of A.C.Romero 88887.356778/2019-00]; São Paulo
Research Foundation (FAPESP) [2019/05043-6].

Using R language to study worldwide Lead
contamination distribution on food using the
GEMSFOODS(WHO) database during 1995-2020
Background/Introduction: Lead (Pb) is the second
most toxic metal after arsenic (As), comprising 0.002%
of the earth’s crust. It is estimated that exposure
to lead was responsible, in 2004, for 143 thousand
deaths and 0.6% of the global disease burden, taking
into account mild mental under development and
cardiovascular outcomes. Beyond health surveillance
actions and analytical monitoring and systematic
evaluation of the data produced is required. Objective:
Investigate the content of Lead in foods using the R
language as a technique for filtering, analyzing and
modeling distributions according to the varieties
of food consumed, countries, analytical conditions
and dates. Find out where and which foods are most
contaminated and create possible hypotheses of
cause. Create a reproducible approach that can be
reviewed and easily shared among the scientific
community. Methods: All calculations and graphs
were made in Rstudio version 1.3.959 and R base 3.6.3
and the GEMS\FOOD database (WHO). Results: Among
developed countries, European Union presents the
highest levels of contamination with most outliers.
Most foods with high lead values have a very limited
intake: “Animal Feed”, “Stimulant beverages”, “Herbs,
spices and condiments”. Only “Products for special
nutritional use” are critical as they may result in a
230
Silva, Fabiano; Couto, Nilton; Almeida, Mariana
Fundação Ezequiel Dias - FUNED - Minas Gerais.
higher intake for longer periods. They show mean
values of There are no indications that imported
samples (to the European Union) are quantified
more frequently than domestic ones, indicating
contamination at source. In the European Union, 91%
of all samples are below the 0,1mg/kg and 99% are
below 1.21mg/kg. Stimulant Beverages, especially tea,
present a high continuous source of Lead in the diet.
Just to address how much, 50% of tea infusion samples
are higher than 0,2mg/kg while 95% of other samples
fall on this range. Discussion/Conclusion: In the 10
years with most data from the European Union and
the most studied sample (Tea Infusion), no difference
was noted in the mean value of contamination. No
difference in the number of reproved samples was
found among those from accredited laboratories and
unaccredited ones. During risk analysis, samples with
a reported LOQ are disregarded. We found that there
are no statistical differences (Tukey HSD, p<0.01) in
quantified results from laboratories with a reported
LOQ below 0.01mg/kg and those who didn’t report the
LOQ. meaning that in practical terms the lack of LOQ
doesn’t mean an inferior result. Acknowledgments:
The authors acknowledge the help of Minas Gerais
State government for supporting
 08 
FORENSIC
 232

An overview of NPS in northeast Brazil:

NMR-based identification and analysis
of ecstasy tablets by GC-MS
Cunha, Ricardo Leal1,3; Oliveira, Celinalva da Silva Lima2; Oliveira, Aline Lima4;
Maldaner, Adriano Otávio5; Pereira, Pedro Afonso de Paula3
1
 Polícia Científica de Sergipe, Aracaju, Brazil; 2 Departamento de Polícia Técnica da Bahia, Salvador,
Brazil; 3 INCT E&A, Universidade Federal da Bahia, Salvador, Brazil; 4 Instituto de Química, Universidade
de Brasília, Brasília, Brazil; 5 Instituto Nacional de Criminalística, Polícia Federal, Brasília, Brazil.

Introduction: The actual illicit market for synthetic
drugs is characterized by a wide variety of psychoactive
substances of different chemical and pharmacological
classes, such as amphetamine-type stimulants and
new psychoactive substances. Knowledge about
its chemical composition, as well as the identity
and quantity of the active substances present, is
important for emergency care in cases of intoxication
by these substances and to establish adequate
chemical and toxicological analysis procedures in
forensic laboratories. Aim: The objective of this work
was to study the prevalence of amphetamine-type
stimulants and new psychoactive substances (NPS)
in the northeast region of Brazil (States of Bahia and
Sergipe), involving seized samples by local police in
the period from 2014 to 2019. Methods: In a total of
121 seized samples (between tablets, blotter papers,
powders, and crystals) in which ecstasy tablets
predominated (n=101), were used GC-MS and 1D-NMR
techniques, in order to unequivocally identify the
nature of classical synthetic drugs and NPS present in
these materials. One-dimensional nuclear magnetic
resonance spectra (1H-NMR and 13C-NMR) were
acquired using two different instruments. Crystal
samples were analyzed on a Bruker Avance III 600
NMR spectrometer, operated at 14.1 T (600 MHz for
1H, 150 MHz for 13C), while the other samples were
analyzed on a Varian INOVA 500 NMR spectrometer
operating at 11.7 T (500 MHz for 1H, 125 MHz for 13C).
On the other hand, the quantity of psychoactive and
adulterant substances present in ecstasy tablets
was determined using an optimized and validated
analytical method by GC-MS (Agilent 7890A/5975
GC-MSD). Results: Twenty-one substances were
identified, of which sixteen NPS of different chemical
and pharmacological classes (25I-NBOH, 25B-NBOH,
methylone, N-ethylpentylone, N-ethylpentedrone,
diphenidine, fentanyl and others) and five classical
synthetic drugs (MDMA, MDA, methamphetamine,
amphetamine and clobenzorex). Analyzes of 101
ecstasy tablets showed that MDMA was the main
component, being found in 57% of the samples,
in amounts between 27.3 and 187.1 mg per tablet.
In addition, mixtures of MDMA, MDA, caffeine and
synthetic cathinones were observed in 34 samples
analyzed. Conclusions: The results obtained with the
identification of several new psychoactive substances
(NPS) in the states of Bahia and Sergipe, in northeast
region of Brazil, demonstrate that the population of
this region is exposed to the illicit trade of these drugs.
These results are somewhat similar to those found in
previous studies carried out in other Brazilian regions.
REFERENCE
1. BRASIL, Ministério da Justiça e Segurança Pública, MJSP
(2020). RELATÓRIO 2019 – DROGAS SINTÉTICAS. SEPLAB/DPER/
INC/DITEC Polícia Federal.
 233

Analysis of diglycolic acid in victims

intoxicated by the consumption of
beer containing diethylene glycol
Goulart, Cristiano O.L.1,2; Bordoni, Leonardo S.1,3,4; Nascentes, Clésia C.2; Costa, Letícia M.2
1
 Instituto Médico Legal André Roquette, R. Nícias Continentino, 1291, Gameleira, 30510-160, Belo
Horizonte, Brasil; 2 LEAQUAA, Departamento de Química – ICEx, Universidade Federal de Minas Gerais,
Av. Antônio Carlos, 6627, Pampulha, 31270-901, Belo Horizonte, Brasil; 3 Universidade Federal de Ouro
Preto, R. Dois, Campus Morro do Cruzeiro, 35400-000, Ouro Preto, Brasil; 4 Faculdade de Medicina
de Barbacena, Praça Presidente Antônio Carlos, 8, São Sebastião, 36202-336, Barbacena, Brasil.

Introduction: Diethylene glycol (DEG) is a toxic,
colorless and odorless reagent, used for the production
of antifreezing devices, among other applications.
In Minas Gerais - Brazil, several people presented
intoxication symptoms related to DEG ingestion, after
the consumption of a brand of beer contaminated with
DEG, possibly from the leakage of an antifreeze liquid.
Clinical manifestations are gastrointestinal symptoms
(nausea, abdominal pain, among others), inebriation,
with altered mental status and metabolic acidosis,
kidney injury and neurological symptoms. Several
victims had a common history of use in previous days
of a specific beer. In the chemical analysis of this beer,
carried out by another laboratory, DEG was detected
on it. The hepatic metabolism of DEG is carried out by
the enzymes alcohol-dehydrogenase and aldehyde-
dehydrogenase. Recent studies demonstrated that
DEG shows its toxic action through diglycolic acid
(DA). Studies indicated that DA is slowly excreted,
being detected even when DEG is not. Objective:
The aim of this study was to detect DA in biological
samples (whole blood, vitreous humor, urine,
cerebrospinal fluid (CSF), liver and kidney) collected
from victims related to beer intoxication, using gas
chromatography coupled to a mass spectrometer
(GC-MS). Methods: Prior to extraction, solid organ
samples (liver and kidney) were homogenized using a
bead tissue disruptor at 20 Hz for 10 minutes. In a 1.5
mL polypropylene microtube, 20 μL of fluids (blood,
vitreous humor, urine or CSF) or 10 mg of tissue
homogenates (liver or kidney) were mixed with 45 μL
of acetonitrile and 50 μL of methanol. The tube was
then vortexed for 15 seconds, sonicated for 5 minutes
and centrifuged at 5000 rpm for 15 minutes. The
supernatant was transferred to a 2 mL vial and dried
at room temperature with air flow. The dried extracts
were derivatized with 50 μL of BSTFA/1% TMCS for
20 minutes at 70°C. After cooling, the samples were
transferred to inserts and injected into the GC-MS.
Results: In the 9 autopsied victims, whole blood, urine,
vitreous humor, CSF, kidney and liver samples were
evaluated. For DA analysis in the samples collected
from these cases, 2 victims presented positive results
in all matrices. Another 4 presented negative results
in all matrices, while 2 presented negative results
only in the liver, and one negative result in blood and
vitreous humor. Although DEG can be detected using
the analytical method presented in the study, DEG
was not found in the necropsied victims. Conclusion:
DA was successfully used to assist in the investigation
of poisoning cases after the consumption of beer
contaminated with DEG. The existence of one case
with a negative result for DA research in the blood,
with the presence of a positive result for DA research
in other matrices, is an indication that in cases of
necropsy, it could be interesting to evaluate other
matrices besides blood. Finally, it was noticed that, in
samples in which DEG could not be observed, DA was
found. Acknowledgments: This study was carried out
with the support of the Civil Police of Minas Gerais and
the Public Ministry of Minas Gerais.
 234

Determination of phosphatidyl ethanol

in dried blood spots by LC-MS/MS
Guterres, Fernanda S.1; Tegner, Mariane2; Ott, Isabela Ritter2; Linden, Rafael1,2; Antunes, Marina Vezon1,2
1
 Institute of Health Sciences, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate Program
on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil.

Background: The harmful use of ethanol has become
a global health problem, associated with increasing
risk for many harmful health conditions as injuries,
violence, risky sexual behaivors, as well as long-therm
health risks as high blood pressure, heart disease,
cancer, mental health problems, social problems and
alcohol dependence. Phosphatidylethanol (PEth), a
direct ethanol biomarker, formed in the membranes
of the erythrocytes with a 4-6 days half-life. It has
been used as a highly specific biomarker for alcohol
abuse and classification of drinking patterns, with
detection window of 2-4 weeks. The used dried blood
microsampling would facilitate logistics for PEth
measurements, with high stability, low invasiveness
of collection and no need for refrigeration during
transport and storage of the samples. Objective: To
develop and validate a method for the determination
of PEth in dried blood samples (DBS) by LC-MS/
MS. Methods: DBS samples were prepared by liquid
extraction from 8 mm punch with 500 µL of methanol
and acetonitrile (80:20, v/v) containing the internal
standard IS (phosphatidyl propranolol 17ng/mL).
Samples were homogenized for 30 minutes at 25 °C
and 1000 rpm. The organic layer was evaporated at
45 °C for 45 minutes. The extract was resuspended
with 100 µL of water and methanol (50:50, v/v), 10 µL
was injected into the LC-MS/MS with electrospray
ionization in negative mode. Chromatographic
separation occurred in an phenyl column (100 x 2.1
mm x 3 μm) at 40 °C. Mobile phase was ammonium
acetate 4mM in water and acetonitrile 28:72 (v/v)
eluted at 0.4 mL/min -1. A electrospray source was
used for ionization in negative MRM mode, with source
temperature of 350 °C, capillary temperature of 200 °C,
auxiliary gas nitrogen at 10 arb and sheath gas 50 arb.
The quantification transition was m/z 781 – 281 for
PEth and m/z 741 – 281 for IS. The validation tests are
following the recommendations of the international
forensic guidelines (SWGTOX, 2013), Results: Total
analytical run time was 6 min, with elution of PEth in
2.1 min and IS in 2.5 min. The method was linear from
10 to 3,000 ng/ml (r2 = 0.99), accurate 87% to 109%,
precise with CV% within 5.4 to 12.3% Mean extraction
yield was 70%. There was no significant impact
of the hematocrit, 25% to 55% in assay accuracy
(85-115%) or recovery (p=0.23). The use of the IS
was satisfactorily compensated the matrix effect
(-2 to +17%). Discussion/Conclusion: The method
presented adequate performance for measuring PEth
and can be used for evaluating patterns of alcohol
consumption in clinical studies. Acknowledgments:
Financial support CNPq and CAPES.
 235

First Report of national database on toxicological

criminal information (ToxCrim system)
Costa, Rony Anderson Rezende1; Costa, Jose Luiz2
1
 Official Expert of Cientific Police Institute of Paraiba State and PhD student of
Pharmacology Program at UNICAMP; 2 Executive Coordinator of Campinas Poison
Control Center and Professor of Pharmaceutical Science at UNICAMP.

Introduction: There are three different database in
Brazil with toxicological information, the Information
System of Grievances Notifications (SINAN) of
Health Ministry, the National System of Toxic and
Pharmacologic Information (SINITOX) of Oswaldo
Cruz Foundation and the National Health Surveillance
Agency and Brazilian System of Intoxication Data
(DATATOX) of Brazilian Association of Information and
Toxicological Assistance Centers. Unfortunately, there
is no collection by any of them of criminal toxicological
information because of the clinical approach to the
toxicology of these systems. Such absence of criminal
toxicological information and identified analyte data
creates a gap of knowledge that is of vital importance
for official laboratories to use in delineating criminal
toxicological investigations. The ToxCrim system, as a
pilot of a national database of criminal toxicological
information, provides an important knowledge of the
scope of intoxication cases in the Brazilian scenario,
especially in the involvement with crimes in these
cases. Objective: To present the development of
a national database on toxicological information
prepared by official forensic laboratories in the
first semester of 2021. Methods: The records in the
ToxCrim system (www.toxcrim.com.br) were based
on data from forensic toxicologists reports issued/
signed (regardless of the examination entry date) and
collected by the participants designated by the official
forensic laboratories. The information required were:
a) Identification, b) State, c) Age, d) Sex, e) Cause of
death, f) Middle of the Cause of death, g) Biological
matrix , h) Analytical technique and i) Analyte. The
data were extracted, filtered and consolidated that
have been identified and/or detected by the official
forensic laboratories of the States of Bahia, Espírito
Santo, Goiás, Sergipe, Paraíba, Rio Grande do Sul, São
Paulo and Rondônia between January and June 2022.
For this first report only data from post mortem cases
of traffic accidents and homicides were exposed.
Results: 2,975 post mortem cases were consolidated
with 10,734 records of analytes detected/identified
in post mortem blood samples. 825 cases were
classified as traffic accidents. Of these 825 cases,
711 records of toxicological tests to quantify ethyl
alcohol in blood (alcohol levels) and 206 records
of other tests were recorded. In 92 cases both
records were recorded. In this legal cause of traffic
accidents, the most detected/identified analytes in
post mortem blood samples were ethyl alcohol (711),
benzoylecgonine (118) and methylecgonine (82).
Homicide cases were all classified directly in this way,
whether intentional or negligent or due to bodily injury
or after robbery or robbery with resistance to arrest
and consisting of the following means: firearm, melee
weapon, other blunt agent, fire or other. There were
461 homicides containing 1117 records of analytes
detected/identified in post mortem blood samples.
The most detected/identified analytes were alcohol
(280), benzoylecgonine (216) and methylecgonine
(182). Several other data were obtained from the
records. Acknowledgments: We would like to thank
the participating Official Experts Ana Cecília Bandeira
(BA), Fabrício Pelição (ES), Mariana Dadalto (ES),
Sophia Wieczorek (GO), Ricardo Cunha (SE), Victor
Gianvecchio (SP), Carolina Lupi (RS), Ana Júlia Frazão
(RO) and Carolina Diniz (RO).
 236

Forensic entomotoxicology: application of

studies in the development and rearing of
Lucilia cuprina flies (Diptera: Calliphoridae)
Chimendes, Nayomi de Andrade1; Bairros, André Valle1; Ugalde, Gustavo Andrade1; Santos, Lara Celestina1;
Pacheco, André Lucas Bezerra1; Reginato, Fernanda Ziegler1; Berlato, Dener Gomes1; Rosa, Victória Gomes1;
Nascimento, Marcelo Henrique Santana1; Pereira, Alessandra de Oliveira2; Monteiro, Silvia Gonzalez2
1
 Nucleus Applied to Toxicology (NAT); 2 Parasitic Disease Laboratory (LAPAVET).

Introduction: Forensic Entomotoxicology is the
application of the study of insects and other arthropods,
associated with forensic procedures involving
toxicological analysis. Among the species of flies of
the Calliphoridae family, the blowfly (Lucilia cuprina)
is commonly linked to post-mortem phenomena. The
larvae, more precisely at the L3 stage, of this species
are used as an alternative matrix in the analysis of
toxicants, especially in bodies in an advanced state
of decomposition and tissue unfeasibility. Objective:
Due to its great importance and influence in the
area of ​​forensic entomotoxicology, the objective of
this study was to monitor the development stages
of these insects, from the egg, larva, pupa to adults.
Methods: For a better understanding of the facts, a
protocol for rearing Lucilia Cuprina flies was started,
meeting all the requirements of their biological cycle,
to be developed in the laboratory. The rearing room
where the larva, pupa and adult stages were kept at
25ºC, relative air humidity of 70% (± 5%) with a 12-
hour photo phase. Entomological cages (30 x 30 x 50
cm) were used for rearing. The adult’s food is made up
of honey, placed on top of rice grains, placed in a Petri
dish, in small glass jars, and water is kept freely on a
filter paper strip so that they can hydrate themselves
without to die by drowning. So that there is egg
laying, a cut of beef liver was placed next to the same
feeding cage as the adults. The postures should be
carefully removed with a damp brush and transferred
to another Petri dish. This should contain wet dog
food (Pedigree® sachet for dogs), so that when the
eggs advance to the larval state, they maintain their
saprophagous and/or carnivorous habits. The plate
containing the substrate and eggs was placed on the
sterilized sand in a plastic pot (350 mL). The pot was
closed with organza tissue and rubber tie. The larvae’s
food was changed every two days and, to do this, the
plastic pot was filled with water, sifted the sand and
the content containing the larvae was poured into a
sieve. Then, larvae at stage L1, L2 and even L3 must
be transferred to a new pot containing sand and food.
Larvae in L3 that had already migrated to the sand and
pupae, should be transferred to pots containing only
sterilized sand. After leaving the pupae, the adults
were changed cage to continue the cycle. Results: As
its life cycle is relatively short, it was easy to monitor
its development, allowing for a continuous collection
of material when the creation of insects is established.
L. cuprina completed its whole life cycle in 29 to 45
days where the eggs hatched within 1–2 days and the
larvae went through three instars with an average
duration of 6.5 days. The pupal stage lasted for 14
days and longevity of moths averaged 36.25 days.
Conclusions: Therefore, through the aforementioned
facts, the advantages of studying this matrix are
understood, as it is considered to be easy to create,
because it has a smaller amount of matrix interfering
when compared to other more complex biological
matrices and for maintaining the preservation of
analytes, that can undergo biological and/or abiotic
alteration in the decomposing tissue to be analyzed.
Acknowledgements: We wish to thank the staff of the
insect rearing center at Parasitic Disease Laboratory
(LAPAVET/UFSM) for providing the space and share
the knowledge about the insects rearing.
 237

Interference of larvae matrix of flies

Lucilia cuprina in the determination of
Azamethiphós and Paraoxon ethyl using
dispersive solid phase extraction (dSPE)
Pacheco, André L.B.1; Ugalde, Gustavo A.1; Chimendes, Nayomi A.1; Santos, Lara C.1;
Berlato, Dener G.1; Nascimento, Marcelo H.S.1; Reginato, Fernanda Z.1; Rosa, Victória
Gomes1; Santos, Rachel1; Cardoso, Leonardo Correa1; Monteiro, Silvia Gonzalez2;
Stainki, Daniel Roulim2; Pereira, Alessandra de Oliveira2; Bairros, André V.1
1
 Nucleus Applied to Toxicology, Center of Health Sciences, Federal University of Santa Maria, Santa
Maria, Brazil; 2 Parasitic Disease Laboratory, Federal University of Santa Maria, Santa Maria, Brazil.

Introduction: Organophosphate pesticides are
one of the main classes of pesticides related to
intoxication. When the human body is in a state
of decomposition, it presents few alternatives of
biological matrices. Therefore, the flies larvae of
the species Lucilia cuprina, a saprofable insect, is
an alternative matrix in these situations. However,
studies with organophosphates in larvae of flies
Lucilia cuprina are scarce. Objective: The objective of
this study was to evaluate the behavior of the larvae
matrix using larvae at the third-stage of development
(L3) in the determination of organophosphates and
their respective biotransformation products using
dispersive Solid Phase Extraction (dSPE). Methods:
The analyzes were performed in GC-MS with RTX®-
5MS column (30 m x 0.25 mm, film thickness 0.25 µm)
using ultrapure helium gas at a constant flow of 1.20
mL/min. The chromatographic parameters used were:
Injector temperature 175°C; 1 µL of injection volume
in splitless mode; Column oven configuration: 50°C
(maintained for 4 min) → 50°C (35°C/min) → 190°C
(maintained for 7 min.) → 190°C (40°C/min) → 300°C
(maintained for 1 min) and total chromatographic
analysis time was 15.75 minutes. The dSPE method
was used using 1g of the L3 homogenized and
weighed in a Falcon tube of 15 mL, followed by the
analytical standards (Azamethiphos, Carbofenotione,
Chlorpyrifos ethyl, Chlorpyrifos methyl, Ethion,
Fenitrothion, Formotion, Malathion, Malaoxon,
Paraoxon ethyl, Paraoxon methyl, Pirimiphós ethyl,
Pirimiphós methyl, Triazophos) and the internal
standard Medazepam were added in duplicate at a
concentration of 50 ng/g. Then, 2 mL of acetonitrile
were added and tubes were manually shaken for
1 minute. Then, 1 g of NaCl and 4 g of MgSO4 were
added. After, it was centrifuged (4,000 RPM) for 10
minutes, 1 mL of the supernatant was transferred to
another 15 mL Falcon tube, with 150 mg of NaCl and 25
mg of previously weighed primary secondary Amine
(PSA). Finally, the tubes were manually shaken for a
further 1 minute, centrifuged at 4,000 RPM and 500 µL
of the supernatant was transferred to a 2 mL flask and
dried in a sample concentrator. It was resuspended
in 50 µL of ethyl acetate and 1 µL was injected in GC-
MS. The analytes were analyzed in their respective
ions: Azamethiphos (109, 125, 215) Carbophenothion
(342, 157, 199), Chlorpyrifos ethyl (314, 199), 197),
Chlorpyrifos methyl (79, 125, 286), Ethion (125, 153,
231), Fenitrothion (125, 260, 277), Formothion (93, 125,
198), Malathion (125, 127, 173), Malaoxon (127, 195, 268),
Paraoxon ethyl (109, 149, 220), Paraoxon methyl (230,
109, 247), Pirimiphós ethyl (318, 304, 168), Pirimiphós
methyl (180, 276, 290) and Triazophos (161, 162, 172).
Results: For the analysis of the results, ions 125 and
109 were used for the quantification of Azamethiphos
and Paraoxon ethyl, respectively. The retention time
(RT) of the analytes were Azamethiphos RT:13.339
minutes and Paraoxon ethyl RT: 9.001 minutes, but
in the blank sample, there are interference peaks
of the matrix shown at the same retention time as
Azamethiphos and Paraoxon ethyl, which makes it
impossible to determine and quantify these analytes.
Thus, the importance of the study of matrix behavior
from larvae of flies Lucilia cuprina is relevant
for toxicological analysis. Acknowledgments:
Acknowledgments to CNPQ for financial incentives.
 238

Nortriptyline overdose and quantitative analysis

in post mortem vitreous humor – a case study
Cunha, Ricardo Leal1,2; Dalbosco, Juliana Santos1; Brito, Maria Carolina Santos Ribeiro1; Costa, José Luiz2,3
1
 Instituto de Análises e Pesquisas Forenses, Polícia Científica – Aracaju, SE - Brasil;
2
 Faculdade de Ciências Farmacêuticas, UNICAMP – Campinas, SP – Brasil; 3 Centro
de Informação e Assistência Toxicológica, UNICAMP – Campinas, SP – Brasil.

Introduction: The vitreous humor (VH) has in recent
years become an important alternative biological
fluid for post mortem toxicology. Due to its anatomical
position, this important biological fluid is protected
by the eyeball, being less affected by post mortem
phenomena such as putrefaction and redistribution. A
case of fatal nortriptyline overdose was investigated
after the death of a 27-year-old woman who used this
pharmaceutical to treat depression. Her body was
embalmed and buried, but after a period of four days,
the exhumation was performed by court order to
clarify the real cause of death. Thus, considering the
formaldehyde used in the preservation of the body,
the VH was collected under appropriate conditions for
carrying out the toxicological analysis. An analytical
method using micro-QuEChERS and LC-MS/MS was
applied to determine antidepressants in VH in order
to evaluate a possible overdose case. Aim: The aim of
this work was to determine nortriptyline in VH as an
alternative biological sample and to correlate with the
concentration in post mortem blood (PB) to confirm
a case of fatal intoxication. Methods: For sample
preparation, 300 μL of acetonitrile was transferred to
a polypropylene tube containing 100 mg of QuEChERS
salt mixture (MgSO4/NaAc 4:1), followed by 200 μL of
ultrapure water, 100 μL of blank post mortem vitreous
(negative samples previous analyzed at laboratory)
and 10 μL of internal standard (citalopram-d6 5 μg/
mL in methanol). The tube was homogenized in vortex
for 5 minutes and centrifuged at 14000 rpm/10 min
in a refrigerated centrifuge at 10°C. The upper layer
(300 μL) was transferred to a microtube, dried at a
gentle nitrogen flow and reconstituted with 40 μL
of methanol in a small insert glass. After this step, 2
μL was injected on LC-MS/MS system model LCMS-
8040 (Shimadzu, Kyoto, Japan). Chromatographic
separation was performed in a Restek Raptor Biphenyl
Column (100mm x 2.1mm, 2.7μm) maintained at 40°C
with mobile phase composed by ultrapure water
(A) and methanol (B), both containing 0.1% formic
acid and 2 mmol/L ammonium formate in a gradient
elution starting at 5% MeOH/95% H2O to 100%
MeOH. The flow rate was set to 0.4 mL/min with 6.0
minutes total run time. The mass spectrometer was
equipped with an electrospray ionization (ESI) source
operating in positive ionization mode. The source
parameters were: heat block temperature of 400°C;
capillary voltage of 4.5 kV, nebulizer gas (N2) flow of
3 L/min, desolvation line (DL) temperature of 250°C,
drying gas (N2) flow of 15 L/min and collision induced
dissociation gas (Ar) pressure of 230 kPa. The analyses
were performed in multiple reaction monitoring
mode (MRM), in which two MRM transitions were
chosen, one for quantitation and one for confirmation.
Results: The calibration curve for nortriptyline was
linear from 10 - 500 ng/mL, achieved an r > 0.99, and
the limit of detection 5 ng/mL. Nortriptyline was
quantified in VH at a concentration 209 ng/mL and the
ratio between VH and PB concentrations (VH/PB=0.13)
proposed by Øiestad et al was used to determine the
concentration in PB (1607 ng/mL). Conclusions: A
sensitive method based on micro-QuEChERS and LC-
MS/MS was applied to quantify nortriptyline in post
mortem VH samples (209 ng/mL) and to correlate
with the concentration in PB (1607 ng/mL). Therefore,
the concentration obtained for PB was very high for
nortriptyline considering the therapeutic (7-200 ng/
mL) and lethal (1500-2000 ng/mL) range, where the
cause of death was concluded as fatal nortriptyline
intoxication.

Optimization of extraction procedure for
benzodiazepines and antidepressants
analysis in vitreous humor using cork sheet
Ossanes, Daniela Souza; Birk, Letícia; Eller, Sarah; Oliveira, Tiago Franco
Background/Introduction: Considering the various
alternative biological matrices used in forensic
toxicology, the vitreous humor (VH) presents
several attractive characteristics including the
absence of metabolic activity; low levels of
endogenous interferers; less susceptibility to
postmortem redistribution; and location in an
isolated compartment, reducing the putrefaction
interference when compared to other matrices. Drugs
and medicines have been already detected in VH,
such as cocaine, opioids, alcohol, antidepressants,
and benzodiazepines. However, a few studies have
developed methods for the analysis of a wide range
of compounds in this biological fluid. Therefore, there
is a need of the implementation of effective, fast, and
automated procedures develop in accordance with
the principles of green chemistry. Objective: The
aim of this study was to optimize a straightforward
solid phase-based microextraction employing cork
sheet stripes for LC-MS/MS determination of 18
pharmaceuticals in VH. Methods: The extraction
methodology was performed of 3 steps, all conduced
in a 96 deep well plate. First, a 1.5 cm x 0.5 cm cork
sheet stripe is submerged in 1 mL of the conditioning
solvent for two minutes at 300 rpm. Second, this stripe
is transferred to 1 mL of a diluted sample solution,
containing 200 μL of vitreous humor and 800 μL of
ultrapure water and extraction occurred for 15 minutes
at 300 rpm. Third, the cork stripe is transferred to 1
mL of the desorption solvent for two minutes at 300
rpm. Subsequently, this solvent was transferred to an
Eppendorf tube, dried with assistance of an air flow,
resuspended in 20 μL of methanol and analyzed. A
239
Graduate Program in Health Sciences, Federal University of
Health Sciences of Porto Alegre (UFCSPA), Brazil.
LCMS-8045 triple quadrupole mass spectrometer
(Shimadzu, Japan) was used for the analysis. For
the optimization studies, three experiments were
planned to determine the optimal conditions for the
extraction procedure. Two experiments were based in
simplex-centroid design to evaluate three different
solvents in the conditioning and desorption steps.
For the conditioning solvent, acetonitrile, methanol,
and acetone were tested, and for the desorption step,
acetonitrile, ethyl acetate and methyl tert-butyl ether
(MTBE) were used. In addition, a Doehlert design
was performed for optimization of pH sample and
extraction time. The time variable was analyzed in
five levels, from 5 to 25 minutes, and the pH factor
in three levels, acid, neutral, and basic (5, 7 and 9).
Results: The optimal conditioning solvent determined
was a 1:1 mixture of acetonitrile and methanol, and
the desorption solvent ideal conditions were a 1:1:1
mixture of acetonitrile, ethyl acetate and MTBE.
Considering sample extraction factors analyzed, the
optimal conditions found were a neutral pH and 15
minutes of extraction time. Discussion/conclusion: In
conclusion, this study was able to develop an efficient
and fast microextraction method for 18 substances
in vitreous humor samples, using small cork sheet
stripes for extraction and a 96 deep well plate as
recipient. Moreover, this approach system could be
possible to automate. In addition, this analytical tool
can be used to support the study of other substances
in these alternative biological sample, supporting
broad applications in forensic toxicology. Keywords:
vitreous humor, benzodiazepines, antidepressants,
cork. Acknowledgements: CAPES.
 240

Prevalence of volatile organic compounds in

forensic toxicology reports of São Paulo State
Rodrigues, Taís B.1; Medeiros, Elvis A.2; Chinaglia, Kauê O.1; Gianvecchio, Victor A.P.2; Costa, Jose Luiz1,3
1
 Campinas Poison Control Center, University of Campinas, Campinas-SP, Brazil;
2
 Superintendence of the Technical-Scientific Police, Institute of Criminalistics, São Paulo-SP,
Brazil; 3 Faculty of Pharmaceutical Sciences, University of Campinas, Campinas-SP, Brazil.

Background/Introduction: Volatile organic
compounds (VOCs) is a group of substances with
different structures presented in a wide variety of
products of domestic and industrial use, therefore
people are frequently in contact and have easy
access. Inhalation or prolonged exposure could lead
to poisoning, and its lipid solubility and volatility
enhance their toxicity, having great importance for
forensic analytical toxicology. In addition, these types
of substances are also attractive as they are legal,
cheap and the effect occurs and disappears quickly.
Depending on the amount inhaled, they produce
depressant effects on the nervous system, which can
cause psychological dependence. Several countries
report the abuse of inhalants among children and
adolescents in order to produce effects, including
pleasure, disinhibition, euphoria and hallucinations.
The main harmful effects presented by the user
occur in the heart, lungs, kidney, neurological system,
liver and bone marrow. Studies show that more
than 50% of the deaths after inhalants consumption
were caused by direct toxic effects of the substance,
such as cardiac toxicity and respiratory depression,
other causes included plastic bag asphyxia,
aspiration of stomach contents and trauma due
to dangerous behavior. Objective: The aim of this
work was to evaluate the prevalence of inhalants
consumption in São Paulo state between January
and June 2021. Methods: The authentic samples
were analyzed by the Sao Paulo State Police, being
VOCs identified by GC–MS, according to laboratories
standard operational procedures and the SWGTOX
recommendations. Reports are entered into the GDL
system (Sistema de Gestor de Laudos, in Portuguese)
and data from the first half of 2021 were evaluated.
Results: More than 11,000 cases were registered in
the period, with 382 (3.5%) of the reports positive
for VOCs used as inhalants. Five different substances
were identified: trichlorethylene (63.5%), chloroform
(30.6%), dichloromethane (5.2%), toluene (0.5%) and
benzene (0.2%). In 46.1% of the cases, more than one
compound was detected, being the most frequently
combination trichlorethylene with chloroform
(94.1%). Prevalence of abuse is most common in males
(63%) and aged between 15 and 24 years old (60%).
The toxicological analyzes positive for inhalants were
requested mainly by the city of São Paulo (68.6%) and
those belonging to the metropolitan region (22.5%).
Discussion/Conclusion: The use of inhalants is not
gender specific but is most common among males,
being most prevalent in young population. Brazilian
data from 2010 report this group as the most used by
high school and the second by university students, just
behind marijuana. Data also shown that consumption
at least once in a lifetime is around 10% higher in the
city of São Paulo than in the rest of the country. In
2015, the inhalants most identified in seized solvents
were trichlorethylene (51%), dichloromethane (29%),
ethanol (15%) and chloroform, ether or ethyl chloride
(4%). As observed six years earlier, in toxicological
analyzes, trichlorethylene still is the most prevalent,
but chloroform appears as the second, being six times
higher than dichloromethane. This data showing
the consumption profile of this group of substances
could help to alert judicial authorities and guide them
to take measures aimed to reduce the access and
consumption of these substances, especially among
young population. To our knowledge, there are few
studies that report the prevalence and profile of
inhalants consumption in Brazil.
 241

Profile of synthetic drugs seized between

January 2019 and December 2021 analyzed by
the Forensic Toxicology Center of the Forensic
Expertise in the State of Ceará (PEFOCE)
Oliveira, Juliana Ribeiro Ibiapina Leitão; Magalhães, Danielle de Paula; Martins, Mayane Emanuella Melo
Lopes; Almeida, Vivian Romero Santiago; Saboya, Andrea Luiza Rocha; Holanda Júnior, Wanderley Pinheiro

Forensic Expertise in the State of Ceará (PEFOCE).

Introduction: The widespread use of synthetic
chemical substances for recreational purposes, allied
to their ease of acquisition, has been considered a
serious global public health problem. According to the
UNODC (United Nations Office on Drugs and Crime)
World Drug Report 2021, about 275 million people used
drugs worldwide in the last year, while more than 36
million suffered from disorders associated with drug
use. The market for these illicit synthetic substances
is considered complex and very dynamic. According
to data from the European Monitoring Center For
Drugs and Drug Addiction in 2019, the production of
synthetic drugs has become more sophisticated and
diversified around the world, with a new trend having
been observed with the use of unusual and more
easily acquired precursors. Traditionally, the term
“synthetic drugs” was used to refer to amphetamines,
substituted amphetamines (MDMA and its analogues)
and LSD, synthetic substances used for abuse. With
the emergence of the so-called new psychoactive
substances (NPS), this concept had to be expanded
to include this new class of substances. Objective:
The aim of this work was to carry out a survey on
the synthetic drugs most commonly identified by the
Forensic Toxicology Center of the Forensic Expertise
in the State of Ceará on the samples seized between
January 2019 and December 2021.Methods: The data
were collected from the Software Catalog/Database
used by the Forensics team – Galileu
​​ System, which
is a platform that holds the record of all information
related to forensics investigations, that also allows
communication and data exchange with the Police
Information System (SIP - Sistema de Informação
Policial) of the Civil Police of the State of Ceará. The
data were extracted from reports issued by Galileu,
referring to samples received by PEFOCE between
January 2019 and December 2021, registered in the
system as suspected synthetic drugs. After identifying
the reports in which synthetic drugs were analyzed,
the substances identified and reported in each report
were also verified. Results: During the study period,
PEFOCE received 199 suspected synthetic drug
samples, of which 137 were analyzed, while the others
are still being analyzed at the laboratory. The most
identified substance was MDMA, with 75 appearances,
followed by its analogue MDA, identified in 33 samples.
Other synthetic drugs identified were: LSD, ketamine,
eutylone, methamphetamine, ethylone, PMK-
glycidate, isosafrole, N-ethyl-pentylone, 4-chloro-
eth-cathinone and derivatives of phenethylamines
2C-I, 2C-E and 25H-NBOME. In 7 samples analyzed, the
presence of any substance prohibited or controlled by
344/98 of ANVISA was not identified. Discussion/
Conclusion: The three substances most identified
in the analyzed samples of synthetic drugs belong
to the group of “Classic Drugs” (MDMA, MDA and
LSD). These findings do not reflect the reality only
in the state of Ceará. Data from the latest Federal
Police Synthetic Drug Reports reveal that, for four
consecutive years (2017 to 2020), the synthetic drug
most seized in Brazil was MDMA; and in the 2020
report, this substance was preceded by MDA and LSD
in the ranking of seizures. Given the data presented, it
is necessary to create policies to monitor the routes
of origin of these substances marketed in Brazil, as
well as to investigate the existence of clandestine
laboratories in the country.
 242

Profile of toxicological analysis of suicide

cases reported by the Santa Catarina
Scientific Police in the last 5 years
Silva, Bruna Espíndola1; Boff, Bruna de Souza2; Silveira Filho, Jair2; Schroeder,
Samilla Driessen2; Rezin, Kéttulin Zomer2; Marchioni, Camila3
1
 Resident Pharmacist, Hospital University, Federal University of Santa Catarina,
Florianópolis, Brazil; 2 Scientific Police of Santa Catarina, Florianópolis, Brazil; 3 Department
of Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

Introduction: According to the World Health
Organization (WHO) suicidal behavior comprises
from the planning of the act, the attempts and even
the success when reaching the purpose, being a
serious public health problem. Around 800,000
people worldwide commit suicide annually, being
the second leading cause of death among young
people aged 15 to 29. In Brazil, Santa Catarina is
in second place in relation to the rate of death by
suicide, with a rate of 8.62 deaths per 100 thousand
inhabitants, according to 2015. Suicide is considered
a form of violent death, therefore, cases of death
by suicide must undergo analysis by the Scientific
Police of Santa Catarina (PCI/SC). Objective: Analyze
the epidemiological profile of suicides committed
between 2015 and 2020 registered by the PCI/
SC, with emphasis on the results of toxicological
analyses. Methods: Study using data obtained from
the PCI/SC forensic toxicology department, based on
an internal case registry worksheet. The analyzed
data were: number of suicides, sex, region of the
state, result of toxicological analysis and alcohol
dosage. Screening of blood, urine or gastric content
samples was performed by immunoassay (Randox®).
The confirmation was made in a gas chromatograph
coupled to mass spectrometry and alcohol dosage in
whole blood by headspace and gas chromatography
with flame ionization detection. Results: 11.44 deaths
per 100,000 inhabitants were observed in the state
of Santa Catarina. The percentage of male cases
increased over the years analyzed, reaching 82.2%
of cases in 2020. Cocaine detection was present in
380 cases (10.7%), with a prevalence of more than
90% in men. Furthermore, an increase in the number
of suicides was observed with increasing age, with a
peak in the age group of 50 to 59 years, followed by
a decrease. The mesoregion with the highest suicide
rate was the west of Santa Catarina, with 15.71 deaths
/ 100 thousand inhabitants and with the lowest rate
the north of Santa Catarina with 7.61 deaths / 100
thousand inhabitants. Toxicological analysis detected
the presence of toxic substances in 43.18% of the
cases and the presence of alcohol was detected in
27.49% of the suicides. Discussion/Conclusion: The
data showed that special attention should be given
to cases of suicide that are increasing in the state,
especially among males. There is a high prevalence
of suicide among men and also most cases involving
cocaine are in this group. In relation to 2020, despite
the consequences of the COVID-19 pandemic, there
was no significant increase in suicide cases compared
to previous years. Almost half of the reports showed
some substance detected, the most frequent being:
antidepressants, anxiolytics and cocaine. Although
the data be limiting in terms of substance dosage
and direct correlation with the cause of death,
they show the need for discussions and preventive
measures and/or public health actions. Keywords:
Suicide. Toxicological Analysis. PCI/SC. Drugs.
Acknowledgments: The Scientific Police of Santa
Catarina for the partnership and the provision of data.
 243

Profile of victims of traffic accidents with blood

samples collected for alcohol tests and analyzed
by the Forensic Expertise of the State of Ceará
(PEFOCE) from January 2020 to December 2021
Magalhaes, Danielle de Paula; Holanda Júnior, Wanderley Pinheiro; Saboya, Andréa Luiza Rocha; Oliveira,
Juliana Ribeiro Ibiapina Leitão; Martins, Mayane Emanuela Melo Lopes; Santiago, Vivian Romero

Forensic Expertise of the state of Ceará (PEFOCE).

Introduction: Several studies worldwide report that
alcohol is major responsible for traffic accidents,
worsening morbidity and consequently increasing
mortality. Since 2008, the Brazilian legislation has
established severe punishment for drivers caught
driving after drinking alcohol or under the influence
of any other psychoactive substance. At the end of
2012, the law was changed, becoming stricter, but
even so, in the global scenario, Brazil accounts for
almost half of all deaths from traffic accidents.
Objective: Describe the demographic profile of
traffic accident victims which had their blood alcohol
content tested at the Forensic Toxicology Center
(NUTOF) of PEFOCE from January 2020 to December
2021. Method: The data extracted from the Gallileu
system, forensics management software developed
and used by PEFOCE, and tabulated in Excel® 2019
and Stata/MP version 2016 programs. In the period
from January 2020 to December 2021, from the total
number of blood alcohol levels (BALs) requested
to the NUTOF, the cases of traffic accidents by
collision/trespassing and with ethanol detection test
performed were considered for the description of the
victims’ profile. The variables analyzed were sex, age,
BALs verification, in vivo and post mortem. It was
considered as detected, values equal or greater than
1,0 dg of ethanol per liter of blood. Results: PEFOCE
received 7,015 requests for BALs tests in the years
2020 (51.0%) to 2021 (49.0%), of which 2,433 (34.7%)
were related to traffic accidents. Of these, 847 (35%)
samples were analyzed, with a higher prevalence of
males (86%; n=729), and ethanol was detected in
47.8% (n=405) of the total analyzed. Most analysis
(92%; n= 782) were performed on post-mortem
samples. Of the samples with the presence of ethanol,
61.8% (n=250) had a value between 15,0 and 29,0 dg/L.
The positivity of the test was higher in males (92%;
n=372). The presence of ethanol was detected in 51%
of males (H) and 28% of females (M). The median age
was 37.2 years, with 38.7 years (M) and 37.1 (H). The
age group 30 to 39 years had the highest prevalence
among the cases analyzed (26%; n=218), as well as
among the positive cases (15%; n=125). Conclusion:
Ethanol was detected in a significant portion of the
cases analyzed, which reflects the involvement of
alcohol consumption in traffic accident cases. A high
prevalence of male gender was observed in cases of
traffic accidents, especially in the young adult age
group, up to 40 years old. We also observed a higher
number of positivity among males. Thus, we conclude
that there is a need for investments in public policies
of awareness and more efficient enforcement.
Acknowledgments: The Forensic Experts of the
state of Ceará and the colleagues from the Forensic
Toxicology Center.
 244

Quantifying ethanol content in

alcoholic beverages by HS-GC/FID
Bigão, Vítor Luiz Caleffo Piva1; Costa, Bruno Ruiz Brandão1; Santos
Júnior, Wilson José Ramos1; De Martinis, Bruno Spinosa2
1
 Faculdade de Ciências Farmacêuticas de Ribeirão Preto – USP; 2 Faculdade de
Filosofia, Ciências e Letras de Ribeirão Preto – Departamento de Química – USP.

Introduction: Counterfeiting alcoholic beverages
can lead to harmful effects on consumers’ health
due to the lack of quality control, which can generate
formulations that contain ethanol content in improper
concentrations or even at hazardous levels. In this
context, the monitoring of ethanolic content may
be used as a possible counterfeiting marker, aiding
the elucidation of possible frauds. Objectives: This
study aimed to develop and validate a method for
quantifying ethanol content in alcoholic beverages.
Methods: Ethanolic solutions of 11, 21, 31, 41 ,51, 61%
v/v (ethanol:water) were used for calibration and
validation of the method. For the ethanol extraction,
50 µL of the sample, 0.25 g of NaCl, 15 µL of acetonitrile
(internal standard, IS), and 935 µL of distilled
water were incubated in a headspace (HS) vial for 5
minutes at 80 ºC with an agitation of 500 rpm. A Gas
Chromatographic with Flame Ionization Detector (GC/
FID) system with a CP Wax 52 CB capillary column was
applied for the analysis. The oven temperature was
set as follows: 50 ºC (hold for 2 min); then ramped
to 70 ºC at 10 ºC.min-1. Next, the temperature was
ramped at 30 ºC.min-1 to 200 ºC. The injection was
performed in the split mode (120:1). Nitrogen 5.0 at 1
mL.min-1 was used as the carrier gas. The parameters
limit of detection (LOD), the limit of quantification
(LOQ), selectivity, linearity, weighting, precision, and
accuracy were evaluated during the validation of the
method following the recommendations of the UNODC
Guidance for Validation of Analytical Methodology.
Precision and accuracy were expressed as relative
standard deviation (RSD%) and relative error (RE%),
respectively. The heteroscedasticity at an α=5%,
carry-over, and stability were evaluated according
to the RDC Nº27/ANVISA. The ethanol stability in the
working solution was assessed during the analytical
process by determining the precision and accuracy
at two concentration levels (21 and 61% v/v) in
triplicate. Freeze-thaw stability was evaluated for 2
cycles of thawing at room temperature followed by
re-freezing at 4°C for 24 h. The short-term stability
was determined from the ethanolic solutions kept at
room temperature for 5 h before processing. Results:
The method presented LOD of 2 µg.mL-1 and LOQ of 5
µg.mL-1. The analyzed interferents did not interfere in
the retention time, identification, and quantification
of the analyte. No residual effect was observed either.
The F-test showed the model is heteroscedastic, so
a 1.Y-2 weighted linear regression was employed and
the method is linear (r>0.999) in the working range of
11 to 61% v/v. The method also showed good precision
(RSD=4.08 to 7.97%) and accuracy (RE=-0.05 to
-1.31%). For short-term stability, the accuracy ranged
from 2.85 to 3.34% with a RSD from 3.35 to 4.41%.
Concerning the Freeze-thaw stability, RE ranged from
-0.66 to -3.05% and RSD ranged from 2.84 to 3.52%.
Discussion/Conclusion: The method developed
and validated met all the criteria required by the
Guidance for Validation of Analytical Methodology
(UNODC, 2009) and the RDC nº27 (ANVISA, 2012). The
method will be applied to forty-four seized alcoholic
beverages (whiskey, rum and tequila) kindly provided
by the Superintendência da Polícia Civil Técnico-
Científica de Minas Gerais. Acknowledgments: This
research was supported by CAPES – Brazil (Financing
Code 001).

Selective determination of 4-aminopyrine in
seized cocaine samples by a reduced graphene
oxide/Prussian blue nanocomposite
Reis, Karsonn B.; Borges, Pedro H.S.; Rocha, Raquel G.; Ramos, David L.O.;
Instituto de Química, Universidade Federal de Uberlândia, 38400-902, Uberlândia, MG, Brazil.
4-aminopyrine (4AP) is a palliative drug generally
recommended for the febrile condition. This substance
usage showed relations to the decreasing of white blood
cells, bone marrow suppression, and carcinogenesis,
reasons which the drug was banned from some countries.
Furthermore, 4AP is usually found as an adulterant in
cocaine samples, either to mimic its pharmacological
properties or to increase the sample volume for higher
trafficking profits.1 One way to monitor this substance is
through the electrochemical method. Among other analytical
techniques, it exhibits some advantages such as low cost,
simple handling, cheap instrumentation, and real-time
response. In addition, the selectivity and sensibility of the
electrochemical method can be improved by modification
of the working electrode surface.2a reduced graphene
oxide sensor associated to the batch injection analysis
system with amperometric detection was investigated for
the simple, fast and sensitive monitoring of sulfanilamide
in lake water and synthetic biological fluid (saliva, sweat
and urine Prussian blue (PB) is a well-known electroactive
inorganic compound, highly employed for electroanalytical
purposes. It can be easily produced by an electrochemical
approach directly over the working electrode surface.
Although PB shows good stability in acid media, the
presence of graphene (and derivatives) as support for PB
species can not only enhance its pH stability range but the
electron transfer signal too.3,4 In this work, we show an all-
electrochemical preparation of a stable reduced graphene
oxide/Prussian blue (rGO/PB) nanocomposite and some
of its spectroscopic and microscopic characterizations.
Moreover, the electroactivity of the material was evaluated
towards the 4AP presence, which led to the application
as a selective electroanalytical sensor of 4AP in seized
cocaine samples. The nanocomposite was produced over a
glassy carbon electrode in a typical three-electrode system
by cyclic voltammetry (CV) immersed in a GO dispersion
with Fe2+ ions. After dried, this precursor was submitted
to CV in an acid ferricyanide solution, obtaining the rGO/
PB nanocomposite. Several electrochemical tests of the
material in the absence and the presence of 4AP were
executed. The determination of the analyte in cocaine-seized
samples was made by amperometry along with a batch
injection analysis (BIA) system. Moreover, the material,
245
Muñoz, Rodrigo A.A.; Richter, Eduardo M.; Nossol, Edson
along with its isolated components, was characterized by
spectroscopic and microscopic techniques. While the CV
of the first step of preparation showed a typical signal of
graphene electroreduction, the second step CV attested
the formation of PB particles by the current growth of its
characteristic redox pair through the cycles. Its composition
was also certified by the presence of several standard
assignments in Raman and FTIR spectra. SEM images
showed well-distributed PB particles over and between
rGO sheets, which also had its elemental constitution
confirmed by EDX. The nanocomposite exhibited higher
stability at pH= 4 (KCl 0,1 mol L-1), which was the media
employed in the entire work. Also, it demonstrated a good
response for the electrooxidation of 4AP by CV around
0.7 V. For amperometry/BIA determination of the analyte,
some parameters were optimized for better results. The
analytical curve of standard 4AP injections was obtained at
0.8 V showing good sensitivity (0,168 µA L mol-1) and linearity
between 0.25–10.0 µmol L-1 (R2 = 0.999). The selectivity test
showed no response (3-fold) of 8 different usually founded
components in cocaine seized samples. Furthermore, the
injection of the seized sample showed no significant signal
difference with and without a known concentration of 4AP,
suggesting that the sample obtained was not filled with
the analgesic. In conclusion, the rGO/PB nanocomposite
can be successfully prepared by an easy two-step
electrochemical method, as attested by electrochemical,
spectroscopic, and microscopic techniques. This material
exhibits excellent response to 4AP electrooxidation, which
provides its application as a highly sensitive and selective
electroanalytical sensor for the analyte in complex cocaine
seized samples. We would like to thank the support
from CAPES, FAPEMIG, CNPq, IQ-UFU, GQMIN, NUPE, INCT
Nanocarbon, Criminalistic Institute of São Paulo, and SBTox.
REFERENCES
1. W. R. de Araujo, A. O. Maldaner, J. L. Costa and T. R. L. C. Paixão,
Microchem. J., 2015, 121, 213–218.
2. L. V. de Faria, T. P. Lisboa, T. A. Matias, R. A. de Sousa, M. A. C. Matos, R. A.
A. Munoz and R. C. Matos, J. Electroanal. Chem., 2021, 892, 115298.
3. S. C. Silva, R. M. Cardoso, E. M. Richter, R. A. A. Munoz and E. Nossol,
Mater. Chem. Phys., 2020, 250, 123011.
4. Y. Yang, Y. Cao, X. Wang, G. Fang and S. Wang, Biosens. Bioelectron.,
2015, 64, 247–254.
 246

Sociodemographic profile of authors

involved in illicit drug arrest in the
city of Betim, Minas Gerais
Costa, Anna Carolina de Moura; Oliveira, Lara Luiza Freitas; Sales, Thais
Lorenna Souza; Sanches, Cristina; Chequer, Farah Maria Drumond

Federal University of São João del-Rei (UFSJ), Dona Lindu Center-

West Campus (CCO), Divinópolis-MG, Brazil.

Introduction: Betim is considered the fifth largest
municipality in the state of Minas Gerais, with a
population estimated by the Brazilian Institute
of Geography and Statistics, in 2021, of 450.024
inhabitants. The rapid industrial growth provided
the migration of a large number of people to the
municipality in search of employment. Most of the
time without professional qualification, these people
started to live in situations of social vulnerability,
opening doors for the growth of drug trafficking,
making Betim one of the most violent cities in the
State of Minas Gerais. Objective: This study aimed to
analyze the sociodemographic profile of the authors
involved in the arrest of illicit drugs by the Civil
Police in the region of Betim-MG. Methods: This is a
descriptive observational study designed according
to the guidelines proposed by the Strengthening the
Reporting of Observational Studies in Epidemiology
(STROBE). A total of 568 reports were analyzed
regarding arrest of illicit drugs (marijuana, cocaine
and crack) issued by the Criminal Investigation from
January 2017 to December 2018, in Betim-MG. The
variables collected were: sex, age, marital status
and education. The database was created using the
Questionnaire Development System (QDS) version
2.6.1.1, and later, the data were exported to the
Statistical Package for Social Sciences (SPSS) version
19, for statistical analysis. Results: According to the
data obtained, the sociodemographic profile of the
authors indicted in the reports of arrest of illicit drugs
(marijuana, cocaine and crack) in Betim-MG is mainly
composed of male individuals (86.6%), with a median
age of 20 years old (IQ25% = 17 ; IQ75% = 24), with
single marital status (88.0%) (including divorced and
separated), and with incomplete primary education
(33.2%). As for the age of the authors, the youngest
was arrest at 14 years old and the oldest at 61 years
old, and 17 years old was the age with the highest
number of individuals apprehended. Discussion/
Conclusion: The sociodemographic profile described
for the municipality of Betim is consistent with that
presented, in general, for the profile of drug users.
These are generally young, male, single and with a
low level of education. These results demonstrate the
need of the studied municipality, as well as the country,
in general, to invest in education, to prevent more and
more young people from entering the world of drugs.
The knowledge of the profile of the perpetrators
of drug arrest in this specific region (Betim-MG),
allows preventive measures for trafficking and use
to be created, thus reducing public health and safety
problems caused by the use and abuse of these
substances. Keywords: Illicit Drugs. Arrest. Prisoners.
Acknowledgments: The present work has carried out
with the support of the Coordination of Improvement
of Higher Education Personnel - Brazil (CAPES) -
Financing Code 001. We thank the Federal University
of São João del-Rei (UFSJ), Dona Lindu Center-West
Campus (CCO) for the support. To the management and
other teams of the Betim Civil Police for the reception
and support during the realization of the research.
 247

Survey of drugs/adulterants found

concomitantly with cocaine, in victims
of violent death, in the state of
Minas Gerais, in the year 2021
Corrêa, Brunna F.1; Bordoni, Leonardo S.1,3,4; Couto, Tauer J.G.1;
Goulart , Christiane G.L.; Goulart, Cristiano O.L.1
1
 Instituto Médico Legal André Roquette, R. Nícias Continentino, 1291, Gameleira, 30510-
160, Belo Horizonte, Brasil; 2 Universidade Federal de Ouro Preto, R. Dois, Campus Morro
do Cruzeiro, 35400-000, Ouro Preto, Brasil; 3 Faculdade de Medicina de Barbacena,
Praça Presidente Antônio Carlos, 8, São Sebastião, 36202-336, Barbacena, Brasil.

Introduction: Cocaine (COC) is a drug that causes
dependence and overdose deaths, which makes this
drug a public health problem in Brazil and worldwide.
To make greater profits from the sale of COC, dealers
mix cocaine with adulterants such as talc, boric acid
and sodium bicarbonate. Dilution decreases COC
activity, therefore, to mask this dilution, various
drugs are added to COC-containing mixtures such
as lidocaine, caffeine, carfentanil (found recently in
adulterated COC in other countries), levamisole and
others. At the same time, the concomitant use of
COC with other drugs such as ethanol increases the
lethality of COC. In this way, it is important to know
which drugs are found along with COC in individuals
who consume it, this can help in the creation of public
policies to contain damage, such as the purchase of
antidotes such as naloxone, to prevent death and/or
other side effects produced by adulterants. Objective:
The objective of this study was to evaluate which
drugs were found concomitantly with COC in the
blood, urine, stomach contents and liver of victims
of violent death in the state of Minas Gerais/Brazil,
in the year 2021. Methods: Retrospective analysis of
283 positive COC cases analyzed in 2021, obtained by
gas chromatography coupled to mass spectrometry
and from liquid/liquid extractions (stomach contents
and liver) and solid phase extraction (urine and
whole blood). Results: Analytes were found in the
analysis: caffeine 174(61%), cocaethylene 82(29%),
lidocaine 51(18%), levamisole 23(8%), theobromine
16(6%), diazepam 13(5%), midazolam 11 (4%),
chlorpheniramine 11(4%), tetracaine 10(4%), tramadol
6(2%), phenobarbital 6(2%), cyclobenzaprine 5(2%),
nortriptyline 5(2%), amitriptyline 5( 2%) (2 cases in
common with nortriptyline), orphenadrine 5(2%),
ketamine 4(1%), probarbital 4(1%), clomipramine
3(1%), codeine 3(1%), propofol 3 (1%), methadone
3(1%), promethazine 3(1%), carbamazepine 3(1%),
methamphetamine 2, propylhexedrine 2, efavirenz 2,
diphenhydramine 2, orphenadrine 2, ethosuximide 2,
phenytoin 2, clopidogrel 2, biperiden 2, metoclopramide
1, hydroxyzine 1, propafenone 1, pheniramine 1,
olanzapine 1, metoprolol 1, mirtazapine 1, fluoxetine
1, fentanyl 1, phenacetin 1, carisoprodol 1, ibuprofen 1,
temazepam 1 (common case with diazepam), adrafinil
1, escitalopram 1, sertraline 1, trazodone 1, bupropion
1, doxepin 1, meprobamate 1, chlorpromazine 1,
ticlopidine 1 , fluconazole 1, phenacetin 1, aminopyrine
1, bupivacaine 1 and lamotrigine 1. Conclusion: A high
number of cases containing the analytes caffeine
174 (61%), lidocaine 51 (18%), levamisole 23 (8%),
theobromine 16 (6%), chlorpheniramine 11 (4%) and
tetracaine 10 (4%) were found. The drugs discussed
are COC adulterants. Cocaethylene was found in 82
(29%) of the cases. This metabolite increases the risk
of heart attack. Few opioids were found (tramadol
6(2%), codeine 3(1%), methadone 3(1%) and fentanyl
(1). This is an indication that the COC adulteration
by opioids had not yet reached Minas Gerais in the
year 2021. Finally, many cases of benzodiazepines
diazepam 13 (5%), midazolam 11 (4%) were found.
This is an indication that COC users may be using
benzodiazepines to treat crises of insomnia possibly
caused by this drug. Acknowledgments: This study
was carried out with the support of the Civil Police of
Minas Gerais.
 248

The increase in the number of incarcerations

in Brazil involving drug crimes and the main
drugs seized over ten years (2009-2019)
Bonfioli, Maysa Guilherme; Coelho, Júlia França Figueredo; Melo, Saulo
Nascimento; Belo, Vinícius Silva; Chequer, Farah Maria Drumond

Universidade Federal de São João del-Rei, Campus Centro-Oeste Dona

Lindu (UFSJ-CCO), Divinópolis, Minas Gerais, Brasil

Introduction: Illicit drugs has been persistently a
global problem. In this sense, the analysis of the
seizures of these drugs can be used as an indicator
to reflect the dimensions of its market. Its existence
in society has the consequences on violence,
corruption and countless health events. In the last
decade, drug trafficking and the use of illicit drugs
has shown a continuous incremental trend, remaining
in the world as a challenging problem in the society.
Objective: To verify the evolution of incarcerations
for crimes related to illicit drugs and the amount of
these substances seized. Methods: A descriptive
documental study was carried out, observing
secondary data from the reports issued by the System
of the National Penitentiary Department (SISDEPEN)
that is available on the website http://antigo.depen.
gov.br/DEPEN, from January 2009 to December 2019,
as well as the data issued by the Federal Police on the
Statistics of Drugs Seized in Brazil, that is available on
the website https://www.gov.br/pf/pt-br/acesso-a-
informacao/estatisticas/diretoria-de-investigacao-
e-combate-ao-crime-organizado-dicor/drogas_
apreendidas_por_uf.pdf/view, in the same period
above. Results: According to the data from SISDEPEN,
in the first semester of 2009, the number of prisoners
in Brazil was 469.546, and after ten years this number
increased by 61%, reaching 755.274 people. In relation
to the group of drug-related crimes, the percentage
increased from 18,21% in 2009, with 85.506
individuals, to 26,56% in 2019, with 200.583 prisoners.
Drug trafficking was the crime responsible for the
most convictions, in which it represented, in 2009,
93,04% in relation to all drug crimes (laws 6,368/76 e
11,343/06); while in 2019, this percentage dropped to
84.3%. As described by the Federal Police Seized Drug
Statistics, in 2009 about 131.4 tons of marijuana were
seized, and in 2019 this number increased to 266 tons,
totalizing a percentage increase of 102%; in relation
to cocaine, 24 tons were seized in 2009, increasing
to 104.6 tons in 2019 (an increase of 336%); 49,457
ecstasy were seized in 2009, growing to 561,951 pills
in 2019, totalizing an increase of more than 1,000%;
and over Lysergic acid diethylamide (LSD) were
48,424 stamps in 2009 dropping to 10,643 stamps in
2019 with a 78% drop. Conclusion: The use of illicit
drugs varies over time, and it is extremely important
to do correctly document data related to this problem
in the community. In Brazil, through a database, it is
possible to observe a growing number of individuals
convicted of drug-related crimes, and an increase in
the seizures of drugs by the Federal Police between
2009 and 2019. It is, therefore, necessary to work on
solutions to the control of the illicit drug market to
contain its advance in society. In this way, it would
be possible to resolve much of the damage caused
by them. Keywords: Illicit Drugs; Drug Trafficking;
Marijuana; Cocaine; Crack; Prisoners; Lysergic acid
diethylamide. Acknowledgments: The authors would
like to thank the Federal University of São João del-Rei
(UFSJ)- Dona Lindu Midwest Campus for its support
and financial support as well. (PIBIC Scholarship).
This work was carried out with the support of the
Coordination for the Improvement of Higher Education
Personnel - Brazil (CAPES) - financing code 001.
 249

The mystery behind the apprehensions of the

selective agonist to cannabinoid type 2 receptor
BZO-HEXOXIZID (MDA-19) as a drug of abuse
Araujo, Karen Rafaela Gonçalves1; Fabris, André Luis1; Neves Júnior, Luiz F.2;
Ponce, Júlio de Carvalho2; Costa, José Luiz3; Yonamine, Mauricio1
1
 School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP 05508-000, Brazil;
2
 Superintendence of the Technical-Scientific Police, Institute of Criminalistics, São Paulo, SP 05507-
060, Brazil; 3 Campinas Poison Control Center, University of Campinas, Campinas, SP 13083-859, Brazil.

Introduction: First synthesized in 2008 by Diaz and
colleagues as a selective agonist to cannabinoid
receptor type 2 (CB2R), MDA-19 or its formal name
BZO-HEXOXIZID (N′-[(3Z)-1-(1-Hexyl)-2-oxo-1,2-
dihydro-3H-indol-3-ylidene]ben-zohydrazide) was
targeted as a potential treatment for neuropathic pain.
Thereafter, several researches have been conducted
and promising results were published evidencing the
therapeutic potential of this compound in different
clinical conditions, such as melanoma, osteosarcoma,
neurodegenerative disorders, and hepatocellular
carcinoma. Notwithstanding this potential as a
medicine, MDA-19 was unexpectedly apprehended
as drug of abuse by law enforcement in Spain and
Germany a few years later, in 2016. Furthermore, this
substance was prohibited in China in July 2021, while in
the United States this compound was seized by police
officers for the first time more recently. Objective:
To report the first apprehension of BZO-HEXOXIZID in
Brazil followed by its chemical characterization and
to discuss pharmacologically the possible reasons
why a CB2R agonist has been incorporated to the illicit
market. Methods: Suspected seized samples were
forwarded to the Laboratory of the Scientific Police of
the State of Sao Paulo. After the screening, samples
were confirmed for the presence of MDA-19 by using
GC-MS, FTIR, and NMR techniques. Results: A total
of 53 samples were analyzed; 25 (47,17%) contained
only MDA-19, whereas 28 (52,83%) were associated
with other illicit substances. ADB-BUTINACA, caffeine,
and cocaine were found associated with MDA-19 in 8
(36.6%), 5 (22.7%), and 9 (30.9%) of these samples,
respectively, representing 22 cases. Cases containing
two or more substances were also found, there
represented 6 cases that are 2 (33.3%) were MDA-
19, cocaine, and caffeine, and one (16.6%) of each
for cocaine, sildenafil; ADB-BUTINACA caffeine and
lidocaine; THC and ADB-BUTINACA; and THC, ADB-
BUTINACA, and cocaine, ever with MDA-19. Most of the
cases (41) were apprehended in Sao Paulo Metropolitan
Area, while the remaining 12 cases were submitted
to the laboratory in coastal cities of the state.
Discussion/Conclusion: We herein have described
the first apprehension followed by identification and
characterization of BZO-HEXOXIZID in Brazil by the
State Police of Sao Paulo. Of those, 47,1% had MDA-19
only, while 52,83 % it mixed with other compounds.
Interestingly, ADB-BUTINACA, a CB1R agonist, was
found in 11 of these samples (only with MDA-19 or
with two other rugs), which could be an attempt to
mimic the effects of cannabis consumption - mixing
a CB1R agonist with a CB2R agonist. However, most
of these seized materials had only MDA-19; hence,
the reason behind its individual use remains unclear.
Speculations go from its isolated use to induce
analgesia states by activation of opioid receptors or
achieve any reward sensation by stimulation of the
mesolimbic pathway when associated with other
synthetic cannabinoids, such as ADB-BUTINACA,
that significantly stimulate the CB1R resulting in a
cannabis-like effect. Acknowledgments: INSPEQT
(Investigation of new psychoactive substances in
Forensic Chemistry and Toxicology) project, funded
by the Coordination for the improvement of Higher
Education Personnel (CAPES), through Public Notice
16/2020- Academic Cooperation Program in Public
Security and Forensic Sciences (PROCAD), process
88887.61228/221-00.
 250

Toxicological findings in cases

attended by Division of Postmortem
Inspection of Porto Alegre in 2021
Birk, Letícia1; Barbosa, Fábio de Souza1; Petry, Adriana Ubirajara Silva1,2; Menezes,
Francisco Paz2; Gonzaga, Alexsandro Pinto2; Eller, Sarah1; Oliveira, Tiago Franco1
1
 Graduate Program in Health Sciences, Federal University of Health Sciences of Porto Alegre (UFCSPA),
Brazil; 2 Division of Postmortem Inspection, Associação Hospitalar Vila Nova - Porto Alegre, Brazil.

Background: The Division of Postmortem Inspection
is a public institution of the National Public
Health System. The main goal of this service is the
elucidation of deaths that occurred of natural causes
but under suspicious conditions, by request of the
responsible physician and the family authorization.
In 2020, there was an expansion of this service in
the country with the building and improvement of
the units, including one in Porto Alegre, Brazil. In
this scenario, toxicological examination can provide
valuable information for the investigation of these
inconclusive deaths. However, the complexity
associated with the analysis of biological matrices
mainly in postmortem cases represents a challenge
for toxicologists. Consequently, the use of sensitive
and specific methodologies is essential in order to
guarantee the correct determination and appreciation
of the results. Objective: This study aimed to
investigate the presence of psychoactive substances
of toxicological interest in the cases attended by
the Division of Postmortem Inspection of Porto
Alegre in the year of 2021. Methods: Blood samples
were prepared through a protein precipitation
methodology according to the article published by
Franco de Oliveira et al. (2019). An aliquot 800 µL of an
acetonitrile:methanol (80:20, v/v) mixture was added
to 100 µL, followed by vortexing for 30 seconds and
centrifugation at 10,000 rpm for 5 minutes. An aliquot
of 50 µL of the supernatant was transferred to a vial
and 3 µL were injected into the analytical system. A
Nexera UFLC system coupled to a LCMS-8045 triple
quadrupole mass spectrometer (Shimadzu, Japan)
was used for the analysis. The analytical method
was developed for the detection of 67 substances,
including drugs of abuse and pharmaceuticals such
as anesthetics, antidepressants, sedatives, and
anxiolytics. Results: During the year of 2021, a total
of 75 blood samples were analyzed resulting in 13
positive samples for the presence of one or more
than one psychoactive substance. Sixteen substances
were identified being as it follows: benzoylecgonine
(12.9% of cases, N=4; 0.113 – 0.396 µg/mL), cocaine
(9.7%, N=3; 0.016 – 0.166 µg/mL), amitriptyline (9.7%,
N=3; 0.079 – 0.203 µg/mL), nortriptyline (9.7 %, N=3;
0.103 – 0.198 µg/mL), cocaethylene (6.5%, N=2; 0.024
– 0.062 µg/mL), diazepam (6,5%, N=2; 0.110 – 0.460
µg/mL), nordiazepam (6.5%, N=2; 0.250 – 0.270 µg/
mL), lidocaine (6.5%, N=2; 0.165 µg/mL), promethazine
(6.5%, N=2; 0.013 – 0.042 µg/mL), ecgonine methyl
ester (6.5%, N=2; 0.027 – 0.078 µg/mL), norcocaine
(3.2%, N=1; 0.001 µg/mL), fluoxetine (3.2%, N=1; 0.087
µg/mL), norfluoxetine (3.2%, N=1; 0.030 µg/mL),
carbamazepine (3.2%, N=1; 3.410 µg/mL), alprazolam
(3.2%, N=1; 0.138 µg/mL) and phenobarbital (3.2%,
N=1; 27.410 µg/mL). One interesting analytical finding
was the presence of promethazine along with cocaine
derivatives in two samples, which can indicate the use
of this pharmaceutical as a cutting agent in cocaine.
Discussion/Conclusion: Considering these findings,
there can be an influence of the use of drugs of abuse
and pharmaceuticals in deaths that occur without
traumatic events. Nevertheless, these data suggest
that it can be an understatement the use of these
substances in the population, since epidemiological
data usually is based on information from the poison
control centers and forensic medical institutes. The
toxicological analysis will continue in the samples
provided by the service, being able to compare data
across the years and identify trends in substance
intake. Acknowledgments: CAPES.
REFERENCES
Franco de Oliveira, S. C. W. S. E., Zucoloto, A. D., de Oliveira, C.
D. R., Hernandez, E. M. M., Fruchtengarten, L. V. G., de Oliveira,
T. F., & Yonamine, M. (2019). A fast and simple approach for
the quantification of 40 illicit drugs, medicines and pesticides
in blood and urine samples by UHPLC-MS/MS. Journal of Mass
Spectrometry. doi:10.1002/jms.4369
 251

Validation of methods for the determination

of cocaine, benzoylecgonine, and
cocaethylene in dried blood using the
hemapen microsampling device
Smidt, Mariana1,2; Bastiani, Marcos Frank1; Hahn, Roberta Zilles1; Linden, Rafael1,2
1
 Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate
Program on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil.

Background/Introduction: Biological matrices can TQS-micro system, using a separation on an ACQUITY

be used to assess human exposure to xenobiotics.
Among these, blood stands out for having a better
relationship with the effects on human performance
and adverse effects. Microsamples bring many
benefits for a minimally invasive quantification of
drugs of abuse and allows stabilization of analytes
in the dried samples. Transport and storage of
specimens is also facilitated as dried blood is not
considered biohazardous. Recently, new alternatives
for volumetric microsampling of blood have
emerged, in addition to the already known dried
blood on paper, such as the hemaPEN device, which
allows capillary collection of exact volumes of
blood from a capillary puncture, regardless of blood
hematocrit. Furthermore, as the sample is safely
protected inside the device, its use is attractive
in the context of forensic toxicology. Objectives:
Develop and validate a method for quantification of
cocaine, benzoylecgonine and cocaethylene using
liquid chromatography associated to tandem mass
spectrometry (LC-MS/MS) and capillary sampling
with the hemaPEN device. Methods: The hemaPEN
devices were opened with a special tool provided
by the manufacturer. Afterwards, the 4 filter paper
discs inserted into the device were separated and
transferred to a 2 mL polypropylene tube. The tubes
with the disks were added with 500 µL of a methanolic
solution of internal standards and homogenized
for 45 minutes at room temperature, at 1000 RPM.
Afterwards, a 440 µL aliquot of the supernatant was
transferred to another tube and evaporated at 45 °C.
The dry extract was recovered with the LC mobile
phase for subsequent injection into the LC-MS/MS.
The analytes were quantified in an Acquity-Xevo
UPLC BEH C18 column (1.7 µm 2.1 x 50mm), kept at
40°C, with mobile phase A composed of 0.1% formic
acid in water and mobile phase B, composed of 0.1%
formic acid in acetonitrile. Calibrators were prepared
at concentrations of 2, 6, 20, 100, 400 and 1000 ng/
mL. The method has been validated according to
international guidelines, including evaluation of
matrix effect, hematocrit effect on accuracy, precision
and accuracy, stability, and extraction yield. Results:
The analytes were stable in the hemaPEN devices for
seven days, both at room and elevated temperature
(45 °C). The accuracy in evaluated in blood with
hematocrits between 25 and 50% presented values
between 89 and 108.8%, demonstrating a small
influence of the blood hematocrit. The matrix
effect was adequately compensated by the internal
standards, presenting values between -0.42 and
9.75%. The intra-assay precision was between 2.57
and 18.15% and the inter-assay precision was between
2.70 and 10.82%. Accuracy ranged from 89.5 to
118.0%. The method was applied to samples obtained
from patients admitted to a chemical dependency
treatment unit. Discussion/Conclusion: A method for
the determination of cocaine, benzoylecgonine and
cocaethylene using LC-MS/MS in dried blood samples
obtained with the hemaPEN microsampling device
was developed and validated. The developed method
presents adequate performance and is being applied
in an ongoing study with patients. The hemaPEN
device can be a safe and convenient alternative for
assessing exposure to drugs of abuse in both clinical
and forensic toxicology. Acknowledgments: CAPES,
Universidade Feevale, and Trajan.
 09 
GENOTOXICITY AND
CARCINOGENESIS
 253

Cellular spheroids as a platform for

toxicogenomic assays in cancer
Queijo, Rodrigo Gonçalves; Carvalho, Larissa Anastacio da Costa; Maria-Engler, Silvya Stuchi

Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University

of São Paulo, Avenida Professor Lineu Prestes, 580, São Paulo 05508-00, SP, Brazil.

Introduction: Monolayer models, widely disseminated
and used, are not able to reconstruct the complex and
heterotypic in vitro cellular microenvironment, and
animal models face increasingly reinforced ethical
barriers. Meanwhile, organotypic culture promotes
physiological parameters of organs and tumors,
and allows to mimic the architecture of the parental
tissue, being important to understand the role of
the microenvironment, transcriptional plasticity
and agents for targeted therapy in heterogeneity,
selection of cells with intrinsic or acquired resistance
and in the reprogramming of oxidative metabolism
by tumor cells. The 3D cell culture model allows the
study of changes in the behavior of cells and the
performance of tests to elucidate the mechanism
of drug action, being an interesting platform for
toxicogenomic tests. Objective: To standardize the
technique for obtaining and isolating the three-
dimensional model of spheroids, based on tests with
different proportions of melanoma and fibroblasts;
to observe the behavior of the 3D cell aggregate in a
reconstructed human skin model; and to investigate
the tumor heterogeneity and expression of proteins
of the melanoma proliferation and invasion pathways
from the analysis of SKMEL-28 clones. Methods:
Clonal subpopulations were stochastic isolated from
the initial parental SKMEL-28 cell line, then 48-well
culture plates were prepared using 1.5% soft-agar,
and stemming from the hanging drop method, 2,5.104
cells were plated in 30 uL, in different proportions of
melanoma and fibroblast. After the incubation period,
3D aggregates were obtained and subsequently fixed
for histological processing. In a model of reconstructed
human skin, protocolled and standardized by the
group, spheroids were added to the dermis and
histologically analyzed. Results: The spheroids
obtained with a higher proportion of fibroblasts
showed higher levels of compaction and density,
highlighting the importance of non-cancerous stromal
cells for the creation of the tumor microenvironment.
The SKMEL-28 cell line, being metastatic, showed
an invasive phenotype in all spheroid proportions
when sedimented, and in the reconstructed human
skin model. Discussion/Conclusion: The size of the
spheroid is not only determined by the number of cells,
but the interaction effects of compaction inducers,
being important for the architecture of the three-
dimensional cell aggregate. The use of organotypic
culture ensures a more accurate understanding of
the cellular interaction with the extracellular matrix
and other cell types, further studies are needed
to compare the results of 2D and 3D models, and
to elucidate the mechanism of drug action and
emergence of resistance, with the possibility of using
this standardized platform for testing antimelanoma
compounds. Acknowledgments: FAPESP (17/04926-
6, 18/14936-1 and 21/05150-7), CNPq (#408769/2018
and #304339/2017-2) and CAPES.

Collaborative analysis of complex nitrosamines
Avila, Carolina Martins; Ponting, David. J.; Werner, Anne-Laure D.;
Background/introduction: The recent discovery of
small-molecule nitrosamine impurities in marketed
drugs, starting with nitrosodimethylamine (NDMA)
in batches of Valsartan in 2018, has led to significant
regulatory response, including drug recalls and
regulatory guidance that requires the evaluation
of all synthetic and formulation routes for the
potential presence of nitrosamine impurities. Due
to the wide range of potential routes of formation
for nitrosamines1, many active pharmaceutical
ingredient (API) structures are themselves liable to
be nitrosated, either during the later stages of the
synthetic process or as the formulated drug product.
Objectives: This work describes the formation of a
data-sharing initiative, coordinated by Lhasa Limited,
which has the following aims: Firstly, since many APIs
are generic drugs and are manufactured by multiple
companies, the reduction of duplicate testing and
the availability of an Ames test result conducted to
the highest standards and in appropriate conditions
should reduce potential uncertainties for regulatory
submissions. Secondly, the consortium aims to
investigate differences between these compounds
and the small molecules which comprise the
majority of publicly available nitrosamine data2
and elucidate any structure activity trends and
significant differences. Methods: Pharmaceutical
industry partners share Ames data on structurally-
complex nitrosamines, which is input by Lhasa (in an
anonymised form) into a shared database to enable
read across and reduce duplicate testing. Results:
The consortium currently consists of 6 respected
members within the pharmaceutical industry,
254
Tennant, Rachael E.; Oliveira, Antonio Anax F.; Heghes, Crina
Lhasa Limited, Leeds, United Kingdom.
with additional pharmaceutical companies in the
process of joining. It is estimated that mutagenic
data from 60 compounds will be donated during Q2
2022. Discussion/Conclusion: Extensive testing of
complex nitrosamines – synthesized on purpose and
fully characterized – is currently underway as part of
the second stage of the regulatory response to the
current crisis. The need for large numbers of Ames
tests, and potential in vivo follow-up assays, leads
to pressure on the few laboratories able to carry out
these tests. As a result, the sharing of testing results
amongst companies – both discovery pharmaceutical
companies and generic manufacturers, leads to a
reduction in the testing burden for each member.
The majority of toxicity data that has been
reported is for small-molecule nitrosamines, with
nitrosodiethylamine and nitrosodimethylamine the
most studied as well as among the most potent, while
a far smaller amount of data is currently available for
more complex structures. The proportion of complex
nitrosamines which are Ames negative may be higher
than the proportion of small molecule nitrosamines,
for reasons such as differences in metabolic potential
or the proposed adducts formed. Full testing of this
hypothesis requires the curation of a large high-
quality dataset, which can only be achieved via cross-
industry collaborations such as this.
REFERENCES
1. Lopez-Rodriguez et al (2020), Org. Process Res. Dev. 24, 1558-
1585.
2. Thresher et al (2020), Regul. Toxicol. Pharmacol., 116, 104749.
3. Brambilla and Martelli (2007), Mutat. Res., 635, 17-52.
4. Schmitsdorff et al (2022), Arch. Pharm. e2100435.
 255

Copper (II) complex as a potential treatment

to overcome BRAF inhibitor resistance
and aggressive NRAS point-mutation
Moraes Junior, Manoel Oliveira1; Carvalho, Larissa Anastácio da Costa1; Nunes, Cleia
Justino2; Ferreira, Ana Maria da Costa2; Maria-Engler, Silvya Stuchi1
1
 Department of Clinical and Toxicological Analyses, School of Pharmaceutical
Sciences, University of São Paulo, São Paulo, Brazil; 2 Department of Fundamental
Chemistry, Chemistry Institute, University of São Paulo, São Paulo, Brazil.

Background/Introduction: melanoma is the deadliest
type of skin cancer due to its high metastatic
capacity. Accumulation of genetic mutations in
melanocytes confers phenotypic changes, especially
in MAPK signaling pathway which might develop point
mutations in BRAF protein (V600E, the most common)
or NRAS protein (Q61K and Q61R). Targeted therapy
has revolutionized the treatment of cancer. Melanoma
is one of the biggest beneficiaries considering the
emergence of BRAF inhibitors (iBRAF, e.g., vemurafenib)
along with MEK inhibitors, in combination or as a single
treatment option against NRAS+ mutation tumors
which present poor prognosis. However, even when
therapy does not initially fail, high heterogeneity of
melanoma cells leads to a selection and emergence of
treatment-resistant cells that culminates in disease
relapse. This fact highlights the need to search for
new therapeutic alternatives, whether isolated or
combined, to overcome these resistance phenotypes.
In this context, metallodrugs (e.g., cisplatin) appear
as anticancer drugs candidates, in which we can
highlight copper(II) complexes. Copper(II) complexes
have already been studied for their ability to trigger
programmed cell death, also claiming an activity with
more tolerable adverse effects. Design, synthesis,
and biological evaluation of dinuclear copper(II)
complexes on metastatic melanoma cell lines
were already reported, showing cytotoxic activity
displaying apoptotic and autophagic profiles, yet
their activity against BRAF inhibitor-resistant cells,
as well as, with different mutations in NRAS have not
been investigated thus far. Objective: The present
study evaluates the cytotoxic activity of the dinuclear
copper(II) complex [Cu2(apyhist)2(dpam)]ClO4) against
strains resistant to iBRAF (vemurafenib) and with
different mutations in NRAS. Methods: MTT assay
and trypan blue (TB) exclusion techniques were used
to evaluate cell viability of naive and vemurafenib-
resistant WM164 and SK-MEL-28 (BRAFV600E) and SK-
MEL-173 (NRASQ61K) metastatic melanoma cell lines.
Concentrations ranging from 0,03µM to 100 µM were
tested in a 24-hour incubation period. Results: The
copper(II) complex demonstrated a concentration-
dependent cytotoxicity in a micromolar range potency,
both assessed by MTT and TB exclusion. IC50 values​​
were slightly higher for vemurafenib-resistant strains
compared to the naive cell lines. Regarding the NRAS+
cell line (SK-MEL-173), the IC50 value was about double
compared to the BRAF mutated cell lines. In addition,
the copper(II) complex was also more potent than
the anticancer metallodrug cisplatin and the first-
line anti-melanoma drug dacarbazine. Interestingly,
the copper(II) complex was 40x more potent against
WM164 cells compared to cisplatin cytotoxicity.
Discussion/Conclusion: These results support the
hypothesis that the copper(II) complex can overcome
the vemurafenib-resistance phenotype, in addition
to the more aggressive cell line NRAS+. This scenario
configures a viable alternative therapy for patients
with BRAF and NRAS mutations with no longer
responsive to existing treatments.
 256

Evaluation of photomutagenic potential

of UV filters and IR3535 combination
Fuzinaga, Thaís Y.T.1; Gluzezak, Ana J.P.1; Tavares, Renata S.N.1; Kawakami, Camila M.1;
Abe, Flávia R.1; Oliveira, Danielle P.1; Maria-Engler, Silvya S.2; Gaspar, Lorena R.1
1
 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo;
2
 School of Pharmaceutical Sciences, University of São Paulo.

The worldwide increased incidence of mosquito-
borne diseases has led the population to look for
preventive measures, as the use of insect repellents.
Although insect repellents are considered safe, there
is an increase of their simultaneous use combined
with UV filters due to high incidence of solar radiation
in Brazil. Consequently, studies that evaluate the
interaction of the combination of these substances
are required to ensure the safety of the population
exposed to these compounds. In the literature we can
find many studies about the undesirable effects of
UV filters and repellents as isolated raw materials.
There are also studies that demonstrate interactions
between UV filters and insect repellents. Thus, this
study evaluated the photomutagenic potential of UV
filters and IR3535 combination in an in-house skin
model, an in vitro assay, based on Reconstructed Skin
Micronucleus (RSMN) assay, recently accepted into
the OECD’s test guideline development program, for
mutagenic potential evaluation. The combination of
UV filters avobenzone, ethylhexyl methoxycinnamate
and octocrylene with IR3535 (UVF+IR3535) were
diluted in sesame oil and applied onto in house
reconstructed human skin models, so as the positive
control 8-methoxypsoralaen (8MOP). After 24 hours
of incubation, the skin models were irradiated and
incubated with cytochalasin B until the isolation
of the cells, that were dropped onto glass slides,
stained, and analyzed by fluorescence microscopy.
Regarding mutagenicity, the results of the frequency
of micronuclei obtained in non-irradiated models
indicate that the compounds did not show mutagenic
potential. Regarding photomutagenicity, it was
observed that the treatment with positive control
8MOP (+UV) showed statistically higher micronuclei
values than the values of UVF+IR3535 and sesame
oil, considered non-photomutagenic. The percentage
of binucleated cells between treatments submitted
or not to UV radiation, did not show any significant
variation. The cytokinesis-block proliferation
index (CBPI) indicated that the irradiation dose,
experimental conditions, and concentrations studied
did not interfere in cell proliferation and were not
cytotoxic, since no variation was observed among
treatments and those pairs submitted or not to UV
radiation. The insect repellent IR3535, according
to TOXNET (Toxicology Data Network) and HSDB
(Hazardous Substances Data Bank) platforms, has
no evidence of genotoxicity or photogenotoxicity.
According to literature, in vitro assays and in vivo
micronucleus assay have also shown that the IR3535
was not mutagenic. In conclusion, the substances
studied did not show any mutagenic potential.
Regarding photomutagenicity, the combination of UV
filters and IR3535 and the vehicle (sesame oil) also did
not show any photomutagenic potential. This work
was supported by “Fundação de Amparo à Pesquisa
do Estado de São Paulo” (FAPESP), “Conselho Nacional
de Desenvolvimento Científico e Tecnológico” (CNPq),
and “Coordenação de Aperfeiçoamento de Pessoal de
Niv́ el Superior” (CAPES).
 257

Gliotoxin and its potential cytotoxicity

against NRAS-mutated melanoma
Noma, Isabella Harumi Yonehara; Carvalho, Larissa Anastacio da Costa; Maria-Engler, Silvya Stuchi

Department of Clinical and Toxicological Analyses, School of Pharmaceutical

Sciences, University of São Paulo, São Paulo, Brazil.

Background/Introduction: Genetic alterations melanoma (SK-MEL-173 and SK-MEL-147), primary

affecting RAS proteins are commonly found in human
cancers. Roughly a fourth of melanoma patients carry
activating NRAS mutations, rendering this malignancy
particularly challenging to treat. Although the
development of targeted as well as immunotherapies
led to a substantial improvement in the overall survival
of non-NRASmut melanoma patients (e.g. BRAFmut),
patients with NRASmut melanomas have an overall
poorer prognosis due to the high aggressiveness of
RASmut tumors, lack of efficient targeted therapies or
rapidly emerging resistance to existing treatments.
Understanding how NRAS-driven melanomas
develop therapy resistance by maintaining cell cycle
progression and survival is crucial to develop more
effective and specific treatments for this group of
melanoma patients. With the goal of improving the
low effectiveness of MEK inhibitors in metastatic
NRAS-mutated melanoma, it is necessary to research
new therapies for these patients. In others studies,
the gliotoxin (GT) exhibits cytotoxic effect and a
therapeutic potential for prevention of naïve BRAFmut
melanoma metastasis. GT is a fungal secondary
metabolite that mimics the peroxiredoxin activity by
reducing hydrogen peroxide and being reduced by
the NADPH electrons from TrxR system. Objective:
Our aim is to evaluate the cytotoxicity of GT in NRAS-
mutated melanoma and its therapeutic potential.
Methods: The cell lines used were NRAS-mutated
melanocytes, and primary fibroblasts as control.
Cell viability assay using trypan blue was used to
calculate IC50 (Inhibitory Concentration) of melanoma
cells treated with GT. The clonogenic assay evaluates
the ability of a single cell to grow into a colony and
determine the effectiveness of cytotoxic agents.
The wound healing assays creates a “wound” in the
cell monolayer and the captured images during cell
migration are followed during the closure or not of
the wound. Results: The trypan blue viability assay
has IC50 values of nM to melanocytes (50), SK-MEL-147
(57), SK-MEL-173 (58) and fibroblasts (73), respectively
after 24-hour GT treatment. In clonogenic assay,
all cell lines (Skmel-147, 173 and fibroblasts) had a
significant decrease of colonies after GT treatment
with concentrations IC25 and IC12,5. The wound healing
assay showed that SK-MEL-147 had a significant
decrease of migration after GT treatment (p<0.05)
in 24-hour. Discussion/Conclusion: Our results
show that gliotoxin proved to be efficient at low
concentrations with higher cytotoxicity and selectivity
to melanoma than fibroblasts and also impacted the
migration process. Therefore, GT has attributes as a
promising molecule that can be further explored for
NRAS-mutated melanoma. Acknowlegments: We
thank to financial support of CNPq (#408769/2018,
#304339/2017-2 and 142058/2020-3), FAPESP
(18/14936-1 and 17/04926-6) and CAPES.
 258

Strategies for overcoming toxicity and

resistance in melanoma progression
and in adaptative resistance to braf
inhibitors using ADK modulation
Silva, Julia Rezende1; Oliveira, Érica Aparecida2; Carvalho, Larissa
Anastacio da Costa1; Maria-Engler, Silvya Stuchi1
1
 Clinical Chemistry and Toxicology Department, School of Pharmaceutical
Sciences, University of São Paulo, São Paulo, Brazil; 2 Centre for Evolution and
Cancer, The Institute of Cancer Research, London, SM2 5NG, UK.

Background/Introduction: Melanoma is a malignant
neoplasm that originates from melanocytes and
although it corresponds to only 3% of skin cancers,
it is responsible for 80% of deaths; mostly due to its
resistance to specific inhibitors of mutated proteins
in the MAPK pathway, such as BRAF. Researchers has
shown an interesting relationship between adenosine
kinase (ADK) activity and cell growth and proliferation,
but its importance has never been investigated in
melanoma. ADK plays a key role in purine metabolism
regulation and studies have shown that one of the
ADK isoforms might be related to cancer biology. In
silico screening studies of our group, using TCGA and
GEO databases, identified ADK among a set of genes
differentially expressed between nevus and invasive
melanoma. Objective: In this study, we investigate
how ADK may contribute to the mechanisms that
lead to melanoma progression and resistance.
Methods and Results: Analysis of a TCGA cohort of
cutaneous melanoma samples revealed that 9% of
the samples had genetic alterations in ADK, mainly
characterized by mRNA increase. To validate this
data, we checked the gene and protein expression
levels in a broad panel of human melanoma cell
lines, with sensitive and resistant cells to a BRAF
inhibitor by RT-qPCR and Western Blotting assays,
in which we observed that metastatic melanoma
showed increased ADK expression when compared
to melanocyte. Furthermore, we observed a decrease
in both genetic and protein ADK expression in cells
resistant to the BRAF inhibitor. Cell proliferation assay
showed that the inhibition of ADK, either by a specific
inhibitor (ABT702) or by genetic knockdown using
shRNA, in ADK-high melanoma cells resulted in an
increase of proliferation compared to their controls.
Discussion/Conclusion: These data suggest that ADK
downregulation might have an impact in melanoma
cell growth and proliferation. Therefore, exploring the
impacts of ADK, both by pharmacological and genetic
inhibition, may contribute to understanding the
role and regulation of ADK regarding the molecular
mechanisms that support the progression of melanoma
and resistance to therapy. Acknowledgments: This
work has been supported by the following Brazilian
research agencies: FAPESP, CAPES, CNPq. The first
author is funded by the grant # 2020/14878-1, São
Paulo Research Foundation (FAPESP).
 259

The peroxiredoxin mimetic gliotoxin overcomes

heterogeneity, intrinsic and acquired resistance
to BRAF inhibitor in metastatic melanoma
Carvalho, Larissa A.C.1; Noma, Isabella H.Y.1; Uhera, Adriana H.1; Siena, Ádamo D.D.2;
Harumi, Luciana3; Mori, Matheus P.4; Goding, Colin5; Pinto, Nadja C. Souza4; Freitas,
Vanessa M.3; Silva-Jr, Wilson A.2; Smalley, Keiran S.M.6; Maria-Engler, Silvya S.1
1
 Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, USP, SP,
Brazil; 2 Department of Genetics, Ribeirão Preto Medical School, USP, SP, Brazil; 3 Department of
Cellular Biology and Development, Institute of Biomedical Sciences, USP, SP, Brazil; 4 Department
of Biochemistry, Institute of Chemistry, USP, SP, Brazil; 5 Ludwig Institute for Cancer Research,
Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; 6 Department of
Molecular Oncology, Comprehensive Melanoma Research Center, Moffitt Cancer Center, FL, USA.

Background/introduction: melanoma is the most
aggressive type of skin cancer and more than 50%
of the patients harbor a BRAF mutation. Although
BRAF inhibitors (BRAFi) has revolutionized therapy,
the minimum residual disease is still refractory.
Microenvironment and transcriptional plasticity
propitiate distinct subpopulations within the tumor
and the use of BRAFi contributes to the selection
of intrinsic resistant cells or acquired resistant
phenotype. MITF is the key biomarker for phenotype
switching (proliferative versus invasive). MITFhigh/
AXLlow phenotype is linked to proliferation and
BRAFi sensitivity and MITFlow/AXLhigh to invasion and
resistance. MITF controls oxidative metabolism by
regulating PGC-1α, responsible for mitochondrial
biogenesis and antioxidant proteins expression.
BRAF mutation suppresses MITF, PGC-1α, OXPHOS,
antioxidant proteins and ROS, while BRAFi increases
it. Naïve cells show multiple transcriptional states,
but it is poorly explored if all cells respond to BRAFi
similarly and what is the role of antioxidant defense at
the single cell level. PRX1 and PRX2 are central players
in redox signaling acting as sensors, antioxidants and
biomarkers. Since the lack of PRX is advantageous
for melanoma progression, we tried to overcome
the resistance associated to the heterogeneity
using gliotoxin, an antioxidant metabolite from
fungi containing a disulfide bridge that mimics PRX.
Objective: understand the role of PRX1 and PRX2 in
heterogeneity and overcome intrinsic and acquired
resistance to BRAFi targeting PRX by using gliotoxin.
Methods: we produced a SK-MEL-28R with acquired
resistance to BRAFi and stochastically isolated clonal
subpopulations (C1, C2 and C3) from naïve melanoma
(SK-MEL-28P). Functional profiles, phenotypes and
survival were analyzed using 2D models and 3D
reconstructed skin. Results: C1 is less proliferative,
more migratory and invasive, less sensitive to BRAFi,
less dependent on OXPHOS and more sensitive
to oxidative stress; C2 is more proliferative, less
migratory and invasive, more sensitive to BRAFi
and less sensitive to oxidative stress; and C3 is less
proliferative, more migratory and invasive, less
sensitive to BRAFi, more dependent on OXPHOS and
more sensitive to oxidative stress. The intrinsically
resistant clones C1 and C3 have lower MITF, PGC-1α,
PRX1 and C1 has higher AXL, linking PRX1 to clonal
heterogeneity, intrinsic and acquired BRAFi resistance.
PRX2 expression is equal among the clones, depleted
in SK-MEL-28R and act as a redox sensor in melanoma.
Gliotoxin exhibited a potent cytotoxicity in 85-160nM,
with different IC50 among the clones and SK-MEL-
28R, inhibiting colony formation and showing to be a
promising agent to overcome intrinsic and acquired
BRAFi resistance. Discussion/Conclusion: our results
illustrate new roles of PRX1 and PRX2 in melanoma
as contributors of heterogeneity and resistance,
demonstrating that redox sensitive reactions could
be a new target in melanoma resistance once it is
linked to tumor heterogeneity and could be overcome
with a PRX mimetic. Acknowledgments: FAPESP
(18/14936-1 and 17/04926-6), CNPq (#408769/2018
and #304339/2017-2) and CAPES.
 10 
IMMUNOTOXICOLOGY

Analyzes of clove essential oil immunotoxicity
based on in vitro and in silico studies
Etcheverry, Bibiana Frasson; Gomes, Gabriela Cristiane Mendes; Rios, Nathália Vieira; Chaves,
Pamella Eduardha Espindola; Sotelo, Êmily Clori; Zuravski, Luísa; Machado, Michel Mansur
Eugenia caryophyllus is a tropical plant belonging to
the Myrtaceae family and is popularly known as the
clove. It is widely applied in culinary and cosmetic
products as a flavoring agent. The essential oil obtained
from buds of Eugenia Caryophyllus L. is generally
used in traditional medicine. Previous studies have
reported its biological activities such as antioxidant,
antibacterial, antifungal, antiallergic, insecticidal,
anticarcinogenic, and antimutagenic effects. There is a
lack of toxicological information on the clove essential
oil. Therefore, this study aimed to analyze the toxicity
of clove essential oil in human lymphocytic cells,
through in vitro and in silico methods. Clove essential
oil was bought commercially from the IBD-certified
firm, BioEssência®. A gas chromatograph linked to
a mass spectrometer (GC/MS) was used to identify
and quantify its main constituents. The identification
process included interpreting chromatogram peaks
and comparing retention times with those found in the
National Institute of Standards and Technology (NIST)
databases. Seven major components were identified
and quantified: eugenol (86.7%), β-caryophyllene
(10.4%), humulene (1.9%), eugenyl acetate (0.3%),
caryophyllene oxide (0.3%), terpinen-4-ol (0.2%),
γ-terpinene (0.1%) and others components (0.2%). To
analyze the toxicity, the lymphocytes were cultivated
and exposed at five different concentrations of
clove essential oil: 1, 10, 100, 500, and 1000 µg/mL,
in addition to negative and positive controls. The
lymphocytes quantification was evaluated using the
Neubauer chamber. For in silico study, the canonical
forms of eugenol, β-caryophyllene, and humulene
identified in clove essential oil were found in the
PubChem database and submitted to computational
261
Grupo de Pesquisa em Imunologia e Genética Aplicada (GIGA),
Universidade Federal do Pampa, Uruguaiana, RS, Brasil.
tests via module DIGEP-Pred from Way2Drug and
GeneCards platforms. Only genes of the species Homo
sapiens were investigated for potential interactions
(upregulation and downregulation with probability
greater than 70%) between the main components of
the oil and genes associated with the cell cycle or
genetic maintenance of the cell. The results in vitro
analysis showed that the minor concentration did not
affect the lymphocytes total count. On the other hand,
the concentrations of 10, 100, 500, and 1000 µg/mL
decreased lymphocytes count as the concentration
increased. The computational analysis of Clove
essential oil compounds demonstrated that the
main component Eugenol induces alterations in the
expression of approximately 21 genes, causing both
upregulation and downregulation. Especially, our
results showed that Eugenol causes downregulation
of the H1FX gene, a protein-encoding that participates
in the formation of histones, which are responsible for
the nucleosome structure of the chromosomal fiber
in eukaryotic cells. To form higher-order chromatin
structures, the chromatin fiber is further compacted
by the interaction of a binding histone, H1, with the
DNA between the nucleosomes. As a consequence,
the organization of DNA in the cell is affected and the
cell cycle is interrupted. This mechanism may explain
the lymphocytes decrease when exposed to higher
concentrations of the clove essential oil. Therefore,
according to our results, Eugenia caryophyllus
essential oil is likely to interfere with gene expression
and, consequently, cell toxicity. However, more
studies are needed to certify the mechanism involved
in the reported effects. Acknowledgments: CAPES,
CNPQ, FAPERGS e UNIPAMPA.
 262

Cigarette smoke and heat-not-

burn tobacco vapor exposures alter
granulopoiesis and neutrophil functions
during experimental arthritis
Scharf, Pablo1; Heluany, Cíntia1; Sandri, Silvana1; Schneider, Ayda Henriques2;
Barbim, Paula Donate2; Fock, Ricardo1; Cunha, Fernando2; Farsky, Sandra1
1
 Department of Clinical and Toxicological Analyses School of Pharmaceutical Sciences University of São
Paulo – Brazil; 2 Department of Pharmacology, Ribeirão Preto Medical School, University of Sao Paulo.

Background: Exposure to combustible products of
cigarette smoke (CS) aggravates rheumatoid arthritis
(RA) symptoms and impairs immune response.
Hence, non-combustible tobacco products, such
as heat-not-burn tobacco (HNBT), which mainly
release nicotine, glycerin and propylene glycol, could
reduce those toxicities caused by CS. Nevertheless,
this hypothesis needs to be proved. Regarding the
immunotoxic effects of non-combustible tobacco
products, further experimental data are required.
Neutrophils are innate immune cells generated by a
process named granulopoiesis in the bone marrow
(BM). Neutrophil delivery into blood is a sustained
process of homeostasis and host defense, also these
cells play a pivotal role during RA course, and its
production and functions can be directly affected by
xenobiotics exposures, including those released by CS.
However, the impact of HNBT in neutrophil generation
and functions, remains unclear. Recently, our group
reported that mice exposed to CS displayed a worsen in
antigen-induced arthritis (AIA) symptoms, with higher
neutrophil migration into synovial cavity; conversely
HNBT did not cause these effects. Objective: We here
investigated the impact of CS or HNBT exposures on
BM and circulating leukocytes profile on AIA model
and the impact of these exposures in neutrophil
functions in vitro. Methods: AIA was induced in C57B6
mice by s.c administration of 500 μg of methylated
bovine serum albumin on day 0; boosted on days
7 and 14 and challenged intra articular (i.a.). on day
21; 24h later, samples were collected. AIA-mice were
exposed to airflow, CS or HNBT from day 14 to 20
after first immunization, for 1h/twice a day under the
Health Canada Intense (HCI) smoking regime (55 mL of
smoke for 2 s puffing, for every 30 s). The cellularity
and profile of BM and circulation leukocytes were
performed by manual counting and morphological
analysis. Neutrophils from naive mice were exposed
to airflow, CS or HNBT (30min, 2 s of smoke/vapor
followed by 58 s of airflow) to evaluate fMLP-induced
chemotaxis and NETs formation by flow cytometry.
Results: AIA increased BM cellularity in airflow-
exposed mice, which was higher in CS-exposed
group, due to the enhanced number of immatures,
banded and segmented neutrophils; conversely,
HNBT exposure did not enhance BM cellularity as CS
did, and reduced the number of immature and blast
cells. Elevated number of circulating leukocytes was
detected in airflow and CS exposed mice under AIA,
which was lower in HNBT exposed animals. In vitro
assays demonstrated that neutrophils exposed to
HNBT or nicotine, but not to CS, showed impaired
fMLP- induced chemotaxis and enhanced activation
state and NETs formation in comparison to airflow
exposure. Conclusion: Associated data obtained
show that CS and HNBT exposures differently affect
granulopoiesis during AIA, and nicotine in either CS or
HNBT impairs pivotal neutrophil functions during the
host defense. Acknowledgments: FAPESP and CNPq.
 263

Cytotoxicity of Marinobufagenin in
Glial Cells Chalenged Whith LPS
Farias, Evelyn Rayani Araújo1; Silva, Rivia Regina Lopes1; Souza, Natacha Medeiros2; Marques,
Ana Maria2; Scavone, Cristoforo2; Quintas, Luís E.M.3; Leite, Jacqueline Alves1
1
 Department of Pharmacology, Institute of Biological Sciences, Universidade Federal de Goiás,
Goiânia, Brazil; 2 Departament of Pharmacology, Institute of Biomedical Sciences, University of
São Paulo; 3 Laboratory of Biochemical and Molecular Pharmacology, Institute of Biomedical
Sciences, Health Sciences Center, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro.

Introduction: Neuroinflammation is a the cytotoxicity effects of different concentrations

pathological factor involved in the development of
neurodegenerative diseases like Parkinson’s and
Alzheimer’s diseases. Lipopolysaccharide (LPS),
in turn, is a membrane element of gram-negative
bacteria, which induces neuroinflammation via
toll-like receptor 4 (TLR-4) and NF-kB activation.
Marinobufagenin is a cardiotonic steroid isolated
from Bufo marinus toad venom. This molecule has the
ability to connect Na+/K+ -ATPase and recent studies
have demonstrated anti-inflammatory effects in vivo
and in vitro based on modulation of cell migration
and production of proinflammatory cytokines
like IL-6 and IL-1β. Aim: The present work seeks
to evaluate the cytotoxic and immunomodulatory
effects of marinobufagenin on glial cells challenged
with LPS. Methods: The initial step was to evaluate
of marinobufagenin (10,100,1000 and 10000 nM) in
primary culture of glial cells. Thereafter, the glial cells
were treated with concentrations of marinobufagenin
mentioned above and challenged with LPS (1ug/ml)
for 18 hours. Lastly, the culture medium was collected
to evaluate the levels of cytokines TNF-α, IL-6 and IL-
10 by ELISA. Results: Marinobufagenin did not show
cytotoxic effects at any concentration tested on glial
cells in the MTT assays. Also did not interfere with
the production of cytokines by glial cells challenged
or not with LPS. Conclusion: Preliminary data suggest
that marinobufagenin did not show cytotoxic effects
in glial cells challenged or not with LPS. However,
further studies are needed to better understand the
effects of marinobufagenin on neuroinflammation.
Financial Support: CNPq.
 264

In vitro and in silico analysis of the

cytotoxicity of Foeniculum vulgare
essential oil in human lymphocytes
Gomes, Gabriela Cristiane Mendes; Etcheverry, Bibiana Frasson; Chaves, Pamella Eduardha Espindola;
Rios, Nathalia Vieira; Campos, Dyene Nascimento; Zuravski, Luísa; Machado, Michel Mansur

Grupo de Imunologia e Genética Aplicada (GIGA), Universidade Federal do Pampa, Uruguaiana, RS.

Foeniculum vulgare, popularly known as fennel, is a
perennial herb that grows in tropical and temperate
regions and receives applications in culinary, food,
and cosmetics industries, as well in traditional
medicine for therapeutic purposes. Computational
methods include a variety of tools that contribute
to processes like molecules design, prediction of
pharmacokinetics and toxicological parameters,
and identification of potential biological targets,
proposing pharmacological activities or mechanisms
of action from the compounds under analysis. Due
to the biological activities attributed to this species
and the relevance to determine the security of
his use, in addition to the advantages of the use
of computational methods, we aimed to evaluate
the potential cytotoxicity of Foeniculum vulgare
essential oil in human lymphocytes and predict the
mechanisms of action through in silico analysis. For
this purpose, Foeniculum vulgare essential oil was
purchased from the company BioEssência, with a
certificate of organic and natural ingredients. The
phytochemical characterization was performed by
Gas Chromatography Coupled to Mass Spectrometry
(GC/MS), the retention times obtained were compared
to data previously described in literature and present
in the databases of the National Institute of Standards
and Technology (NIST). Lymphocytes cultures were
prepared from venous blood obtained by venipuncture
from a donor who did not ingest alcohol, medicines,
or smoke in the last 72 hours. Human peripheral
blood mononuclear cells (PBMC) were isolated using
Histopaque® 1.077 g/mL and maintained in RPMI 1640
supplemented with fetal bovine serum and antibiotics
for 24 hours at 37ºC in a 5% CO2 environment. In the
sequence, lymphocytes were isolated and stimulated
with phytohemagglutinin-M. Lymphocytes were
exposed to Foeniculum vulgare essential oil at a range
of concentrations (1 µg/mL - 1000 µg/mL), while
the negative control contained only supplemented
medium and the positive control received colchicine
(1 µg/mL). After 24h of exposure, was performed the
cell count. For the in silico analysis, the canonical
form of the majority compounds of Foeniculum
vulgare essential oil was searched in the platform
PubChem and posteriorly submitted to in silico tests
in the platforms Way2Drug using the DIGEP-PRED
module and GeneCards for evaluation of the gene
interactions (upregulation and downregulation with
probabilities above 80%) associated to the cell cycle
or with genetic aspects of the cell. The statistical
analysis was realized in specific software. The CG/
MS analysis detected and quantified 99.6% of the
total constituents, being trans-anethole the majority
compound identified in the essential oil. The results
in vitro showed that Foeniculum vulgare essential oil
affects the total lymphocytes count with LC50 of 501.2
µg/mL, and it is possible to predict the mechanisms
related to this effect through in silico simulation.
According to these analyses, Foeniculum vulgare
interferes in the cell cycle promoting a downregulation
of H1FX, a gene that encodes the protein H1x and it is
related to chromosome condensation and regulation
of DNA replication. Therefore, our findings showed
that Foeniculum vulgare essential oil is cytotoxic
for human lymphocytes. Moreover, the majority
compound trans-anethole interferes in gene
expression of H1FX affecting the histones replication,
as predicted through in silico platforms. Subsequent
studies are necessary to elucidate the role of H1FX
in cytotoxicity and also the potential activity against
tumor cells. Acknowledgments: CAPES, CNPq,
FAPERGS and UNIPAMPA.

Lymphotoxicity of Brazil mint essential
oil: an in vitro and in silico study
Chaves, Pamella Eduardha Espindola; Rios, Nathália Vieira; Gomes, Gabriela Cristiane Mendes;
Etcheverry, Bibiana Frasson; Campos, Dyene Nascimento; Zuravski, Luísa; Machado, Michel Mansur
Mentha arvensis, popularly known as Brazil mint,
Japanese mint, or menthol mint is a herbaceous,
perennial, and aromatic plant belonging to the
Lamiaceae family. It is widely cultivated in tropical
and subtropical regions. The essential oil obtained
from the leaves of Brazil mint is extensively used
in culinary, cosmetics, and traditional medicine as
a therapeutic resource for fever, colds, asthma,
digestive and cardiovascular disorders, among other
diseases and conditions. Recent studies with this
oil mainly aimed to prove the effectiveness of its
biological properties in preventing diseases and/or
maintaining health and not verify the safety of its
use. In that regard, the present study had as objective
analyze the lymphotoxicity of Mentha arvensis
essential oil. Initially, the compounds of the oil were
identified and quantified through the analytical
method of Gas Chromatography Coupled to Mass
Spectrometry (GC/MS) by retention times obtained
which compared with those reported in the literature
and present in the databases of the National Institute
of Standards and Technology (NIST). In the sequence,
for in vitro tests, the peripheral blood was collected
by venipuncture of a healthy self-declared individual,
the Peripheral Blood Mononuclear Cells (PBMC) were
separated by density gradient using Histopaque-1077®
(1:1) and incubated in RPMI 1640 supplemented with
SFB, penicillin-streptomycin, and gentamicin at 37°C
in 5% CO2 overnight. The lymphocytes that remained
in suspension were separated from monocytes
which were adhered to the contact surface. Human
purified lymphocytes were exposed to a range of
concentrations of Brazil mint (1, 10, 100, 500, and
265
Grupo de Pesquisa em Imunologia e Genética Aplicada (GIGA),
Universidade Federal do Pampa, Uruguaiana, RS, Brasil.
1.000 μg/mL) and incubated at the same conditions
for 24h, the protocol was performed in triplicate.
After this period, the total lymphocytes were counted
in Neubauer’s hemocytometer and the results were
expressed as lymphocytes/mL. In addition, we
submitted the majority of compounds identified in
the oil to in silico platforms. For this, the canonical
form of the main constituents found were submitted
to the platforms Way2Drug using the DIGEP-PRED
module and GeneCards. We analyzed the possible
interactions (upregulation and downregulation
with probabilities above 70%) between the majority
compounds and the genes related to the cell cycle
or with the genetic maintenance of the cell. The
in vitro assay results were expressed as mean ±
standard deviation and the LC50 value was calculated
by nonlinear regression method. We used a one-way
analysis of variance (ANOVA), followed by Tukey’s
posthoc test. The results were considered statistically
for p<0.05. The Menthol (49.6%), Menthone (24.1%),
and Isomenthone (12.4%) represent the majority of
compounds found in the oil analyzed, totalizing 86%.
The LC50 value calculated was 9.752 µg/mL to human
lymphocytes exposed to Brazil mint for 24h. The used
platforms in our study predicted probabilities higher
than 77% for downregulation of genes CDCA4, CENPJ,
and HAUS8 which were related to cell multiplication
and can be possibly involved in the observed effects.
Therefore, we can conclude that Brazil mint has a
high probability of interfering with gene expression
inducing lymphotoxicity. CAPES, CNPq, FAPERGS, and
UNIPAMPA supported the study.
 266

Mitochondria as a target for imidacloprid

toxicity on RAW 264.7 cells
Cestonaro, Larissa V.1; Schmitz, Felipe2; Ferreira, Fernanda S.2; Conte, Fernanda M.1;
Piton, Yasmin V.1; Wyse, Angela T.S.2; Garcia, Solange C.1; Arbo, Marcelo D.1
1
 Laboratory of Toxicology (LATOX), Department of Analysis, Faculty of Pharmacy,
Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil;
2
 Laboratory of Neuroprotection and Neurometabolic Disease, Department of Biochemistry,
Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.

Introduction: Imidacloprid is a neonicotinoid
insecticide that appeared on the market due to the
resistance related to other classes of insecticides and
the low-risk potential for mammals due to the greater
specificity for insects. Mitochondria are responsible
for synthesis of almost all cellular ATP necessary
to maintain cellular structure and function through
oxidative phosphorylation. Oxidative phosphorylation
occurs in the inner mitochondrial membrane. Electrons
removed from the citric acid cycle by NADH and FADH2
are used to drive the pumping of protons from the
matrix to the intermembrane space. These organelles
represent a preferred and critical target for the
action of drugs, toxins, or their reactive metabolites.
Complex II consists of two types of prosthetic groups
and four different proteins, and the enzyme succinate
dehydrogenase (SDH) is one of them. In this complex,
there is the reduction of ubiquinone to ubiquinol with
FADH2 electrons. Objectives: This study aimed to
investigate the potential involvement of mitochondria
in imidacloprid-related toxicity in RAW 264.7 cells.
Materials and Methods: Cells were seeded in 6-well
plates at a density of 1x106 cells/ml (final volume 0.2
ml/~260,000 cells/cm2). The effects of complex II was
evaluated after 24 and 96-hours incubation-time of
150, 500, and 1000 mg/L of imidacloprid (MUCH 600
FS) at 37 °C. Cell suspensions were analyzed in a FACS
Calibur flow cytometer to determine the mitochondrial
mass and membrane potential. Single dye controls
were used to set compensation. Ten thousand
events corresponding to intact cells were analyzed.
Results: It was observed a significant increase in
complex II activity as well as in SDH activity after
24h of incubation time with 1000 mg/L imidacloprid
(ANOVA/Bonferroni p < 0.001). Interestingly, this
increase was more significant after 24 h compared
to 96 h in SDH (two-way ANOVA/Bonferroni p < 0.001)
and complex II activity (two-way ANOVA/Bonferroni
p < 0.05). Discussion/Conclusion: Mitochondrial
complex II represents one of the intracellular targets
of imidacloprid. Overall, the results pointed to a
potential immunotoxic effect of imidacloprid in RAW
264.7 cells. Acknowledgements: Financial support
from CNPq/Brazil and FAPERGS.
 267

New pyrazoline compounds: effects on

neutrophils and macrophages functions
Goldoni, Fernanda C.1; Benvenutti, Larissa1; Vaz, Milena Menegazzo1; Carlos
Rafael1; Buzzi, Fátima C.2; Quintão, Nara L.M.1; Santin, José Roberto1
1
 Postgraduate Program in Pharmaceutical Science, Universidade do Vale do Itajaí, Itajaí, Santa Catarina,
Brazil; 2 Pharmacy, Courses, Universidade do Vale do Itajaí, Itajaí, Santa Catarina, Brazil; Postgraduate
Program in Pharmaceutical Science, Universidade do Vale do Itajaí, Itajaí, Santa Catarina, Brazil.

Background/Introduction: Inflammation is a form pharmacological characteristics such as molecular

of defense against tissue damage or pathogens
entry. Although being essential for the homeostasis,
this process must be stopped, avoiding this way its
exacerbation. Non-steroidal anti-inflammatory drugs
play an important role stopping the inflammation,
although they have described many adverse effects
that can range from nausea to gastric ulceration and
renal hypertension. In this context, it is necessary
to keep continuously searching for new bioactive
compounds. Pyrazolines are molecules characterized
by having a five-membered heterocycle with high
structural versatility, presenting many biological
activities such as antioxidant, anti-inflammatory,
anti-carcinogenic, among others. Objective: this study
aims to evaluate the toxicological parameters using
in silico methods and anti-inflammatory potential
of new pyrazoline compounds. Methods: Initially,
a predictive pharmacological and toxicological
evaluation of the compounds was performed in silico,
using the SwissADME and Qsar Toolbox platforms. To
evaluate its anti-inflammatory potential, different
methodologies were performed, such as dosage of
nitric oxide (NO), pro-inflammatory cytokines levels
such as IL-1β and TNF, using the ELISA method, and
a chemotaxis assay was performed as previously
described using an agarose gel and fMLP as
chemotactic agent, all of them using macrophages
and neutrophils, the main cells involved in the acute
inflammatory response. Results/Discussion: The
results obtained in the MTT assay showed that none
of the four compounds (PH0, PH3, PH4 and PH7), at
all concentrations evaluated, showed cytotoxicity.
In the in silico analysis of compounds PHO, PH3, PH4
and PH7, it was possible to observe using SwissADME,
an online platform, that all compounds have good
weight lower than 500 g/mol, only one of the four
compounds, PH0, demonstrates to be able to pass the
blood brain barrier, also PH0 and PH7 has shown a
good probability to be absorbed by GS, different from
PH3 and PH4, that seems to have a lower probability
to be easily absorbed by GS due to the presence of an
chloride its stucture. Using the Qsar Toolbox software,
parameters related to the prediction of toxicological
potential of the compounds were observed, such as
genotoxicity, carcinogenicity, hepatotoxicity, effects
on the endocrine system, among others. Analyzing
the results, it was possible to conclude that both
PH3 and PH4 compound has alerts to hepatotoxicity
and carcinogenicity related to the presence of one
halogenated benzene in the molecule. The others
compounds showed no predictive potential to
have negative or toxic effects. The results of nitric
oxide measurement, both in macrophages and
neutrophils, showed that all of the four compounds
were able to significantly decrease nitric oxide levels
in the supernatant of cell culture stimulated with
Escherichia coli lipopolysaccharide (LPS), with PH4
showing the highest inhibition rates. As in the nitric
oxide dosage, the compound that also showed a
satisfactory effect in decreasing TNF levels was PH4,
with a higher average inhibition. The chemotactic
assay was performed using only the compound PH4,
considering that it presented the best initial results,
and also showed satisfactory results regarding in vitro
chemotaxis of neutrophils. Conclusion: The results
obtained in the study so far has shown the promising
effects of the compound PH4, but further analyzes
will be performed to determine if the compound has
potential to go to in vivo rodent tests.
 268

Patchouli (Pogostemon cablin)

essential oil: safe or a hidden risk?
Rios, Nathália Vieira; Chaves, Pamella Eduardha Espindola; Gomes, Gabriela Cristiane Mendes;
Etcheverry, Bibiana Frasson; Sotelo, Êmily Clori; Zuravski, Luísa; Machado, Michel Mansur

Grupo de Imunologia e Genética Aplicada (GIGA), Universidade Federal do Pampa, Uruguaiana, RS, Brasil.

The practice of health care and prevention through
natural products such as aromatherapy is something
that has been drawing attention over the years.
Aromatherapy is a practice that consists of the
use of pure essential oils to prevent and treat
pathologies. One of the most sought-after essential
oils in this practice is Pogostemon cablin essential
oils. Pogostemon cablin is a branched, perennial,
aromatic herb of the Lamiaceae family, native
to Southeast Asia and widely cultivated in many
countries. It is commonly known as Patchouli and
has recently gained prominence in academia due to
its antidepressant, anti-inflammatory, antiseptic,
immunomodulatory, aphrodisiac, and astringent
properties. Despite having numerous biological
effects and being used in ethnopharmacology for
many years, little is known about its cellular toxicity.
Thus, this work aimed to evaluate the cytotoxicity
potential of the major compounds from the essential
oil of Pogostemon cablin. For this, human peripheral
blood was collected by venipuncture (UNIPAMPA
Ethics Committee, nº 27045614.0.0000.5323), and
peripheral blood mononuclear cells (PBMC) were
isolated by density gradient with Histopaque®
1.077 g/mL and maintained overnight in RPMI 1640
supplemented with 10% fetal bovine serum and 1%
penicillin-streptomycin in an atmosphere of 37ºC
and 5% CO2. After this period, the lymphocytes were
separated from the monocytes that adhered to the
contact surface. Lymphocytes were treated with
concentrations of Patchouli ranging from 1 to 1000
µg/mL and stimulated by the addition of PHA-M (1
mg/mL) for 24 hours and then the total lymphocytes
were counted in Neubauer hemocytometer. The
determination of the LC50 was realized using non-
linear regression analysis. For the in silico tests, a
previous analysis was performed by GC-MS (gas
chromatography coupled to mass spectrometry) and
the major compounds in the oil were determined. The
canonical form of the major compounds Pogostol
and α-gurjunene was searched in the PubChem
database and submitted to computational tests
on the Way2Drug platform using the DIGEP-PRED
Module and later the GeneCards website was used to
correlate the genes found. These tools investigated
gene expressions, where up and downregulation
were considered with probabilities above 70%. In
this study, the calculated LC50 value was 66.1 µg/
mL for human lymphocytes exposed to Pogostemon
cablin essential oil. This toxicity can be explained in
part by in silico forecasts, which showed a reduction
in the CENPJ gene caused by the major compounds of
Patchouli (Pogostol and α-gurjunene). CENPJ is a gene
capable of encoding a protein that belongs to the
centromere family of proteins. During cell division,
this protein plays a structural role in maintaining
centrosome integrity and normal spindle morphology
and is involved in microtubule disassembly in the
centrosome. Alterations, in quantity or quality, in this
protein interfere with the cell’s ability to proliferate,
as well as remain metabolic active. It is concluded
that from the results presented, new studies related
to this essential oil are of paramount importance
for the construction of its toxicological profile and
determination of its safety. Agradecimentos: CNPQ,
CAPES, FAPERGS e UNIPAMPA.
 269

Trilobolide-6-O-isobutyrate shows in silico

and in vitro potential for antitumor activity
of A549 and NCI-H460 lung cancer cell lines
Miranda-Sapla, Milena Menegazzo1; Concato, Virginia Marcia2; Gonçalves, Manoela Daiele3; Matos,
Ricardo Luiz Nascimento3; Conchon-Costa, Ivete2; Arakawa, Nilton Syogo3; Pavanelli, Wander Rogério2
1
 Laboratory of Toxicology, University of Vale do Itajai, Itajaí, SC, Brazil; 2 Laboratory of
Immunoparasitology of Neglected Diseases and Cancer, State University of Londrina, PR, Brazil;
3
 Laboratory of Biotransformation and Phytochemical, State University of Londrina, PR, Brazil.

Introduction: Lung cancer is the world’s leading cause
of cancer due to its death aggressive manifestation,
high ability to promote metastasis, and late diagnosis.
Despite the development of several antitumor drugs,
the prognosis of lung cancer is relatively low. The
search for new compounds with antiproliferative
potential that respond selectively to tumor cells with
less toxicity to non-tumor cells have been the focus
of many studies. Trilobolide-6-O-isobutyrate (TBOI)
is a natural eudesmanolide lactone isolated from
Sphagneticola trilobata Pruski (Wedelia paludosa
D.C.) and W. prostrata which have a wide range of
pharmacological activities reported as microbicidal,
anti-inflammatory, and antitumor, but the study
of this natural drug against lung carcinoma is still
limited. Objective: The present study aims to evaluate
the TBOI in silico pharmacological and toxicological
characteristics and the in vitro action of this
compound on normal cell lines and against A549 (lung
adenocarcinoma) and NCI-H460 (large cell carcinoma)
lung cancer cells. Methods: The TBOI structure in
silico study was carried out to evaluate theoretical
drug-likeness and toxicological parameters using
the SwissADME and Qsar Toolbox platforms. MTT
assay was performed to determine the cytotoxic
effects against lung cancer cells A549 and NCI-H460,
and LLC-MK2, VERO, and AMJ2-C11 normal cell lines.
The cells were treated for 24-72 h with different
TBOI concentrations (12 -100 µM) and the cytotoxic
concentration of 50% (CC50) and the tumor selectivity
index (TSI) was determined. The TBOI hemolytic
activity was also evaluated (3-200 µM). Results: In
silico predictions showed good drug-likeness potential
for TBOI with high oral bioavailability and intestinal
absorption, no blood-brain barrier penetration, and
no interaction with the main five CYP isoforms. The
presence of any pan assay interference compounds
(PAINS) was also not identified. Also, the TBOI was
predicted as non-genotoxic, non-carcinogenic, non-
mutagenic, and non-hepatotoxic. TBOI structure also
predicts the in vivo mutagenicity (Micronucleus) alerts
due to the oxalane (tetrahydrofuran) moiety, which
acts as nucleoside analogues, a common chemical
characteristic present in cancer chemotherapeutic
agents, by inhibiting the function of DNA polymerase
and/or being incorporated into DNA as nucleosides
fraudulent. The in vitro analyses showed that TBOI
treatment of lung cancer cell lines was able to reduce
cell viability at all concentrations tested in a dose- and
time-dependent manner defining the CC50 after 24h
for A549 (73µM ±0.06) and NCI-H460 (54µM ±0.03).
The CC50 for LLC-MK2, VERO, and AMJ2-C11 was also
determined as 335 µM (±0.01), 69 µM (±0.02), and 83
µM (±0.01), respectively. It was determined in vitro
TSI A549 of 4.6, 1, and 1.1 for LLC-MK2, VERO, and
AMJ2-C11 respectively, and TSI NCI-H460 of 6.2, 1.2,
and 1.5 for LLC-MK2, VERO, and AMJ2-C11 respectively.
The TBOI tested concentrations also showed no
hemolytic activity in vitro. Conclusion: This study
revealed that TBOI may be a promising candidate for
the development of antitumor drugs, targeting lung
carcinoma.
 11 
NANOTOXICOLOGY
 271

Cytotoxicity and effectiveness in

macrophage polarization of Annexin A1-
surface-functionalized metal-complex
multi-wall lipid core nanocapsule
Broering, Milena F.1; Leão, Matheus C.2; Scharf, Pablo1; Sandri, Silvana1; Uchiyama, Mayara
K.3; Araki, Koiti3; Guterres, Silvia S.4; Pohlmann, Adriana R.5; Farsky, Sandra H.P.1

 Department of Clinical & Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of Sao
1

Paulo, SP, Brazil. 2 Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences,
University of Sao Paulo, SP, Brazil. 3 Department of Fundamental Chemistry, Institute of Chemistry,
University of Sao Paulo, SP, Brazil. 4 Department of Production and Control of Medicines, Faculty of
Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. 5 Department of Organic
Chemistry, Chemistry Institute, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.

Background/introduction: Nanotechnology is the (3-(4,5-Dimethylthiazol-2-yl) assay (MTT assay) was

principle of engineering nanomaterial with different
compound and structures allowed to be used in
several areas of science, including in pharmaceutical.
The interaction of nanoparticles (NPs) with the
immune system is a key element both in the
assessment of nanotoxicity and in the development
of nanomedicines. Annexin A1 (AnxA1) is a protein with
many anti-inflammatory properties and is capable to
act on macrophages, promoting anti-inflammatory
macrophage phenotype in in vitro models polarization,
also improving the efferocytosis. Multi-wall lipid core
nanocapsules (MLNC) and surface-functionalized
metal-complex multi-wall lipid core nanocapsule
(MLNC functionalized) are able to carry efficiently
lipid and protein molecules in the core and in the
polymeric wall, according to the characteristic of
the substance. The metal complex with Zn2+ provides
better binding of the protein to the MLNC structure
by interacting with histidine residues present in the
C-terminal region in the proteins. This supramolecular
structure optimized and enhancing bioaviability and
driving medicines to the pharmacological targets.
Herein, we aimed to functionalize recombinant
AnxA1 on MLNC to investigate the cytotoxicity and
effectiveness on macrophage polarization. MLNC
was prepared by covering lipid core nanocapsule
(LNC) with chitosan, which coordinates metals to the
specific protein chemisorption site. Therefore, MLNC
was linked to Zn2+ and rAnxA1 was added to form
the MLNC-AnxA1. The cytotoxicity of four different
concentrations of recombinant AnxA1 (rAnxA1), MLNC
and MLNC-AnxA1 was evaluated in Raw 264.7 cells and
assess as a screening test. Only the concentrations
that showed mitochondrial activity less than 70%
in the MTT assay were tested by the LDH activity,
necrosis, apoptosis and late apoptosis assay by
flow cytometer in 24 and 48 hours. The uptake of
nanoparticles by Raw 264.7 cells was performed in a
dark field hyperspectral microscopy (CytoViva®). The
polarization of macrophages was evaluated using
primaries cells obtained from the bone marrow of
C57Bl/6 mice. Primaries macrophages were then
differentiated into inflammatory (M1) by the addition
of LPS (100 ng/mL) and treated with AnxA1, MLNC or
MLNC-AnxA1. After 48 hours the cell phenotype was
evaluated by flow cytometry (F480, CD80 and CD206)
and the cytokines of the supernatant were quantified
(TGF-β and IL-10). LNC, MLNC and MLNC-AnxA1
presented 129,5, 152,47 and 162,87 nm, respectively,
and equivalent polydispersity index; incorporation
of chitosan inverted the negative potential zeta; the
encapsulation efficiency of AnxA1 was 92.22%, and
transmission electron microscope photomicrograph
showed MLNC-AnxA1 spherical shape. MLNC-AnxA1
was able to be taken up by Raw 264.7 cells at a
concentration that did not generate cytotoxicity and
both MLNC and MLNC-AnxA1 was able to differentiate
M1 macrophages in anti-inflammatory macrophages
(M2). Altogether the results improve the knowledge
about the cytotoxic capacity of MLNCs and showing
MLNCs actively act on macrophages, an important
cell type present in the innate immune response.
Acknowledgments. USP, CNPq and FAPESP.
 272

Preclinical evaluation of short-term

gold nanoparticles toxicity
Plautz, Katherine1; Gastaldi, Alessandra Betina1; Maia, Thayná Patachini1; Pereira,
Eduardo Manoel3; Ferreira, Gabriela Kozuchovski4; Borgmann, Gabriela1; Cabral,
Heloisi1; Delwing-Dal Magro, Débora5; Delwing-De Lima, Daniela1,2

 Programa de Pós-graduação em Saúde e Meio Ambiente - UNIVILLE, Joinville, SC, Brasil;

1

2
 Departamento de Medicina - UNIVILLE, Joinville, SC, Brasil; 3 Departamento de Farmácia
– Universidade da Região de Joinville – UNIVILLE – Joinville, SC; 4 Departamento de Saúde -
UniSociesc – Joinville, SC; 5 Departamento de Ciências Naturais - FURB, Blumenau, SC

Introduction: Nanotechnology comprises the science
that works with materials at molecular levels,
whether for manipulation and/or creation of new
materials and products. The properties of materials
at the nanoscale can be very different from those
at a larger scale. Metal nanoparticles have been
widely explored for biomedical purposes, especially
gold nanoparticles, due to their low cytotoxicity
and with this, they are used clinically to improve the
potential of drugs, altering the pharmacokinetics,
biodistribution and cellular absorption. Although
nanoparticles modify the physical properties of
several compounds and result in several benefits, the
evaluation of the toxicity of these materials is poorly
understood. Objectives: To evaluate the degree of
acute renal (urea and creatinine) and hepatic (AST and
ALT) toxicity of gold nanoparticles in 60-day-old male
mice. Methods: Adult male mice (20 - 30 g) of the
Mus musculus species were divided into groups and
treated once a day, for four days, with nanoparticles
(doses of 0.025; 0.07 and 0.22 mg/kg) or saline
solution (0.1 mL/10g) intraperitoneally. One day after
the last administration, a blood sample was obtained
for plasma separation and spectrophotometric
measurement of the level of hepatic and renal
biomarkers was conducted. Data are presented
as mean ± standard error. After the synthesis, the
AuNPs solution was characterized by the authors
using the ultraviolet-visible (UV-Vis) spectroscopy
technique. The values ​​obtained were 10 nm, as
expected, and this was confirmed by the Biological
and Analytical Transmission Electron Microscopes
(TEM) image, which showed predominantly spherical
gold nanoparticles (GNP). Results: Gold nanoparticles
doses of 0.025; 0.07 and 0.22 mg/kg and saline resulted
in a blood level of oxaloacetic transaminase of 79.8 ±
4.7; 108.0 ± 9.9; 100.1 ± 6.3 and 99.3 ± 9.0 U/L; glutamic
transaminase of 20.3 ± 1.6; 17.2 ± 1.2; 15.8 ± 1.4 and 16.9
± 2.1 U/L; of urea, 52.1 ± 6.3; 56.2 ± 1.2; 52.8 ± 1.2 and
55.6 ± 2.0 mg/dL and creatinine of 0.27 ± 0.01; 0.28 ±
0.01; 0.30 ± 0.01 and 0.29 ± 0.01 mg/dL, respectively.
Discussion/Conclusion: The data found allow us to
infer that nanoparticles, in short-term treatment,
are not significant promoters of hepatotoxicity or
nephrotoxicity, being well tolerated in this sense
regarding the safety of their use in this period. It is
possible that longer periods of administration imply a
different profile of toxicity, which may motivate other
studies, as well as investigations of toxicity linked
to mutagenesis, carcinogenesis and teratogenesis,
which are essential in the screening of adverse effects
of new compounds with biological activity. Keywords:
Toxicity; Metallic Nanoparticles; Acute Toxicity; Gold
Compounds.
 273

Predictive analysis of ocular and lung

damage in cells exposed by zinc oxide or
cerium dioxide nanoparticles using electrical
impedance compared to MTT test
Alexandre, Angela de Oliveira1,2; Souza, Wanderson2,3; Dal-Cheri, Beatriz Kopke de
Assis1,2; Granjeiro, Jose Mauro1,2,3; Pereira, Leonardo da Cunha Boldrini1,2,3
1
 Postgraduate Program in Translational Biomedicine, University Grande Rio, Duque de
Caxias, Brazil; 2 Directory of Life Sciences Applied Metrology, National Institute of Metrology
Quality and Technology, Rio de Janeiro, Brazil; 3 Postgraduate Program in Biotechnology,
National Institute of Metrology Quality and Technology, Rio de Janeiro, Brazil.

Introduction: nanotechnology products are being
increasingly used in the consumer industry; such
as pharmaceuticals, food, medicine, cosmetics,
pesticides. Zinc oxide (ZnO) and cerium dioxide (CeO2)
nanoparticles (NPs) can be highlighted, due to their
high consumption in these industries. ZnO NPs are
used due to their antimicrobial action and it’s the
raw material for sunscreens and cosmetics. CeO2
NPs are used in sunscreens, cosmetics, in polishing
agent (mainly for the production of ophthalmic
lenses), in diesel additives and cigarettes. The NPs
can be atmospheric pollution agents, which can
cause adverse effects on human health (causing lung
diseases and eye irritation or corrosion). NPs toxicity
studies still use animals, however, animal welfare,
financial and legal issues encourage the consolidation
of alternative predictives methods to cytotoxicity is
necessary to promote the reduction and replacement
the animal use. Objective: the present study evaluated
the cytotoxicity of ZnO and CeO2 NPs using SIRC cells
(to assess eye irritation and A549 human alveolar
epithelial cells to assess lung toxicity. Methods: the
average size of NPs were evaluated by dynamic light
scattering (DLS); the surface charge by zeta potential;
morphology by transmission electron microscopy
(TEM); cytotoxicity was evaluated by electrical
impedance and MTT assay for 72h. Results: ZnO and
CeO2 NPs have an average size (in water) of 241.20 ±
11.49 nm and 212.29 ± 15.38, and with a polydispersion
index (PDI) of 0.20 ± 0. 03 and 0.35 ± 0.07 nm,
respectively. After 24h of stabilization, the ZnO NPs
had an average size in water of 188.90 ± 42.68 nm and
a PDI of 0.13 ± 0.03, whereas the CeO2 NPs was 140.02
± 4.26 nm and 0.18 ± 0.05 nm. The average size of the
NPs after contact with the culture medium (Dulbecco’s
Modified Eagle’s Medium low glucose (DMEM)
supplemented with 10% fetal bovine serum (FBS) and
stabilized with bovine serum albumin (BSA) specific
for the cells of this study was evaluated. The ZnO and
CeO2 NPs had an average size of 192.8 ± 13.62 nm and
167.49 ± 22.62 nm, with PDI of 0.18 ± 0.09 and 0.12 ±
0.01 nm, respectively. In relation to Zeta potential,
the NPs demonstrate stability in water, presenting a
surface charge of 22.6 ± 0.63 for the ZnO NPs and 37.71
± 7.63 mV for the CeO2 NPs; there was a significant
difference in the surface charge of NPs after contact
with the culture medium, with a surface charge of
-8.23 ± 0.69 mV for ZnO NPs and 3.81 mV for CeO2 NPs,
probably due to protein corona formation. NPs have
characteristic spheroidal morphology. The potential
for irritation and cytotoxicity will be analyzed after
exposure of NPs in cells at concentrations of 2.5;
25; 50; 75; 100 µg/mL, for 72h. Discussion: electrical
impedance is a non-colorimetric technique, applied to
cell viability analysis, provides kinetic information in
a non-invasive way and with high temporal resolution
through growth curves recorded in real time. The
performance of this technique can be efficient to
evaluate the cytotoxicity of NPs, being able to be used
as an alternative test system for the toxicity of NPs in
vitro, interference-free, allowing the consolidation of
an alternative test to the use of animals. Conclusion:
cell viability tests without colorimetric interference
appear as a promising and important alternative for
the nanotoxicology field. Mainly to assess the safety
of ZnO and CeO2 NPs, reducing the potential irritation
risk and eye and lung corrosion. Acknowledgments:
thanks to the Ministry of Science and Technology
and Innovations of Brazil, Foundation Carlos Chagas
Filho Research Support of the State of Rio de Janeiro
(FAPERJ), the National Institute of Metrology, Quality
and Technology. The Unigranrio and UEZO universities.
 274

Study of the acute and chronic toxicity of

chrome III oxide nanoparticles on the marine
microcrustacean Mysidopsis juniae (Silva, 1979)
Plautz, Katherine1; Fugazza, Jonas1; Gastaldi, Alessandra Betina1; Kleine, Tamila1;
Matias, William Gerson2; Oliveira, Therezinha Maria Novais1
1
 Programa de Pós Graduação em Saúde e Meio Ambiente – UNIVILLE, Joinville, SC, Brasil;
2
 Departamento de Engenharia Sanitária e Ambiental - UFSC, Florianópolis, SC, Brasil.

Introduction: Nanotechnology, the science of creating is formation of large agglomerates of suspension

devices and materials, from the control of matter
on the nanometer scale is currently considered an
innovative area of scientific and economic growth;
arouses great interest from various sectors and
its use, due to its characteristics, especially its size
(0-100 nm) is already widespread in many areas,
including the pharmaceutical, electronics, paint
industry and manufacturing, among others. However,
toxicological studies are insufficient to accurately
identify the effects to health and to the environment
resulting from the use of nanocomposites. Among
the extensive use of nanoparticles, the Chromium (III)
Oxide nanoparticle (Cr2O3 NP) have a wide variety of
applications. Objectives: the aim of this study was
to evaluate the toxicity of Cr2O3 NP in reconstituted
seawater (RSW) through acute and chronic
toxicological tests using microcrustacean marine
Mysidopsis juniae. Methods: the characterization of
Cr2O3 NP was carried out by different physicochemical
techniques, in different means, through toxicological
testing toxicity of Cr2O3 NP, acute exposure to defining
the LC50(96h) and chronic and transgenerational test
evaluating: birth, mortality and biometrics during
the period of 63 days. Finally, it was conducted the
analyzing of the concentration of Cr (III), Cr (VI) ions
and total chromium to the concentrations of acute
test (25, 50, 75, 100 and 125mg/L-1) and chronic (5mg/
L-1). Results: The results of the characterization of
Cr2O3 NP by Transmission Electron Microscopy (TEM),
Scanning with Field Emission (SEM-FEG) and X-ray
Diffraction showed that the particle diameter is in
nanometer scale (21,8 nm), showing further that there
NP, especially in saline, which was confirmed by
analysis of the hydrodynamic diameter. For acute
toxicological evaluation, it was observed that
concentration below 5 mg L-1 have no mortality and
from the concentrations of 25mg/L-1, always occurred
mortality, being the concentration of 125 mg/L-1
the responsible for overall mortality of organisms
in 96 h of exposure. The LC50(96h) found for the test
organism exposed to Cr2O3 NP was 58.86 mg/L-1.
Chronic and transgenerational test showed that only
the parameter mortality of 2nd generation exposed
to concentration of 5mg/L-1 of Cr2O3 NP was affected
by the studied substance (p<0.05). Finally, it was
possible verify on analysis of chromium ions for both
concentrations of acute test as to the chronic test, the
presence of Cr (VI), and already in the concentration
of 5 mg L-1 of Cr2O3 NP the concentration of Cr (VI)
was 0.25mg /L-1, a result above the standard limit for
discharge of effluents defined by Resolution CONAMA
430/2011, exponentially increasing for the other
evaluated concentrations. Discussion/Conclusion:
this study shows that Cr2O3 NP when dispersed
in saline and when found in very small diameters
tend to have low stability and can contribute to
the agglomeration of NP, being a possible cause of
decrease toxicity which would hinder the absorption
by the organism, however, this same instability also
contributes to the release of ions and in this study,
it was found the presence of Cr (VI), which can be, in
this case, the most responsible for the toxicity found
Keywords: Nanoparticles; Chromium oxide; Acute
toxicity; Chronic toxicity; Mysidopsis juniae.
 275

Study of the interaction between a potential

labeled nano-enabled pesticide and aquatic plant
Forini, Mariana M.L.1; Antunes, Débora R.1; Cavalcante, Luiz A.F.1; Pontes, Montcharles
S.2; Santiago, Etenaldo F.2; Sanches, Alex O.1; Martins, Aline R.1; Grillo, Renato1
1
 São Paulo State University (UNESP), Faculty of Engineering, Ilha Solteira, SP, Brazil; 2 Center for
Natural Resources Study (CERNA), Mato Grosso do Sul State University (UEMS), Dourados, MS, Brazil.

Nano-enabled pesticides have been developed with
the potential to promote more sustainable agriculture
since they improve efficiency and reduce the number
of pesticides in the field. However, little is known
about the interaction of these nanomaterials (also
called nanopesticides) with plants. In this sense,
nanostructured lipid carriers (already used as
nanopesticides) associated with hydrophobic gold
nanoparticles were synthesized using the solvent
emulsion/evaporation method, to study the interaction
of these nanocarriers in aquatic macrophytes of the
Lemna valdiviana species. The labeled nanocarrier
(ca. 250 nm) presented a polydispersion index below
0.2, zeta potential of -17.3 ± 1.5 mV, and excellent
encapsulation efficiency (99.9%) of hydrophobic gold
nanoparticles with the lipid core of the nanocarriers.
Moreover, the interaction of nanoparticles with
aquatic plants was evaluated using Atomic Absorption
Spectroscopy (AAS), and the detection of labeled
nanocarriers was concentration- and time-dependent
in macrophytes. Also, X-ray diffraction (XRD) and
infrared spectroscopy (FTIR) analysis showed changes
in the spectrum of plants when exposed to water (as
standard) and labeled nanocarriers, which indicates
the presence of nano-enabled materials associated
with the aquatic macrophytes. Furthermore,
histological sections of the plants allowed detecting
aggregates of nanocarriers by optical microscopy
analysis, corroborating with the X-ray fluorescence
analysis (FRX) that showed the presence of 0.37% of
gold ions in the plant. Therefore, this study reveals a
new labeled nanopesticide able to comprehend the
interaction of nanopesticides with plants as well as
can help to better understand their mechanisms of
action and toxicological effects in the environment.
Acknowledgements: The authors acknowledge
the FAPESP (#2020/12769-0, #2019/20124-
2, #2017/21004-5), CNPq (#161360/2021-1,
#427498/2018-0), CAPES for the financial support.
Also, the Environmental Nanochemistry Lab, UNESP-
Sorocaba for the support.
 276

Survival rate of Drosophila melanogaster

exposed to nanoparticles containing Naringenin
and in vitro determination of inhibition
kinetics against cholinergic enzymes
Cunha, Viviane Augusta de Medeiros Garcia1; Rossi, Bruna Franzon2; Leimann, Fernanda
Vitória1,3; Ineu, Rafael Porto1; Foleis, Vanessa Kaplum1; Gonçalves, Odinei Hess1,3
1
 Post-Graduation Program of Food Technology (PPGTA), Federal University of Technology
– Paraná – UTFPR Campus Campo Mourão, Campo Mourão, PR, Brazil; 2 Food and Chemical
Engineering Department (DAAEQ), Federal University of Technology – Paraná – UTFPR Campus
Campo Mourão, Campo Mourão, PR, Brazil; 3 Centro de Investigação de Montanha (CIMO),
Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal.

Introduction: Flavonoids are bioactive compounds
that have protective effects on the neuronal circuit due
to their antioxidant properties, free radical scavenging
and their possible impact on intracellular redox impact.
Studies have reported anti-inflammatory, antioxidant
and inhibitory action against acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) enzymes.
The encapsulation of bioactive compounds through
nanotechnology techniques has helped in the
stability, improvement of the physical property and
in the absorption of these bioactive compounds such
as naringenin. Although naringenin in its free form
does not present a high level of toxicity, studies are
needed to promote the investigation of the toxicity
of its encapsulated form. Objective: To investigate
the toxicity of naringenin loaded with nanoparticles
using the survival rate of Drosophila melanogaster.
In addition, the activity of naringenin against the
cholinergic enzymes AChE and BChE was evaluated by
enzymatic kinetics assays. Methodology: Poloxamer
407 (0.9g, Sigma-Aldrich, encapsulant), ethanol
(37.5mL, Dynamic), Tween80 (0.009g, Dynamic) and
naringenin (0.09g, 95% purity, Sigma-Aldrich) were
used to obtain the nanoparticles. Poloxamer 407 and
Tween80 were solubilized in ethanol for 5 min under
gentle agitation at 40°C, naringenin was added and
solubilized for 1 min, then the solution was sonicated
(Fisher Scientific 120, 120W, 1/8” tip) for 3 min. in a
pulsed regime (30s on, 10s off) in an ice bath. The
obtained dispersion was evaporated at 40°C for 3h.
The nanoparticles were characterized by Differential
Scanning Calorimetry (DSC) and Fourier Transform
Infrared Spectroscopy (FTIR). The survival test was
performed as follows: 100 flies were exposed for
five days to nanoparticle-loaded naringenin (160,
210, and 250 µg naringenin.mL-1) of nanoparticles
and also to free naringenin (n=5 for each treatment).
The enzymatic kinetics of naringenin against AChE
and BChE enzymes was determined by the method of
Ellman et al (1961) with modifications. Results: The
same thermal transition temperatures were found
in free naringenin and nanoparticles, while the FTIR
showed absorption peaks related to the chemical
groups of naringenin. The survival rate curve did
not show significant changes in relation to controls
(water or ethanol) at the concentrations evaluated.
No significant changes were detected in the mortality
of flies when compared to the control group (water
or ethanol) at all concentrations. The mechanism of
AChE and BChE enzyme inhibition was determined
using the Lineweaver-Burk plot and the type of
inhibition was determined by the position at which the
lines in the plot intersected. Discussion/Conclusion:
DSC and FTIR analyses detected the interaction
between Poloxamer and naringenin, suggesting that
encapsulation occurred satisfactorily. No toxicity was
detected in the Drosophila melanogaster survival
rate test for pure naringenin (as expected) and also
for the nanoparticles, pointing towards the safety of
the nanoparticles. Naringenin acted as an efficient
inhibitor following a reversible inhibition of the
non-competitive type behavior. In fact, naringenin is
identified as a neuroprotective agent, acting as an
inhibitor of cholinesterase enzymes by catalyzing
the hydrolysis of the neurotransmitter acetylcholine.
Acknowledgments: This work was in part financed by
CAPES/Brazil - Financial Code 001. Authors also thank
CPNq for the support.
 277

Survival rate of Drosophila melanogaster

exposed to Silymarin-loaded nanoparticles and
the ex vivo inhibition of cholinergic enzymes
Souza, Daniela Cristina1; Rossi, Bruna Franzon2; Leimann, Fernanda Vitória1,3;
Gonçalves, Odinei Hess1,3; Appelt, Patrícia1; Ineu, Rafael Porto1

 Post-Graduation Program of Food Technology, Federal University of Technology

1

– Paraná – UTFPR/Campo Mourão, PR, Brazil; 2 Food and Chemical Engineering

Department, Federal University of Technology – Paraná – UTFPR/Campo Mourão,
Campo Mourão, PR, Brazil; 3 Centro de Investigação de Montanha (CIMO), Instituto
Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal.

Introduction: The increased use of nanoparticles in
pharmaceuticals and also in the food industry is in
part explained by the growing demand for bio-based
additives and nutraceuticals by consumers. However,
most naturally occurring bioactive substances present
low water solubility and thus low bioavailability and
nanotechnology may overcome this challenge. This is
the case of silymarin, which is extracted from Silybum
marianum, consisting of a mixture of flavonolignans
with several biological activities. Although silymarin
presents low toxicity, it is important to shed light on
the possible toxicity of silymarin-loaded nanoparticles
and also on how they could affect key metabolic
systems. Objective: To determine the survival
rate of Drosophila melanogaster (DM) exposed to
silymarin-loaded nanoparticles and to evaluate the
activity of silymarin-loaded nanoparticles against the
cholinergic enzymes acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE). Methods: Poloxamer
407 (9 g, Sigma-Aldrich) was used as an encapsulant,
ethanol (375mL, Dinânica) as the solvent, and
Tween80 (0,09g, Dinânica) as surfactant. Silymarin
(0,9g, 95% purity) was acquired from Sigma-Aldrich.
Encapsulant and Tween80 were solubilized in ethanol
for 5 min under gentle stirring at 40°C. Then, silymarin
was added and solubilized for 1 min, then the solution
was sonicated (Fisher Scientific 120, 120W, 1/8” tip)
for 3 min in a pulsed regime (30sec on, 10sec off). The
obtained dispersion was evaporated at 40°C for 24h.
The nanoencapsulated compound was characterized
by Differential Scanning Calorimetry (DSC) and Infrared
Spectroscopy Fourier Transform (FTIR). The effect of
silymarin and silymarin-loaded nanoparticles on DM
was evaluated by a survival rate test (five days, 100
flies each treatment, n=5). AChE and BChE activities
were determined ex vivo (Ellman et al., 1961 with
modifications). Results: The DSC of silymarin showed
peaks at 218.0°C and 281.8°C. In the physical mixture of
silymarin and Poloxamer, the silymarin melting peak
appeared at 290.9°C, while in the nanoparticles this
peak was shifted to 311.6°C. The FTIR signal referring
to C=O (1640 cm-1) present in silymarin was attenuated
in the physical mixture and in the silymarin-loaded
nanoparticles. No significant changes in the mortality
of flies were detected when compared to the control
group (water or ethanol) in all concentrations. The
AChE and BChE activity was significantly inhibited
at 160 and 250 µg.mL-1 for silymarin in water and
ethanol. Nanoparticles did not significantly influence
the activity of the enzymes. Discussion/Conclusion:
DSC and FTIR indicated that interaction occurred
between the Poloxamer and silymarin, suggesting
encapsulation took place as already demonstrated
by our group for curcumin. Microscopy and UV-Vis
analyses (not shown here) confirmed the formation
of the nanoparticles containing silymarin. No toxicity
was detected in the survival rate test in Drosophila
melanogaster for silymarin (as expected) and also for
the nanoparticles, suggesting that encapsulation is a
safe strategy to improve the technological properties
of silymarin. Silymarin is a neuroprotective agent,
acting as an inhibitor of cholinesterase enzymes by
catalyzing the hydrolysis of the neurotransmitter
acetylcholine. It is worth noting that encapsulation
does not affect silymarin activity meaning that
these particles deserve to be further investigated
as a pharmaceutical tool as a reversible cholinergic
inhibitor. Acknowledgments: CAPES/Brasil-Finance
Code 001; and CPNq.
 12 
RISK ASSESSMENT
 279

Animal-Free Safety Assessment of Cosmetics:

a global education and training program
Marigliani, Bianca1; Willett, Catherine2
1
 Research & Toxicology Department, Humane Society International, Brazil.
2
 Research & Toxicology Department, Humane Society International, USA.

Introduction: Great strides have been made in the
ability to use data from non-animal assessment
methods to establish safety of cosmetics and
cosmetic ingredients. However, a lack of familiarity
with these data and processes inhibits uptake, both
for product developers and for those who must
assess the safety of those products. Objective: To
increase confidence and enable regional capacity to
perform non-animal safety assessment of cosmetics
and cosmetic ingredients while addressing the needs
of the regulated and regulatory communities and
other stakeholders. Methods: We facilitated the
creation of the AFSA collaboration, a group of like-
minded industry add non-profit organizations with
expertise in non-animal safety assessment to create
an education and training program based on scientific
knowledge and experience. Results: The Animal-Free
Safety Assessment (AFSA) Collaboration has created
an Education and Training Program that covers the
entire risk assessment process of cosmetics and
cosmetic ingredients in eight modules: Problem
Formulation, Consumer Exposure, In silico Tools,
Exposure-based waiving, Internal Exposure, In vitro
Data Synthesis, and the Overall Risk Assessment.
A ninth module covers the regulatory landscape of
chemicals and consumer products. The program is
built on established principles and processes and
illustrated throughout with case examples from our
members’ experience. Discussion: The first step in
this approach for risk assessment is to understand the
product, its use, and the safety question that needs
to be addressed. The assessment is exposure-led, so
that consumer exposure is the first consideration. If
exposure suggests there is a possibility of risk, hazard
assessment is carried out, followed by refinement
of the exposure estimate if necessary. Finally, all
this information is combined in the risk assessment
which includes estimates of uncertainty. The AFSA
Cosmetics Education and Training course covers
this process and will be freely accessible to all.
Conclusion: Broad dissemination of this program is
intended to familiarize global stakeholders in using
modern, non-animal predictive tools to assess the
safety of cosmetics and cosmetic ingredients, aiming
to better protect people and our planet and hasten
the end of animal testing.
 280

Biomonitoring of occupational exposure

to triazoles and the disruptive effects
of steroidogenesis on androstenedione
biosynthesis in humans
Marciano, Luiz Paulo de Aguiar1; Costa, Luiz Filipe1; Freire, Josiane Oliveira1; Feltrim, Fernando1;
Machado, Simone Caetani2; Silvério, Alessandra Cristina Pupin2; Martins, Isarita1
1
 Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences,
Federal University of Alfenas - Unifal-MG, Gabriel Monteiro da Silva St. 700, 37130-
000, Alfenas, MG, Brazil; 2 University José do Rosário Vellano - UNIFENAS.

Introduction: The use of pesticides to support
agricultural productivity varies between different
crops, in terms of frequency of application, dose and
proximity to harvest time. Fungicides triazoles are
potent inhibitors of the CYP51. In humans, a product
derived demethylation mechanism catalyzed by CYP51
is cholesterol, which is required for the synthesis of
bile acids, mineralocorticoids, glucocorticoids and sex
steroids. Biological monitoring plays an important
role in estimating human exposure, allowing
the health of workers to be assessed. Objective:
Verify the dependence between the presence of
triazoles fungicides in the urine of rural workers
in southern Minas Gerais and the decrease in the
hormone androstenedione. Methods: Cypronazole,
epoxiconazole, metconazole, propiconazole and
triadimenol were quantified in urine samples with
a vortex-assisted liquid-liquid microextraction
analyzed in gas chromatography-mass spectrometry.
The dosage of androstenedione in serum samples
was performed by chemiluminescence. Statistical
analysis of test G were performed with a level of
5% for significance, using the BioEstat version 5.0.
This study was approved by the Ethics Committee of
the Federal University of Alfenas-MG-Brazil (CAAE:
34644620.2.0000.5142). Results: The dependence
between the presence of triazoles in the urine and
the androstenedione dosage equal to or below the
reference value (0.6 ng mL-1) for adult men was tested
(n=30). Then, with only the samples in which triazoles
were detected, another test was performed to assess
whether the relationship could be dose-dependent.
Samples with concentration of triazoles in urine
above the limit of quantification were compared with
samples that presented concentrations between
the detection and quantification limit of the method
and the androstenedione dosage equal to or below
the reference value (n=16). For both cases, an
association was found, p=0.0174 and p=0.0086,
respectively. Conclusion: Therefore, the two
variables are dependent, so there is a concentration-
dependent relationship between the presence of
triazoles in the urine and a trend towards lower
doses of androstenedione in the serum of workers
exposed to fungicides. The next step of the study is
to analyze a greater number of samples from workers
exposed to fungicides in southern Minas Gerais to
assess occupational exposure and check verified
if the association between exposure to triazoles
and decrease in the hormone androstenedione is
increasingly stronger. Acknowledgments: This
study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil
(CAPES) - Finance Code 001.
 281

Buccal micronucleus cytome assay as

a biomarker of genotoxic effect rural
workers exposed to triazole fungicides
Costa, Luiz Filipe1; Marciano, Luiz Paulo de Aguiar1; Freire, Josiane Oliveira1; Feltrim,
Fernando1; Silvério, Alessandra Cristina Pupin2; Martins, Isarita1
1
 Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences,
Federal University of Alfenas - Unifal-MG, Gabriel Monteiro da Silva St. 700, 37130-
000, Alfenas, MG, Brazil; 2 University José do Rosário Vellano - UNIFENAS.

Introduction: The use of triazole fungicides is
widespread in rural areas, due to the favorable
conditions for the proliferation off fungi in different
cultures. These pesticides, despise being efficient,
do not present specificity in relation to the target
organisms, and may cause toxic carcinogenicity and
also endocrine disruption. Objective: The present
study, in a pioneering way, aims to assess the risk
of exposure of rural workers to triazole fungicides,
using the micronucleus test, as a biological indicator
of genotoxic effect, in a sample of the oral mucosa, in
order to assess whether there is correlation between
the results of this test and exposure, through the
investigation of urinary triazoles, subsidizing the risk
assessment and implementation of public polices
to monitor workers exposed this class of pesticides.
Methods: 87 farmers (men) occupationally exposed
to triazole fungicides and 55 women living in rural
areas in the Southern region of Minas Gerais are being
evaluated. In this population, a questionnaire was
applied to collect epidemiological and clinical data,
and the collection of cells from the oral epithelium and
urine are also performed for the relevant analyses. For
the micronucleus test, the slides are being analyzed
under a fluorescence microscope, with 0,001% acridine
orange, staining, observing the presence of cellular
alterations such as the presence of micronucleous,
karyolitic cells, karyorhexis, binucleated and pyknotic.
The investigation of urinary triazoles is being carried
out using gas chromatography- mass spectrometry
with previous miniaturized liquid-liquid extraction.
Statistical analysis was performed with a level of
5% for significance, using the BioEstat version 5.0.
This study was approved by the Ethics Committee
of the Federal University of Alfenas-MG-Brazil
(CAAE: 34644620.2.0000.5142). Results: Based on
the recommendations of national and international
validation guides, the method used showed linearity
in the range of 10 to 200 µg L-1 for cyproconazole,
metconazole and triadimenol and 30 to 200 µg L-1
for epoxiconazole and propiconazole. Of the 86 urine
samples analyzed, 2 had more than one pesticide, 10
samples had concentrations between 10 µg L-1 and
70 µg L-1 and 27 samples were between the detection
limit (2 µg L-1 for cyproconazole, metconazole
and triadimenol; 5 µg L-1 for epoxiconazole and
propiconazole) and quantification (10 µg L-1 for
cyproconazole, metconazole and triadmenol; 30
µg L-1 for epoxiconazole and propiconazole). Partial
results showed statistical differences for kariolytic,
binucleated, pyknotic and karyorrhexis cells (p < 0,05)
more frequently in men (n= 43) when compared to
women (n = 19), both living in rural areas. In addition,
a higher frequency of karyorrhexis cells was found in
men with urinary triazoles above the LQ (n = 8), when
compared to men with urinary triazoles below the
LQ (n= 11). Conclusion: At the end of the study, it will
be confirmed, or not, the hypothesis that exposure
to triazole fungicides increases the risk of genetic
mutations, which can trigger serious diseases, thus
contributing to the area of occupational health, in
the prevention of intoxications from the occupational
exposure. Acknowledgments: This study was financed
in part by the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior - Brasil (CAPES) - Finance
Code 001.
 282

Changes in biomarkers of exposure and

potential harms in adult smokers following
180 days of gloTM tobacco heating product use
Esteves, Iuri1; Zebele, Patricia1; Gale, Nathan2; Hardie, George2;
McEwan, Michael2; Gaca, Marianna2; Goodall, Sharon2
1
 BAT Brasil, a member of British American Tobacco, Republica do Chile 330, Rio de Janeiro,
Brazil; 2 British American Tobacco (Investments) Ltd, R&D, Southampton, SO15 8TL, UK.

Background/Introduction: Cigarette smoking is
indicated as one of the risk factors for a number
of serious diseases, including lung cancer and
cardiovascular disease. It is generally accepted that
smoking-related diseases are associated to exposure
to the thousands of chemical constituents created
during the process of burning tobacco. There has
been significant growth in the number of smokers
using next generation tobacco and nicotine products
including e-cigarettes and Tobacco Heating Products
(THPs). As THPs heat tobacco at temperatures of 200-
300°C instead of burning (900°C), the toxicants within
the aerosol are greatly reduced (~95%) compared to
cigarettes and hold promise as reduced risk products.
Using various guidelines for assessing the modified risk
status of a novel tobacco products, BAT has recently
published a 9-step framework proposing the use of
pre-clinical, clinical and population studies to assess
the reduced risk potential of THPs at the individual and
population level. Objective: To investigate changes
over a six-month period in biomarkers of exposure
and potential harm in smokers switching to the THP
gloTM or quitting. Methodology: An ambulatory four-
arm switching study enrolling 500 participants was
carried out at 4 UK clinical sites conducted with the
ethical principles of the Declaration of Helsinki, Good
Clinical Practice. Regular smokers were randomised
to either continue to smoke their own brand
cigarettes, or to switch to using gloTM for the period
of the study. A further group, (cessation) consisted
of regular smokers who had intentions to quit and
were provided with assisted smoking cessation .
A final control group was made up of participants
who have never smoked and were followed during
the study period. Participants attended the clinic
approximately every 30 days for one year, though the
never smokers attended less frequently. Breath, blood
and urine were collected and analysed for a range of
exposure and potential harm biomarkers, in addition,
physiological, questionnaire and safety measures
were also performed. Clinical Trial Registration:
https://www.isrctn.com/ISRCTN81075760. Results:
In smokers who switched to using gloTM, exposure to
harmful cigarette smoke toxicants were significantly
reduced, in many cases to the same levels observed
in smokers who quit smoking (cessation), or in
never-smokers. The reduction in these exposure
biomarkers were rapid and sustained for the 6-month
period. In cigarette smokers who switched to using
gloTM, a number of biomarkers of potential harm
were significantly changed in a favourable direction
including biomarkers associated with cardiovascular
disease and lung cancer. Discussion/Conclusions:
These data demonstrate gloTM has much lower
toxicant levels, resulting in significantly lower
levels of biomarkers of exposure to toxicants and
a favourable change in a number of biomarkers of
potential harm compared to cigarettes when used by
smokers over a six-month period. These data suggest
that the negative health impacts of cigarette smoking
may be reduced in smokers who completely switch to
using gloTM. This conclusion is consistent with the UK
Committee on Toxicology which found that “it is likely
that there is a reduction in overall risk to health for
conventional smokers who switch to heat-not-burn
tobacco products.” Acknowledgments: The authors
wish to acknowledge Covance (Leeds, UK), Celerion
(Belfast, UK), Richmond Pharmacology (London,
UK) and Simbec Orion (Merthyr Tydfil, UK) for their
management and professional conduct of the clinical
study.
 283

COVID-19 and susceptibility of firefighters

exposed to smoke: A scope review
Silva, Rafael Araújo; Marciano, Luiz Paulo de Aguiar; Costa, Luiz Filipe; Nunes,
Rafaella Ferreira Nascimento; Sakakibara, Isarita Martins

Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences, Federal
University of Alfenas - Unifal-MG, Gabriel Monteiro da Silva St. 700, 37130-000, Alfenas, MG, Brazil

Introduction: COVID-19, caused by the SARS-CoV-2
virus, has been impacting a large number of people due
to high mortality and variable susceptibility among
individuals. It is crucial to assess chemical-exposed
groups that may be more predisposed to severe
COVID-19. Firefighters are often exposed to a range
of occupational hazards due to the broad spectrum
of activities and in the COVID-19 pandemic, these
professionals are considered essential. As a result,
firefighters were more exposed than the general
population. One of the most frequent activities in the
routine of firefighters is fighting fires, whether urban
or forest. In these situations, workers are exposed to
high levels of toxic substances and low oxygen supply.
Objective: In this context, the objective of this review
is to explore the current literature on a possible
relationship between susceptibility (severe signs and
symptoms) to COVID-19 and the occupational exposure
of firefighters to smoke. Methods: The scoping review
protocol used followed the methodology proposed
by the Joanna Briggs Institute (JBI). Published and
unpublished studies were included. The databases
used were MEDLINE (via Pubmed), CINAHL, Scopus,
LILACS, Embase and Web of Science. Regarding gray
literature, they were consulted via Google Scholar or
relevant research organizations/groups. Studies in
English, Spanish or Portuguese were included. Two
independent reviewers performed the screening, data
extraction and selection of articles. A third reviewer
was used in cases where there was disagreement
regarding the inclusion of a study. Results: Articles
focusing on firefighters or smoke exposure in the
context of susceptibility to COVID-19 were included
in this review. As it is a current topic, studies are
still limited, so studies that evaluated respiratory
infections in the general public from exposure to
smoke were also reviewed. It is understood that the
general population may present different health risks
and lower exposures when compared to firefighters,
who generally have physical fitness and training,
but there is no availability of a similar occupational
group. It has been observed in some studies that
increased concentrations of PM2.5 in atmospheric
air due to fires, especially wildfires, have correlated
with increased COVID-19 cases and deaths in some
locations. Conclusions: The results obtained allowed
us to conclude that exposure to smoke from forest
fires can cause lung irritation, inflammation and
alteration of immune function, which can increase
susceptibility to respiratory infections such as
COVID-19. In addition, some studies already show a
possible relationship between exposure to smoke and
cases of COVID-19. This information is important for
the development of policies to protect firefighters
who often work in conditions of high smoke exposure.
 284

Determination of urinary triazoles as

reliable biological indicator for the
application in coffee growers
Marciano, Luiz Paulo de Aguiar1; Costa, Luiz Filipe1; Freire, Josiane Oliveira1; Feltrim, Fernando1;
Machado, Simone Caetani2; Silvério, Alessandra Cristina Pupin2; Martins, Isarita1
1
 Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences,
Federal University of Alfenas - UNIFAL-MG, Gabriel Monteiro da Silva St. 700, 37130-
000, Alfenas, MG, Brazil; 2 University José do Rosário Vellano - UNIFENAS.

Introduction: The harmful effects of pesticides on
human health have been increasingly studied. The
lack of action selectivity represents a risk for man and
other forms of life present in the environment. Brazil
is one of the biggest consumers of pesticides in the
world and more than 90% of rural workers depend
on these substances for pest management. Triazole
fungicides represent the second largest group of
pesticides used in the southern region of Minas Gerais.
These fungicides are potent inhibitors of CYP51, its
main mechanism of action, thus actively interfering
with the production of steroid hormones in humans
and other health hazards. Currently, occupational
exposure monitoring is performed through biological
parameters, called bioindicators, which can be
measured and evaluated expressing a correlation with
exposure. Objective: Determination of triazoles in the
urine of rural workers for application in biomonitoring
of occupational exposure. Methods: Cypronazole,
epoxiconazole, metconazole, propiconazole and
triadimenol were chosen utilizing as criteria the
most used in coffee growing in the southern region
of Minas Gerais, Brazil. This study was approved by
the Ethics Committee of the Federal University of
Alfenas-MG-Brazil (CAAE: 34644620.2.0000.5142).
Sample preparation was optimized with 1 mL of urine
and 100 μL β-glucuronidase enzyme (diluted 1: 28 in
0.5 mol L-1 acetate buffer, pH 5.0). The sample was
incubated at 38 ºC for 12 h. At room temperature, 20 µL
tebuconazole-(tert-butyl-d9) internal standard was
added, followed by the addition of 1 mL of acetonitrile,
2 mL of phosphate buffer pH 7.0 and 200 μL of toluene,
vortexed for 1 minute, then centrifuged at 1134 g for
5 minutes. Next, 200 μL of the toluene fraction was
collected, dried at 30 ºC and resuspended in 100 μL of
toluene and analyzed by gas chromatography–mass
spectrometry. Statistical tests were performed with a
level of 5% for significance, using the Computational
System R. Results: Confidence parameters were
evaluated and linearity (coefficients of determination
higher than 0.98), precision (below 15% for quality
controls and 20% for lower limit of quantification),
accuracy and residual effect were satisfactory
according to the validation guidelines. The calibration
curve ranged from 10 to 200 µg L-1 for cyproconazole,
metconazole and triadimenol analytes and from 30
to 200 µg L-1 for epoxiconazole and propiconazole.
Linear regression model was significant (p <0.0001)
with normality (Shapiro-Wilk), independence (Box-
Pearce) and homoscedasticity (Breusch-Pagan) were
verified and non-significant. To show the applicability
of the method, samples of rural workers who reported
exposure to triazole fungicides were analyzed (n=10).
Epoxiconazole was found in five samples (70.0;
49.99; 47.74; 43.67 and 40.18 µg L-1); Triadimenol
in one sample (45.46 µg L-1); and four samples with
cyproconazole, a sample was quantified (14.98 µg
L-1) and three between the limit of detection and
the lower limit of quantitation (2 µg L-1 - 10 µg L-1).
Conclusion: With these data it is possible to conclude
that the validated method permitted to determine
urinary triazoles in farmers exposed to these
fungicides and the bioindicator of the internal dose is
a reliable tool to assess the occupational exposure.
Acknowledgments: This study was financed in part
by the Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior - Brasil (CAPES) - Finance Code 001.
 285

Dispersive liquid-liquid microextraction

to determinate parabens in body
cream by liquid chromatography
Ramos, Thalita da Silva1; Barbosa, Alyne Maria da Costa1; Rath, Susanne2; Martins, Isarita1
1
 Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences,
Federal University of Alfenas - Unifal-MG, Gabriel Monteiro da Silva St. 700, 37130-
000, Alfenas, MG, Brazil; 2 Institute of Chemistry, Department of Analytical Chemistry,
University of Campinas, P.O. Box 6154, 13084-971 Campinas, SP, Brazil.

Background: The p-hydroxybenzoic acid esters, or
parabens, are preservatives in foods, medicines and
personal care products, cosmetics and perfumes
(PCPCP), belonging to the class of emerging
contaminants that have the ability to promote
xenoestrogenic and oncogenic effects. Studies in
the literature have shown that they act as endocrine
disruptors, as they mimic the functions of estradiol,
and have been associated with the incidence and
increase of breast cancer in animals and humans. Thus,
the ability to interfere with the endocrine system is
the main question to be clarified regarding the safety
of parabens in human exposure. Objective: In this
context, the objective of this study is to develop an
analytical method, applying Dispersive Liquid-Liquid
Microextraction (DLLME) technique for the sample
preparation of body cream and analyze performed by
liquid chromatography, with UV detection. Methods:
To development method, the best conditions of
extraction and detection/ quantification were
evaluated. Blank sample was diluted in Milli-Q water:
methanol (4:1, v/v) and centrifuged. Subsequently, the
supernatant was extracted with acetone (dispersing
solvent): chloroform (extracting solvent) (2:1, v/v).
The extract was analyzed by liquid chromatography
using a chromatographic NST 08 100 A (250 mm x 4.6
mm) column, in isocratic mode, at 35ºC, with water:
acetonitrile (70:30, v/v) as mobile phase in a flow
of 1 mL min-1, and detection at 254 nm. The range
evaluated was between 60 to 480 µg mL-1, covering
the Brazilian limit (0.4%, m/m, to isolated paraben). To
assess the applicability of the method, four samples
of body cream from different brands were analyzed.
Results: The method was linear and showed precision
and accuracy. Average concentration of parabens in
the four samples analyzed was 0.011% m/m for MeP,
and below LoQ for EtP and PrP (LoQ: 0.0058% and
0.0051% m/m, respectively). Discussion/Conclusion:
The results demonstrated that the method is very
efficient in recovering and improving the detectability
of analytes studied. DLLME was efficient to prepare
the samples and pre-concentrate the analytes, as
well as being a practical and fast sample preparation
method, respecting the Green Chemistry principles, as
the decrease the spend of toxic solvents. The method
demonstrated satisfactory analytical performance
to be applied as a potential tool to analyze parabens
from body cream. Acknowledgments: This study
was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil
(CAPES) Finance Code 001.
 286

Evaluating the cytotoxicity of

quantum dots using two different
keratonocyte-cell based systems
Souza, Isisdoris Rodrigues1; Cruz, Juliana Varella1; Thá, Emanoela Lundgren1;
Gagosian, Viviana Stephanie Costa1; Araujo-Souza, Patrícia Savio1; Cestari,
Marta Margarete1; Faustman, Elaine M.2,3; Leme, Daniela Morais1,4
1
 Graduate Program in Genetics, Department of Genetics, Federal University of Paraná
(UFPR), Curitiba, PR, Brazil; 2 Department of Environmental and Occupational Health
Sciences – University of Washington, Seattle, WA, USA; 3 Institute for Risk Analysis and
Risk Communication – University of Washington, Seattle, WA, USA; 4 National Institute for
Alternative Technologies of Detection, Toxicological Evaluation and Removal of Micropollutants
and Radioactives (INCT-DATREM), Institute of Chemistry, Araraquara, SP, Brazil.

Background/introduction: Quantum dots (QDs) -
fluorescent types of semiconductor nanoparticles
with unique electrical and optical properties – have
clinical, electronic and energy applications. However,
QDs uses raise health concerns due to toxic effects
previously reported, of which some are related to
surface chemistry of this nanoparticle. Objective: The
objective of this study was evaluate the cytotoxicity of
a cadmium-selenium containing QD (ITK™ – carboxyl
functional group) on keratinocyte-based systems
because the skin is a potential route of exposure
especially in the occupational setting. Methods:
Immortalized human keratinocytes (HaCaT cell line)
and neonatal primary keratinocytes (HEKn) were
exposed for 24 h to different concentrations of QDs
(2.5, 5, 10, 20 and 40 nM), and the cytotoxicity was
measured by the MTT assay. Intracellular reactive
oxygen species (ROS) were also quantified (by
labeling with H2DCFDA and quantifying fluorescence
by flow cytometry) to verify whether the generation
of ROS is related to cytotoxicity. Results: The tested
QD caused cytotoxicity in both keratinocyte-based
systems; however, the level of cytotoxicity differed
between the two systems. While HaCaT showed an IC50
(half-maximal inhibitory concentration) of 32.75 nM,
the HEKn showed a lower value of IC50 (23.14 nM). A
significant increase in ROS generation was not verified
under the conditions tested. Discussion/Conclusion:
The values of IC50 of HaCaT and HEKn, demonstrate
that the primary cells are more prone to the effects
of QDs than the cell line. The cytotoxicity observed
for this nanoparticle is probably not mediated by
oxidative stress, which is an important mechanism of
QD-induced toxicity; however, additional investigation
is needed to elucidate this hypothesis. In conclusion,
our data showed that ITK™ QD is cytotoxic for skin
keratinocytes. This result is in concordance with
the cytotoxicity previously verified to human neural
progenitor cells. Additionally, these findings draw
attention to the correct choice of the keratinocyte-
based test system because cell line- and primary
cell-based systems have different sensibilities, and
it can affect the determination of safety levels and
protection of human health. Acknowledgments:
CAPES, CNPq.
 287

Exposure driven data generation strategies

for dietary and non-dietary risk evaluation
of crop protection products to inform safety
and minimise unnecessary animal testing
Catalano, Shadia M.I.1; Pais, Mariana Castello Novo2; Latorre, Andreia Oliveira3;
Faria, Patricia Miranda3; Soares, Daniel4; Freeman, Elaine5
1
 Corteva Agriscience, Barueri, SP, Brazil; 2 Syngenta Crop Protection, Sao
Paulo, SP, Brazil; 3 BASF SA, Sao Paulo, SP, Brazil; 4 Bayer SA, Sao Paulo, SP,
Brazil; 5 Exponent International Limited, Washington, DC, USA.

Human health risk assessment (HHRA) involves
characterization of both exposure and hazard.
The traditional approach defines the hazard from
mammalian toxicity studies, many of which do not
contribute data to the HHRAs, only then evaluating
exposure to determine risk. To address this challenge,
we have explored ways of predicting exposure
based primarily on the use scenario and comparing
the exposure to reference dose values derived
by international regulatory agencies to identify
mammalian toxicity studies that are relevant to HHRA.
The premise is that exposure can be predicted based
on the use scenario, regardless of the chemistry or
pesticidal mode of action. The use scenarios were
defined based on registered agricultural uses for
fungicides, insecticides, and herbicides as they
are applied in USA and Europe. For human dietary
exposure, values were based on existing residue data
for substances with comparable use on the same
or similar crops. To provide a point of comparison
for the exposure predictions, data were collated
for acute, chronic and occupational reference dose
values derived by US EPA, JMPR and EU Commission
that are publicly available. The exposure predictions
and range of hazard endpoints were compared using
the ILSI HESI Risk 21 risk matrix plots (https://risk21.
org/) in order to visualise and contextualise the level
of potential concern for the exposure prediction. In
addition, an approach is proposed to categorise the
likelihood of acceptability of risk based on where the
exposure sits relative to the distribution of reference
dose values. The approaches proposed in this study
allow for exposure prediction based on the Good
Agricultural Practice (GAP) in conjunction with the use
of existing hazard data for crop protection products in
order to make an initial determination on acceptability
of risk and to identify key studies that are required for
HHRA and also opportunities for study waivers. This
work is sponsored by Crop Life International Exposure
Based Assessment Team (CLI EBT) to Exponent and
the manuscript is accepted as Parsons et al., 2021
in Critical Review in Toxicology. Acknowledgments:
Wolf D (Syngenta Crop Protection, USA), Aggarwal
M (Corteva Agriscience, USA), Rupprecht JK (Bayer,
USA), Mehta J (ADAMA, UK), McEuen S (FMC, USA),
Lautenschalaeger D (Bayer, Brazil), Ramanarayanan
TS, Weidling R (Exponent US), Gill P (Exponent
International), Byron N (Exponent International),
Williams G (Exponent International).

GHS classification of chemicals frequently
used in laboratories: A risk assessment
Chemical Safety is achieved when all activities
involving chemical products are carried out in a
way that guarantees the safety of human health
and environment. It is a science composed of many
scientific and technical components, including
toxicology, ecotoxicology and the chemical risk
assessment process. Chemicals are part of our day-
to-day and are present in any analysis laboratory. The
UN, aware of it, in order to harmonize the criteria for
classifying and communicating hazards worldwide,
has developed the GHS (Globally Harmonized System
of Classification and Labeling of Chemicals), which
has been adopted and become mandatory in Brazil for
any chemical product used in the workplace. Within
this context, the objective of this work is identifying
the main chemical products used in laboratories,
discussing the GHS classification of these compounds
and evaluating the knowledge of professionals who
work in laboratories regarding the dangers and
potential risks these products present. For this survey,
there carried out an exploratory research using the
BABBIE methodology, in a group of 28 laboratory
workers, applied in an anonymous online format.
The group answered three open questions: (1) What
type of laboratory do you work in?; (2) What is(are)
the main chemical product(s) used in your daily work?;
(3) Do you know if the mentioned product is classified
as dangerous according to the GHS?. According to
the answers of the first question, more than 50%
of the interviewees worked at a physical-chemical
laboratory, while the other interviewees diversify
at school laboratory, research and development,
organic and inorganic chemistry, toxicology and
microbiology and parasitology. The second question,
which allowed an answer with up to 3 components,
provided the identification of the main chemical
products used in these laboratories, the most present
288
Silvério, Kérolyn Aparecida; Pinheiro, Fabriciano
Intertox.
ones were Ethanol (29%), Methanol (18%), Hexane
and Hydrochloric Acid (14%). The last question
resulted in an index of 46.4% of then answered that
the chemicals used were classified according to the
GHS, another 28.6% ones replied yes was classified,
but they could not say if it was according to the GHS
, while almost 10% of them informed they had never
observed the issue of hazard classification and almost
15% reported that the chemicals handled were not
classified as hazardous. Based on that, it was possible
to observe that chemicals classified as hazardous
by the GHS are present in a large proportion in
laboratory environments and many of them can even
pose significant risks to human health, such as skin
corrosion or damage to the central nervous system
and optic nerves, if handled without proper protection.
The survey also showed a concern about knowledge
and training about GHS in Brazil, given that some of
the participants were not able to identify the danger
communications that should be easily visible and
adhered to the bottles of handled chemical products.
The results of this research raise the discussion
that such information may not has been transmitted
to professionals during their academic training or
even through training provided for in legislation.
Regardless of the reason, this knowledge gap
increases the risk of accidents in laboratories, since
safety precautions may not be carefully followed due
to the lack of danger knowledge. Thus, it is necessary
to narrow the approach to the safe management of
chemical products, the dangers and potential risks
that professionals from Brazilian laboratories may be
involved, encouraging the discussion of the topic in
educational institutions, in order to contribute to the
strengthening of the chemical safety culture in public
and/or private institutions. Keywords: GHS. Chemical.
Hazards and Risks
 289

How can nitrosamine risk assessments

be supported by in silico systems
and data sharing initiatives?
Waechter, Fernanda; Kocks, Grace; Avila, Carolina Martins; Burns, Michael J.; Ponting,
David J.; Tennant, Rachael E.; Oliveira, Antonio Anax F.; Heghes, Crina

Lhasa Limited.

Introduction: Nitrosamines are a class of potentially
mutagenic impurities which were found to be present
above the permitted limits in several batches of drug
products in the past few years, leading to global
recalls of several drugs. As a result, regulatory
agencies have issued guidance for industry to
ensure all drugs on the market will be assessed for
the risk of nitrosamines, however this is a complex
evaluation needing a range of different aspects to be
considered. Objective: Here we aim to evaluate how
in silico systems and data sharing initiatives can be
applied to a nitrosamine risk assessment. Methods:
The assessment was done by using in silico models
and proprietary data shared within a consortium.
Results/Discussion: A nitrosamines risk assessment
should start by considering all the potential sources
of nitrosamines within the manufacturing and storage
of a drug product, including the drug substance,
excipients, and their interaction. Nitrites can be
found within excipients, and under specific conditions
may form nitrosating agents that can react with
vulnerable amines in drug formulations, potentially
forming nitrosamines. A data sharing initiative in
which companies share nitrite levels with each other
can help in this stage. Such a database containing
typical results could reduce duplicate testing by the
industry and increase scientific knowledge. If there
is a theoretical risk of forming a nitrosamine, then
a risk assessment should follow as outlined by ICH
M7, the guideline for the assessment and control of
mutagenic impurities in drug products. This guideline
describes that in silico systems can be used to predict
the mutagenicity potential of impurities, in lieu of
performing the Ames test, if two complementary
systems are used – such as Derek and Sarah Nexus.
However, in silico systems may not always be
conclusive and performing the Ames test may be
necessary. Such a test is time-consuming, requires
resources, and can be challenging for low level
impurities which are not easily obtained in their pure
form. An alternative to overcome these challenges are
data sharing initiatives where different companies
can share Ames test results for their nitrosamines and
have access to results shared by other companies.
This then avoids rework and allows for the science
to advance quicker. Lhasa Limited, as a not-for-profit
organisation, acts as an honest broker to allow the
industry to benefit from such initiatives. Finally, if a
nitrosamine is found to be mutagenic, the acceptable
limit must be defined, followed by assessing the
levels of the impurity. Purge calculations are a way of
assessing this through in silico systems, which avoid
the need for analytical procedures to be developed and
validated. Purge assessments are described by ICH M7
and can also be used for nitrosamines. Conclusion: If
the risk is mitigated with either approach – finding
that nitrite levels are not of concern, that the
nitrosamine is not mutagenic, or that it is sufficiently
purged during the synthetic process – the need for an
analytical test is avoided. Therefore, by applying the
use of in silico systems and data sharing initiatives,
unnecessary testing can be avoided using a science-
based and risk-driven approach.
 290

Human health risks assessment by iron mining

dam failure (VALE S.A) in Brumadinho-MG
Domingos, Líllian Maria Borges; Castilhos, Zuleica Carmen

CETEM- Centre for Mineral Technology.

Field work and literature data show that the tailings
disposed on the soil after the failure of Dam I (VALE
S/A), in Brumadinho, are rich on iron, silica, aluminum
and manganese; they are also composed by fine
particles (from 10 to 100 µm), ultrafine particles
(from 10 at 1 µm) and colloidal (less than 1 µm)
ones. Inhalation of particles and their consequent
deposition in the airways is an important risk
parameter for human health. This work presents the
conceptual model of human environmental exposure
to iron ore mining tailings after dam failure and the
preliminary estimate results of particle deposition in
the lung by inhalation exposure using MPPD Multiple-
Path Particle Dosimetry Model. After the selection
of values for the input parameters based on primary
data and literature, the preliminary results indicated
that the concentration, the density and size of the
particles are important factors for the differential
deposition of the particles in the respiratory system.
It is important to emphasize that the decrease in
uncertainties in the results obtained in this work,
from a preliminary approach, will be possible from the
availability of air quality data in the monitored areas
in Brumadinho. This information will be forwarded to
local institutions and other stakeholders as a subsidy
for discussions on the environmental impacts of the
iron mining dam I failure.

Occurrence and dietary exposure to
histamine by the Brazilian population
Diniz, Fabiana Barbosa1; Braga, Douglas Evangelista2; Custódio, Flávia Beatriz1; Gloria, Maria Beatriz Abreu1,2
Background: Histamine is a biogenic amine naturally
present in different foods or produced due to the
activity of histidine decarboxylase from added or
contaminating microorganisms. At low concentrations
histamine is a vasoactive and neuroactive substance,
which is involved in three primary areas of the body,
e.g., immune system function, digestion and brain/
nervous system as a neurotransmitter. However, at
high concentrations, histamine can cause intolerance,
with symptoms like itching, hives, sneezing, watery
eyes, asthma, headaches, abdominal pain, diarrhea,
tachycardia, and hypotension. The European Food
Safety Authorities (EFSA), in 2011, established no
observed adverse effect levels (NOAEL) for histamine
per meal for normal (50 mg) and for histamine
intolerant (0 mg) individuals. Objective: To investigate
the dietary exposure to histamine by the Brazilian
population. Methods: The levels of histamine
in different Brazilian foods were compiled from
literature and used to calculate dietary exposure. The
food consumption profile of the Brazilian population
was obtained from the Family Budget Survey (POF)
2017-2018 (IBGE). The daily portion of each food
with detectable levels of histamine was considered.
The risk characterization was evaluated by the
relationship between Brazilian daily acute exposure
to histamine from the consumption of the main
histamine containing foods and the acute reference
dose per meal (50 mg). Results: The Brazilian foods
containing higher histamine levels were in mg/100 g:
291
1
 Faculty of Pharmacy; 2 Veterinary School, UFMG.
grated cheese (115); Parmesan cheese (65.5); soy sauce
(39.5); Gouda cheese (19.5); Italian sausage (18.5);
eggplant (12.5); bean sprout (8.8); tuna with tomato
sauce (8.4); canned grated tuna (5.6); sausage (4.7).
The total intake of histamine by Brazilian population
can reach acute toxic levels. Cheese was the main
contributor to total histamine intake. Considering
the consumption of grated cheese and the other
foods, the total histamine intake was 94.9 mg. If it the
consumption of Parmesan cheese was contemplated
instead of grated cheese, the total histamine intake
(67.3 mg) would still be above the acute reference
dose. Discussion/Conclusion: Histamine legislation
is only available for Scombroid fish (tuna, bonito)
and other histamine formers. However, fermented
products including cheese, sausages and soy sauce,
as well as germinated products were the food items
which showed higher levels of histamine. Cheese,
mainly grated and Parmesan, accounted for a high
intake of histamine. Histamine intolerant individuals
must be careful regarding histamine free food
products. The NOAEL can be easily reached in a meal,
especially when fermented products are present. One
must also consider that alcoholic beverages (wine
and beer), which were not included in the survey, also
contribute with significant amounts of histamine
and can potentiate its toxic effects. Acknowledge:
We thank Capes, CNPq and Fapemig for the financial
support.

Occurrence and exposure to aspartame from
soft drinks by the Brazilian population
Sousa, Roberto Cesar Santos; Custódio, Flávia Beatriz; Gloria, Maria Beatriz A.
Graduate Program in Food Science, Faculty of Pharmacy, UFMG, Belo Horizonte, MG.
Background: The prevalence of obesity and other
diet-related chronic noncommunicable diseases
is increasing worldwide. In this scenario many
consumers started to choose foods containing
sweeteners instead of sugars, one of the largest
contributors to the high prevalence of these diseases.
Non-nutritive sweeteners are used in small amounts
to reduce the caloric and sugar contents of foods and
beverages. One of the main sweeteners in the market
is aspartame, a synthetic one 200 to 300 times
sweeter than sucrose and with a low caloric value.
Aspartame has been incorporated into over 6,000
products available in the market, mainly beverages.
It is therefore consumed by millions of people across
the world. Objective: With the increasing availability
of products containing aspartame in its formulation
in the market, the objective of this study was to
investigate the exposure of the Brazilian population
to this sweetener through consumption of soft
drinks. Methods: Soft drinks sold in wholesale and
retail stores in the city of Belo Horizonte, Minas
Gerais, Brazil were included in this study. The
labels of these products were analyzed to obtain
information regarding the brand, presence or absence
of aspartame, and the amounts declared (when
available). Exposure was estimated using the mean
levels of aspartame declared in labels and the mean
consumption of beverages divided by the mean body
weight of Brazilian population. The food consumption
profile and the mean body weight of the Brazilian
population (≥10 years old) was obtained from the
Family Budget Survey (POF) 2017-2018 (IBGE). The risk
characterization was evaluated by the relationship
between Brazilian daily exposure to aspartame
from the consumption of soft drinks and the chronic
292
toxicological reference value, described for aspartame
as the Acceptable Daily Intake (ADI) of 40 mg/kg
body weight for neurological damage related to the
generated metabolites: phenylalanine, aspartic acid
and methanol. Results: Overall, 15 different types of
soft drinks from eight different brands were available
in the market. Among them, only six (40%) had
aspartame. The declared aspartame contents varied
from 0 to 0.35 mg/mL, with an average of 0.1 mg/mL.
All the samples were within the limit established in
the legislation for beverages with total replacement
of sugars, which corresponds to 0.75 mg/mL. All
risk assessment scenarios for aspartame showed
that the daily consumption of aspartame through
soft drinks is not greater than 5% of the ADI for this
sweetener, even in the most pessimist consumption
scenario. The subpopulations with the highest
aspartame intake were adolescents (by age group),
male (by gender) and residents in the South of Brazil.
Discussion/Conclusion: Soft drinks are the non-
alcoholic beverages widely consumed in the modern
diet and aspartame is an important sweetener used
in them. Low-sugar or sugar-free beverages account
for about 30% of the total non-alcoholic beverage
market. Consequently, this has led to an increased use
of sweeteners in product development. This whole
scenario requires the protection of consumers’ health
by carrying out a risk assessment. The estimated
daily intake of aspartame by the Brazilian population
showed levels below the ADI indicating low risk. The
results of the present study are in agreement to other
works in the scientific literature. Acknowledgement:
This study was financed in part by the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), Brasil – Finance Code 001.
 293

Permitted Daily Exposure (PDE) from preclinical

studies of ginkgo biloba dry extract
Lamb, Liliane Weber Bolfe1,3; Rodrigues, Gabriela Zimmermann Prado2; Saraiva, Thalia Emmanoella
Sebulsqui1; Souza, Douglas1; Berna, Gabriel da Costa1; Garcia, Ana Letícia Hilário2; Bertoldi,
Fernando4; Kayser, Juliana Machado1,2,3; Ferreira, Julia Gabriele de Jesus1,3, Veiverberg, Andriele1;
Marco, Mariana1; Gehlen, Günter2,4; Betti, Andresa Heemann1,3; Mattos, Cristiane Bastos1,3
1
 Bioanalysis Laboratory, Health Sciences Institute, Universidade FEEVALE, Novo Hamburgo-
RS, Brazil; 2 Compartive Histoly Laboratory, Health Sciences Institute, Universidade
FEEVALE, Novo Hamburgo-RS, Brazil; 3 Postgraduate Program on Toxicology and
Analytical Toxicology, Universidade FEEVALE, Novo Hamburgo, Brazil; 4 Postgraduate
Program on Environmental Quality, Universidade FEEVALE, Novo Hamburgo, Brazil.

In Brazil, the publication of RDC 301/2019 and the
14 Normative Instructions became the regulatory
framework for Good Manufacturing Practices.
This regulation establishes general guidelines
for good drug manufacturing practices, in which
ANVISA requires companies to have a Quality Risk
Management that should include precautions to
mitigate cross-contamination, in addition to a
toxicological evaluation of all active ingredients
present in multipurpose manufacturing areas. Within
this context, the determination of the Permitted Daily
Exposure (PDE) emerged, defined as the specific
dose of a substance that is unlikely to cause adverse
effects to an individual exposed daily during lifetime
based on data from preclinical and clinical studies.
Thus, this study aimed to determine the PDE of the dry
extract of Ginkgo biloba leaves, utilized as an input
in a pharmaceutical solid dosage form administered
orally in preclinical studies in mice. Ginkgo biloba
was chosen because this plant is the fourth
greatest in reported adverse effects, which include
gastrointestinal disorders, allergic skin reactions,
hypotension, palpitations, hemorrhages, among
others. For that reason, acute oral toxicity tests were
performed based on the OECD 423 guideline, exposing
three male and female mice to a single dose of 2000
mg/Kg body weight of Ginkgo biloba dry extract and
monitoring them for 14 days. In addition, the repeated
dose toxicity test was performed based on the OEDC
407 guideline, with the administration of Ginkgo biloba
dry extract at doses of 120, 240 and 480 mg/Kg to 10
mice in each dosing group and evaluated for 28 days.
Finally, a genotoxicity assessment was carried out
based on comet and micronucleus assays. The former
evaluated the DNA damage index in lymphocytes, and
the latter determined the polychromatic immature
lymphocytes of the bone marrow and treatment
differentiated polychromatic and normochromic
erythrocytes. The results indicated that the dry extract
of Ginkgo biloba leaves causes low acute toxicity, with
no liver toxicity or alteration of blood glucose levels
in males and females. Changes in weight gain were
not demonstrated in the groups tested, but in males,
the food intake was lower during the first week of
treatment at the highest dose of extract (480 mg/
Kg). The hematological parameters were not altered
in males, whereas in females receiving 480 mg/Kg
there was a reduction of leukocytes and lymphocytes
and an increase of neutrophils. The lipid profile of
males was not altered by the doses administered,
whereas females treated with 480 mg/kg presented
higher total cholesterol levels. In the genotoxicity
tests, no significant difference was observed in the
micronucleus and comet assays. The results found
in this work permitted calculation of the PDE, which
was estimated at 0.1 mg/day; when correlating to the
maximum residual concentration of 250 µg/mL, it is
concluded that the current cleaning process is safe
and does not need to be re-evaluated.
 294

Thyroid and sex hormonal profile and

manganese exposure in pregnant women from
a Brazilian birth cohort: preliminary results
Santos, Nathália Ribeiro1,2; Bah, Homegnon Antonin Ferréol1,3; Gomes-Júnior, Erival Amorim1,4;
Martinez, Victor Otero1,2; Costa, Daisy Oliveira1,2; Menezes-Filho, José Antonio1,2,3,4
1
 Laboratory of Toxicology, College of Pharmacy, Federal University of Bahia; 2 Postgraduate
Program of Pharmacy, College of Pharmacy, Federal University of Bahia; 3 Postgraduate
Program of Collective Health, Institute of Collective Health, Federal University of Bahia;
4
 Postgraduate Program of Food Science, College of Pharmacy, Federal University of Bahia.

Thyroid function during gestational age is a
determinant factor for fetal neurologic development
since studies have been demonstrating that prenatal
thyroid dysfunction may result in an impairment
of cognitive and behavior functions and also
other outcomes such as birth weight, fetal stress,
malformations and prematurity. Pesticides are
of an increasing concern about their effects on
human and environmental health. Mancozeb (MZ),
an ethylene bisdithiocarbamate manganese (Mn)-
based, is the most used fungicide in Brazil, Europe
Union and United States of America and considered
as an important endocrine disruptor chemical.
The aim of this study is to assess the Mn exposure
and thyroid and sex hormones concentrations in
pregnant women of Recôncavo Baiano - Brazil, and
to evaluate their correlation. Blood, occipital hair
and toenails were collected from volunteers in the
second trimester. Mn in hair (MnH) and Mn in toenails
(MnTn) were determined by graphite furnace atomic
absorption spectrometry (GF-AAS). Thyroid hormones
such as thyroid stimulant hormone (TSH), thyroxin
(T4), triiodothyronine (T3) and the sex hormones
estradiol (E2) and testosterone (T) were measured
by chemiluminescence. Sociodemographic and
pesticide-related questioners were applied. A total of
146 pregnant participated, with mean of 26.5 years-
old and 18.1 gestation weeks at enrollment. Median
(minimum and maximum) of hormones were: TSH 1.11
µIU/mL (0.14-3.41), FT4 0.91 ng/dL (0.71-1.24), T3 132.15
ng/dL (84.11-207.46), E2 5263.82 pg/mL (450 – 9030)
and T 0.56 ng/dL (0.26-2.52); and for Mn biomarkers
of exposure: MnH 0.20 µg/g (0.05-3.04) and MnTn
0.68 µg/g (0.05-22.35). A total of 16 (11.3%) women
reported that have already worked with pesticide
before and 28 (20.1%) husbands/partners reported
that are now working with them. MnTn levels were
significantly different in those pregnant that work with
pesticide (p=0.026), and also T concentrations were
different in this group (p=0.015). Significant difference
of MnH levels in women that have already worked
with pesticides before (p=0.03) and in those who still
work with those (p=0.011) were observed. However,
these results are controversy, showing higher levels
of these biomarkers and hormones in groups with
no pesticide contact. No correlation was observed
between biomarkers of Mn exposure and thyroid or sex
steroid hormones. It could be a result of our number
of volunteers that is still not enough. This study is still
in progress as well as a panel of pesticides analysis
and the recruitment of more pregnant women. The
present work is one of the fields of investigation of
the Brazilian Birth Cohort called Socio-Environmental
Determinants of Neurodevelopment at 12 months-
old (DSAN-12m). The authors would like to thank
the participation of all pregnant and nursers in the
project, and CNPq Universal Edital 421550/2018-0 and
FAPESB/PPSUS EFP_00019898 for funding. Keywords:
pesticide exposure, pregnancy, neurodevelopment,
neuroendocrine.
 295

Total and inorganic arsenic in fish and exposure

to inorganic arsenic by fish consumption in Brazil
Ramos, Vitor Serrão1; Vasconcelos Neto, Milton Cabral2; Custódio, Flávia Beatriz1
1
 BioTox - Laboratório de Bioquímica e Toxicologia de Alimentos, Departamento de Alimentos,
Faculdade de Farmácia da UFMG; 2 Instituto Octávio Magalhães - IOM, Fundação Ezequiel Dias - Funed.

Background: Arsenic (As) is a widely distributed
element in nature and its exposure can cause
carcinogenic effects to humans. One of the sources
of arsenic dietary exposure is fish consumption. The
main compound found in fish is arsenebetaine, a non-
toxic and non-carcinogenic organic arsenic compound.
Current speciation analyses are still expensive and
not widely available. However, to evaluate the real
risks of fish consumption, it is necessary arsenic
speciation or to distinguish non-toxic and toxic
arsenic. Objective: The objective of the work was to
identify the occurrence of total and inorganic arsenic
in fish and crustaceans and to evaluate inorganic
arsenic exposure of Brazilian population by fish
consumption. Methods: All data of arsenic in fish
available in GEMS/Food database (Global Environment
Monitoring System - Food Contamination Monitoring
and Assessment Programme from World Health
Organization) up to 2021 was downloaded for the
first analysis. Global data from fish and crustaceans
that had clearly defined name or species were
selected. Fish were categorized by species, family or
commercial name and crustaceans were categorized
in four groups: shrimps and prawns, lobster, crabs
and crayfish. Descriptive analyses were done for each
category and for fish and crustaceans. The intake of
inorganic arsenic was estimated by fish consumption
and global inorganic arsenic occurrence estimated
from GEMS/Food. Fish and crustaceans consumption
data were obtained from the Family Budget Survey
2017-2018 (POF) of Instituto Brasileiro de Geografia e
Estatística (IBGE). Risk characterization was evaluated
by calculation of margin of exposure (MOE), i.e., the
ratio of the toxicological reference dose (BMDL0.5 = 3
µg/kg bw day for lung cancer) and estimated intake.
Results: It was evaluated 12941 results of total arsenic
(92% of total data) and inorganic arsenic (8%) in fish
(89%) and crustaceans (11%). Tuna (1,339 results), cod
(3,608 results), salmon (1,455 results) and shrimps
(1,018 results) were the most representative groups
in database. For fish, the global range of total As
level was 0-110.0 mg/kg (mean= 2.92 mg/kg) and the
global inorganic As levels varied from 0-0.16 mg/kg
(mean=0.0028 mg/kg). For crustaceans, the global
range of total As level was 0-100.4 mg/kg (mean=3.08
mg/kg) and global inorganic As levels were 0-0.90
mg/kg (mean= 0.05 mg/kg). The proportion of total
As and inorganic As obtained for fish ranged for 0.02
to 16.4% (mean=0.10%) and for crustaceans, 0.14%
to 2.2% (mean=1.50%). Brazilian fish and crustacean
consumption varied from 8.8 to 49.5 g/day and
0.2 to 1.2 g/day respectively. Brazilian intake of
inorganic As from fish and crustaceans consumption
was estimated in 0.0004-0.0022 µg/kg bw and
0.0001-0.0009 µg/kg bw respectively. MOE values
ranged from 83,370 to 468,959 and 14,031 to 83,901
respectively. Discussion/Conclusion: Total As levels
were at most above Brazilian legislation of 1 mg/
kg for fish and crustaceans, although the inorganic
species, the main toxic forms, in all samples were
largely below this value. This reinforces the need for
As speciation and/or revision of Brazilian legislation.
By the MOE calculation it was possible to conclude
that, at first sight, the ingestion of inorganic As from
fish consumption does not present substantial risk
to the Brazilian’s health. The obtained results are
important to understand and evaluate risks of arsenic
intake by fish consumption in Brazil, being a base to
risk analysis and decision making in food safety area.
Acknowledgments: CAPES
 13 
TOXICOLOGIC HAZARD
IN THE WORKPLACE
 297

Assessment of serum levels of DDT and

metabolites in workers in malaria control
campaigns in the state of Pará, Amazon, Brazil
Rocha, Thiago L.1; Rocha, Cássia C.S.2; Miranda, Antônio M.M.2; Mendes, Rosivaldo de A.2
1
 Postgraduate Program in Epidemiology and Health Surveillance/Instituto
Evandro Chagas; 2 Environment Section/Instituto Evandro Chagas.

Introduction: Dichlorodiphenyltrichloroethane opinion nº 5.178.481. Results: The detection frequency

(DDT) was the most used chemical substance in
the 20th century, due to characteristics such as
high environmental persistence, toxicity and low
degradability. In Brazil, it was widely used to control
malaria in the period 1945-1997, with workers in
health campaigns exposed to DDT for years. In the
Amazon, full coverage was reached at the end of the
1960s. In the 80s and 90s, the incidence of the disease
grew alarmingly in Brazil, with 99% of cases located
in the Amazon region. During public health campaigns
using DDT, workers involved in home spraying were
vulnerable to contamination due to high exposure due
to factors such as lack of training, lack of use of PPE or
resistance to its use by workers. Objective: Evaluate
the distribution of serum levels of pp’-DDT and pp’-
DDE in serum samples from health agents who
worked in public health campaigns to combat malaria
until 1997, attended at Instituto Evandro Chagas from
2009 to 2015. Methods: This study is epidemiological,
longitudinal and retrospective , where 460 health
workers were treated, exposed to activities such as
transport, preparation, handling, storage, disposal
and vaporization of DDT in the internal and external
areas of residences in endemic areas by the minimum
period of up to 5 years. Serum samples were extracted
with n-hexane and analyzed by Gas Chromatography
electron capture detection (GC-ECD) model CP 3800
Varian with extraction Data were analyzed using
the following applications: StatisticalPackage for
the Social Science (SPSS), version 20.0. This work
was submitted to the Research Ethics Committee
of the Instituto Evandro Chagas under the
Certificate of Presentation of Ethical Appreciation
nº 52618121.4.0000.0019 and approved through the
of p,p’-DDT and pp’-DDE corresponded to 40% and
87.82% of the samples, respectively. The average
levels of pp’-DDT were 1.70 ± 3.92 μg/L, while pp’-
DDE presented 9.78 ± 10.99 μg/L. The DDE/DDT ratio
found was 5.75 μg /L. The predominant age group in
the research was from 41 to 50 years old (38.9%). The
variables significantly associated with p,p’-DDT levels
were: > 20 years of work, fish intake and symptoms
of cramps and low sexual performance (p<0.05). The
variables significantly associated with p,p’-DDE levels
were: > 20 years of work, working in mining areas and
eating fish (p<0.05). Of these, < 20 years of work was
the variable that most influenced the concentration of
p,p’-DDE (β=5.68; p<0.001). Discussion/Conclusion:
The high means of p,p’-DDE in relation to p,p’-DDT
indicate that the concentrations in the study are
the result of old exposure, probably until 1997 when
spraying officially ended in Brazil. The high index of
statistical significance of p,p’-DDE concentrations
achieved in the multivariate analysis for working time
(p<0.001) is a strong indication that the concentrations
in the study are the result of a high occupational
exposure. Despite the indication of old exposure, the
ratio between p,p’-DDT and p,p’-DDE was relatively
low when compared to other studies, indicating a
natural decline in DDT levels, considering that the
last spraying occurred in 1997 and the Participants
only carried out the collections from 2009 onwards.
Despite the absence of more specific clinical data, the
results lead to the creation of a health care program
for agents, due to the persistence of DDT in the body.
Acknowledgments: CAPES and FUNASA for their
logistical and financial support.

Case Studies: challenges on implementing
the GHS classification of pesticides in
the reevaluation of active substances
Aguiar, Larissa Muratori; Braz, Juliana Machado; Moreira, Camila Queiroz;
Background: The hazard classification of the active
substances (AS) used in pesticides is a fundamental
step on the reevaluation process. Recently, the
Brazilian Health Regulatory Agency (Anvisa)
approved a legislation setting new parameters for
the classification of pesticides for a better alignment
between the Brazilian assessment and the Globally
Harmonized System of Classification and Labelling
of Chemicals (GHS). Objective: To detail Anvisa’s
challenges and approach on the hazard classification
of pesticides during the reevaluation of AS, in order
to comply with both GHS criteria and the legislation
adopted by the normative RDC n⁰ 294/2019. Methods:
All toxicological data about the AS from regulatory
and non-regulatory (scientific literature) studies
were considered in a weight of evidence approach
(WoE) to select the endpoints with toxicological
relevance for humans and their corresponding lowest
observed adverse effect level (LOAEL). Then, the AS
were classified according to the hazard categories
of the GHS, by comparing the available data with
the criteria from GHS and RDC n⁰ 294/2019. When
gaps on the classification criteria were identified,
scientific judgment was used to decide whether
a category should or not be attributed to the AS.
Results: Case study #1: substance A was classified in
Category 1 for Specific target organ toxicity – single
exposure (STOT-SE) due to testicular toxicity in rats
after single oral exposure at doses ≥ 50 mg/kg body
weight. In addition, this test substance was also
placed in Category 1B for reproductive toxicity – as a
presumed human reproductive toxicant – due to its
adverse effects on reproductive physiology and on
embryofetal and neonatal development. Although the
GHS defines that the same endpoint should not be
298
Santos, Thiago Santana; Freitas, Daniel Roberto Coradi
Brazilian Health Regulatory Agency (Anvisa).
used for the classification in two distinct categories,
in this case, it was considered necessary to inform
specifically the occurrence of testicular effects after
a single dose exposure. Moreover, the chronic effects
used to the reproductive classification included but
were not limited to the testicular effects. Case study
#2: substance B was not classified for Specific target
organ toxicity – single exposure (STOT-SE) since the
thyroid effects (altered hormone levels) were not
considered relevant in this kind of exposure. On the
other hand, sub chronic and chronic studies showed
disruption of thyroid homeostasis with relevant
histological damages and changes on the hormone
levels, which led to a classification in the Category 2
for Specific target organ toxicity – repeated exposure
(STOT-RE). This AS was also classified in Category
1B for reproductive toxicity due to the likelihood of
the changes on the thyroid hormone levels to cause
neurodevelopmental impairments of the offspring
exposed during critical windows of development.
Although both classifications were supported by the
damage observed in the same organ, the outcomes
used for the classifications were considered distinct
and important to be communicated. Discussion/
Conclusion: To properly communicate the hazard
to the user in the AS reevaluation, a careful analysis
of all available scientific data is critical to select
the toxicological endpoints that will be used to
classify the substances within the different hazard
categories of GHS and RDC n⁰ 294. However, for
some AS the classification criteria cannot promptly
be used, because of a potential overlap on the
endpoints assigned to each Category. When facing
these situations, Anvisa is relying on a case-by-case
scientific judgment.
 299

Clinical and laboratorial evidenced organic

damage due to the incorrect use of PPE in
rural producers of tomato and onion crops
in the contestado region - Santa Catarina
Boff, Everton; Kampmann, Micheli Gabardo; Benvenutti, Régis Carlos

Universidade do Oeste de Santa Catarina - UNOESC.

Brazil is one of the world leaders in pesticide use.
The adverse effects caused to the health of workers
in contact with these substances depend on the
toxicological profile of the product and mainly on
the intensity and time of exposure. Eventual damage
due to individual exposure can be minimized with the
correct use of personal protective equipment (PPE).
The study aimed to analyze the adverse effects due
to exposure to pesticides used in tomato and onion
crops without the correct use of PPE. Forty workers
who plant both crops on their rural properties and
commonly do not use PPE completely and correctly
were clinically and laboratory analyzed. Were
measured plasma acetylcholinesterase levels, the
mutagenicity parameter evaluated through the
micronucleus test and investigation of oxidative
stress through measurements of the parameters of
superoxide dismutase (Sod), myeloperoxidase (MPO),
glutathione-S-transferase (GST), vitamin C (VIT C),
thiobarbituric acid reactive substances (TBARS),
protein carbonyl (PC) and DNA damage. All results
were compared to the control group consisting of 20
professional accountants from offices in the urban
area of t​​ he city of Caçador (SC). The clinical evaluation
was performed by an occupational doctor registered
at the Regional Council of Medicine of Santa Catarina
(CRM/SC). The ethical opinion approved has protocol
No. 4,255,852. All the laboratory parameters of the
farmers showed significant differences in relation to
the control group. In the clinical evaluation, no apparent
alterations were observed in any of the exposed and
non-exposed individuals. Referring to the significantly
lower plasma levels of acetylcholinesterase in
exposed workers, demonstrates that incorrect,
incomplete and depending, even non-use, are
compromising the exposed bodies. The micronucleus
test with significantly higher results in rural producers
demonstrates a greater possibility of mutagenicity
development. Finally, all parameters evaluated for
oxidative stress were significantly different between
both groups, being unfavorable results for the group
of farmers exposed to pesticides, which have a greater
tendency to diseases and/or injuries due to the excess
of Radicals Oxygen Species (ROS). It was evident that
the incorrect, incomplete or even non-use of PPE can
bring numerous damages to the exposed worker’s
body, especially in the case of chemical substances
with biocidal powers, which is the case of pesticides.
used in tomato and onion crops. Keywords: personal
protective equipment, pesticides, health of workers.
We thank all participants and collaborators of this
research, as they contributed to a scientific and social
well-being.
 300

Evaluation of proteins and genes

expression in workers exposed to
crystalline silica in Southern Brazil
Peruzzi, Caroline Portela1,2; Göethel, Gabriela1,2; Flesch, Ingrid1,2; Nascimento,
Sabrina1,2; Nardi, Jessica1,2; Arbo, Marcelo Dutra1; Garcia, Solange1
1
 Laboratory of Toxicology (LATOX), Department of Analyzes, Faculty of Pharmacy,
Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; 2 Postgraduate
Program in Pharmaceutical Sciences, Federal University of Rio Grande do Sul (UFRGS),
Porto Alegre, RS, Brazil; 3 FUNDACENTRO - Jorge Duprat e Figueiredo Foundation, Porto
Alegre, RS, Brazil; 4 Regional Worker Health Unit, Ametista do Sul, RS, Brazil.

Introduction: The great challenge of occupational
toxicology is the prevention of chronic non-
communicable diseases, since these diseases are
often disabling, reducing workers time and quality
of life. Studies to evaluate the use of biomarkers,
especially those with an early effect, are essential for
technical-scientific advancement and the prevention
of irreversible chronic effects in exposed workers.
Occupational exposure to crystalline silica (CS), which
is classified as a group 1 carcinogen by the International
Agency for Research on Cancer, it can trigger several
pathological processes, such as silicosis, pulmonary
fibrosis, chronic obstructive pulmonary disease and
cancer. In the vast majority of cases, the diagnosis
of these diseases is late, and ceasing exposure to
the causative agents does not guarantee that they
will stop developing, neither that the damage caused
to stabilize, as it is a progressive disease. In this
line, biomarkers, including genes and proteins, can
be detected and/or quantified in biological fluids
as a response to pathological changes in the face
of chronic exposure to different xenobiotics, thus
contributing to early diagnosis and/or indicating the
severity of diseases. Objective: This study aimed to
investigate potential early peripheral biomarkers of
inflammation through protein and gene expression
in workers occupationally exposed to CS. Methods:
The non-occupationally exposed workers at CS (NEW)
group (n=22) and CS exposed workers (CSEW) were
divided into two groups: workers diagnosed with
silicosis (CSEW I, n=26) and workers without silicosis
(CSEW II, n=28). Serum reactive C-protein (RCP) was
measured by immunoturbidimetry. Lymphocytes and
monocytes protein expression of L-selectin, CD18
and CD54 by flow cytometry and gene expression
of CXCL2 and IL-8 by RT-PCR were performed. CEP:
1.868.122. Statistical analyses: IBM SPSS (version
22). Significance was accepted at p≤0.05. Results:
Lymphocytes protein expression of L-selectin was
significantly decreased in the exposed groups (CSEW
I: 25.73 ± 1.51 %; CSEW II: 40.56 ± 1.73 %) compared
to the NEW group (48.51 ± 2.18 %) (p<0.05) and
was significantly different between CSEW I and
CSEW II groups (p<0.001). No significant difference
was observed between the CD18 and CD54 groups.
Significant higher values of RCP in CSEW I (16.36 ± 5.08)
than in CSEW II (1.91 ± 0.54) and NEW (2.40 ± 0.97) (p
<0.05), indicating an increase of inflammatory activity.
Otherwise, CESW groups presented significant lower
gene expression of CXCL2 and IL-8 (CSEW I: 0.15 ±
0.03 and 0.53 ± 0.07, respectively; CSEW II: 0.27 ± 0.06
and 0.52 ± 0.07, respectively) when compared to the
NEW group (1.88 ± 0.33 and 1.03 ± 0.03, respectively)
(p<0.001). Also, were observed that the more T cells
were activated, the lower were the gene expression
of these inflammatory markers (L-selectin vs. IL-8
r= -0.293 p<0.05; L-selectin vs. CXCL2 r= -0.420
p<0.001). Discussion/Conclusions: As the exposure
to CS causes an excessive inflammatory response,
increasing adhesion molecules levels in plasma,
we can suggest that the downregulation of IL-8 and
CXCL2 chemokines is a negative feedback mechanism
for organism protection against inflammation. Our
findings help us to increase our understanding of the
complex mechanism of silicosis pathogenesis and to
identify new early biomarkers of precocious diagnosis.
These results may be considered for designing new
studies with more details in the future. Funding: This
work receives financial support by FAPERGS (PqG
02/2017) and CNPq. Acknowledgments: BANRISUL.
 301

Evaluation of residual contamination in

anticancer drug packaging using UHPLC-MS/MS
Silva, Luciana Stein1; Silva, Laura Cé2; Machado, Cibele da Silva Barbosa1; Capp,
Edison1; Linden, Rafael2; Ness, Sandro Luis Ribeiro1; Antunes, Marina Vezon2
1
 Department of Gynecology and Obstetrics, Federal University of Rio Grande do Sul, Porto Alegre-
RS, Brazil; 2 Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo-RS, Brazil.

Background: The manipulation of anticancer drugs
must follow strict safety rules in order to minimize
the risks of occupational exposure to professionals.
Contamination of the external surface of medication
bottles is recognized as a health risk. The identification
of potential sources of contamination becomes
important for the adequacy of protection measures
and effective monitoring. Objective: To determine if
there is residual contamination on the surface of vials
and packages of the anticancer drugs doxorubicin
(DOX) and cyclophosphamide (CP) using ultra high
performance liquid chromatography coupled to
a mass spectrometer (UHPLC-MS/MS). Methods:
Samples were collected using a Whatman® Qualitative
Filter Paper Grade 2 moistened with 1 mL of methanol
and kept in a test tube until analysis. Samples were
prepared by solid-liquid extraction. 5 mL of methanol
and ethyl acetate (1:1, v/v) were added to the tube
containing the filter paper and homogenized by
vortex for 30 minutes at room temperature. After
centrifugation, 2 mL of supernatant was filtered to a
new tube and evaporated at 60 ºC. The dried extract
was recovered with 200 µL of water with 0.1% formic
acid and methanol (1:1, v/v). After homogenization,
an aliquot of 10 µL was injected on UHPLC-MS/MS
system. Chromatographic separation occurred in an
Acquity UPLC BEH C18 (50 x 2,1 mm; 1.7 μm) at 40 °C.
Mobile phase was water with 0.1% formic acid (eluent
A) and acetonitrile with 0.1% formic acid (eluent B)
eluted in gradient mode from 90:10 to 50:50 (A:B, v/v)
at flow rate of 0.3 mL min -1. Monitored transitions
for quantitation were 261.9/141 for CP and 544/360.8
for DOX. Retention times were 2.7 and 2.8 minutes
for CP and DOX, respectively. Calibration and control
samples were analyzed in all batches, with linear
intervals ranging from 1 to 1000 ng/patch. Results:
A total of 198 samples were analyzed, 66 of CP and
132 of DOX. All CP samples belong to the same
manufacturer, while DOX samples are from two
different manufacturers. For each manufacturer, 33
samples of vials and 33 samples of packages were
collected. CP levels were detected in 9 samples
(13.63%), 3 were below the lower limit of quantification
(LLQ) and the other 6 showed contamination levels
ranging from 1.24 to 28.04 ng/patch. DOX levels were
detected in 25 samples (18.93%), 2 were below the
LLQ and the others showed levels between 1.32 and
664.84 ng/patch. 85.29% of samples with residual
contamination were from vials. Conclusion: This
study demonstrated that cytotoxic drug vials and
packaging may contain residual contamination. These
results highlight the need to implement strategies
to prevent and monitor the effectiveness of safety
measures, in order to minimize the risk of exposure
of professionals involved in the preparation of
medicines. Acknowledgments: FIPE/HCPA.

Green tobacco sickness occurrence and
evaluation of life quality among tobacco
workers in Paraná countryside
Bortoli, Stella; Schamne, Tatiane; Kalv, Danielle Crystiane; Pedroso, Bruno; Vellosa, Jose Carlos Rebuglio
Brazil is one of the largest tobacco producers in the
world, the production is concentrate at country south,
where 97% of the crops come from and employ more
than 600.000 people. In tobacco production, the
tobacco workers expose themselves to substances
such as nicotine, present in tobacco leaf and pesticides.
The exposure to high nicotine concentration can occur
during several stages of tobacco chain production,
mainly at harvesting the leaves, arranging the leaves
for drying and also while sorting leaves for quality
selection. Handling tobacco leaves cause nicotine
poisoning, an acute intoxication also known as Green
Tobacco Sickness (GTS). The mains symptoms are
nausea, vomiting, weakness, dizziness, abdominal
pain and sometimes fluctuations in blood pressure
or heart rate. Risk of nicotine poisoning increases
when the leaves or skin is wet, allowing nicotine to
get onto the skin and pass into the bloodstream more
easily. The objective of this study was to observe
the symptoms associated to GTS occurrence and to
evaluate the life quality of tobacco workers. This is a
cross-sectional descriptive study with a quantitative
approach, using two questionnaires as instruments:
(i) sociodemographic and health conditions data of
tobacco workers, mainly those associated to GTS;
and (ii) life quality information (WHOQOL-bref). The
majority of the participants were men, under the
age of 40 (39.9 ±11.92 years old) and incomplete
elementary school education. Few workers reported
chronic disease as depression, obesity and asthma.
Most of them (93.55%) were non-tobacco smokers. In
regarding of GTS symptoms, most of the participants
reported dizziness (41.93%), headache (38.71%),
weakness (38.71%), abdominal pain (35.48%),
vomiting (22.58%), nausea (32.26%), drooling (12.9%)
and diarrhoea (9.68%) after the harvest phase, which
reinforces the evidence of green tobacco sickness
occurrence. Those results were in accordance with
302
Universidade Estadual de Ponta Grossa - UEPG.
studies conducted in Rio Grande do Sul at 2014 and
2016. It is important to emphasize that symptoms
related to GTS and pesticide poisoning are similar,
as well the tobacco workers are exposed to both,
nonetheless tracked symptoms were reported
only after harvest phase. In addition, 77.42% of the
questionnaire respondents reported using personal
protective equipment (PPE), however, participants
did not specify whether the PPE used were regular
cotton clothing or the full standard garment
consisting of waterproof pants or long sleeve shirt
and gloves. Some home measures are adopted by
41.94% of tobacco farmers in an attempt to relieve
these symptoms, such as the use of teas and cola
drinks. The descriptive results of the WHOQOL-bref
by domains, facets and total quality of life, revealed
that tobacco growers presented good averages in all
domains: physical (68.45%), psychological (66.85%),
social relations (73.12%) and environmental (59.30%).
Environmental domain showed the lowest satisfaction
index, highlighting greater interference mainly from
the facets “Health care”, “Recreation and leisure” and
“Financial resources”. The “Health Care” demonstrate
a strong influence of the performed activity: the
production of tobacco on the health of the producer,
which is directly affected by DFVT, also to pesticide
exposure and the physical effort required. Similar
was observed in other studies regarding countryside
populations, especially exhaustive workload and
pesticide poisoning. The present study demonstrated
evidence of GTS among tobacco farmers in south
Brazil. The tobacco workers activity directly interferes
with their life quality. Many are unaware that the
symptoms experienced after harvesting are GTS
or nicotine intoxication, so the need for access to
information is emphasized, as well as the importance
of using PPE correctly.

Pesticide poisoning in banana cultivation
in Corupá city, Santa Catarina
Borgmann, Gabriela1; Plautz, Katherine1; Zimmath, Michel2, Tenfen, Adrielli2
1
 UNIVILLE, Programa de Pós-Graduação em Saúde e Meio Ambiente; 2 UNISOCIESC Jaraguá do Sul.
Introduction: In Brazil, health professionals face
difficulties in diagnosing, registering and referring
patients with pesticide poisoning1. Across the national
territory, countless varieties of products from agriculture
are marketed. The southern region is considered one of
the country’s agricultural granaries - in Santa Catarina
(SC), mainly in the micro-region of Corupá, agriculture
is family-owned - and the number of cases of poisoning
in the region is proportionally high compared to the rest
of the country2. In 2017, 1.196 cases of poisoning and
25 deaths were reported in the southern region. This
points to the importance of protection through personal
protective equipment (PPE)3,4. Objectives: Determine
the main pesticides used in banana cultivation in Corupá
- SC and raise awareness among banana growers
about the importance of taking care with the use of
pesticides and the proper use of PPE. Methods: This
study is a literature review with subsequent extension
activity through the lecture “Poisoning by Pesticides in
Banana Farming” at the event “Prevention and Care in
the Use of Agricultural Chemicals and Water Quality,
promoted by the Association of Banana Growers of
Corupá (ASBANCO). Results and Discussion: According
to acquired information from ASBANCO at the event,
the compounds most used in banana plantations in
Corupá are thiazides and strobilurin. Thiazides are
herbicides and are used to control weeds and broadleaf.
Strobilurin, on the other hand, are fungicides derived
from a secondary metabolite produced by the fungus
Strobilurus tenacellus and, therefore, they are so
called, and are often used with thiazides5. The event
highlighted the importance of extending scientific
knowledge to banana communities, demonstrating the
need for the correct use of PPE: water repellent cotton
jumpsuit with long sleeves, waterproof wide-brimmed
hat, waterproof apron, face shields, disposable mask,
covering the nose and mouth, gloves and rubber boots.
In addition, according to VSPEA-SC data from 2020, 251
cases of poisoning were reported in Santa Catarina,
however only one of them happened in Corupá6. In 2021,
also according to VSPEA-SC data, there weren’t any
303
notifications of poisoning in Corupá7. Considering that
Corupá is a large banana producer, the low amount of
poisoning events in this city could be justified by the
use of natural based pesticides - such as strobilurin
- and the correct use of PPE by the banana growers.
Conclusion: There is a large amount of annual poisoning
in Santa Catarina and it is necessary to intensify
prevention and control actions. However, specifically in
Corupá, the pesticides used are of light classification,
which is a positive point for the region and contributes
to minimize poisoning cases. Even so there are risks in
their use, if the necessary precautions are not taken.
Keywords: Pesticides; Poisoning; Banana growers;
Personal protective equipment.
REFERENCES
1. LONDRES, F. Agrotóxicos no Brasil: um guia para ação em defesa
da vida. Rio de Janeiro: AS-PTA - Assessoria e Serviços a Projetos em
Agricultura Alternativa, 2011.
2. Instituto Brasileiro de Geografia e Estatística. Área plantada,
área colhida, quantidade produzida, rendimento médio e valor
da produção dos produtos das lavouras temporárias, segundo
a Unidade da Federação, suas Mesorregiões, Microrregiões e
Municípios. Acesso em: 10 out. 2021. Disponível em: https://www.
ibge.gov.br/estatisticas/economicas/agricultura-e-pecuaria/9117-
producao-agricola-municipal-culturas-temporarias-e-permanentes.
html?=&t=resultados
3. Ministério da Saúde: Sistema Nacional de Informações Tóxico-
Farmacológicas. Casos Registrados de Intoxicação Humana por Agente
Tóxico e Centro. Região Sul, 2017. Acesso em: 10 out. 2021. Disponível
em: https://sinitox.icict.fiocruz.br/
4. Ministério da Saúde: Sistema Nacional de Informações Tóxico-
Farmacológicas. Óbitos Registrados de Intoxicação Humana por Agente
Tóxico e Centro. Região Sul, 2017. Acesso em: 10 out. 2021. Disponível
em: https://sinitox.icict.fiocruz.br/
5. RODRIGUES, MAT. Classificação de fungicidas de acordo com o
mecanismo de ação proposto pelo FRAC. 2006. 249 f. Dissertação
(mestrado) - Universidade Estadual Paulista, Faculdade de Ciências
Agronômicas, 2006. Disponível em: http://hdl.handle.net/11449/97224
6. Governo de Santa Catarina: VSPEA-SC. Informativo VSPEA-SC
Setembro/2020. Acesso em: 15 fev. 2022. Disponível em: http://www.
vigilanciasanitaria.sc.gov.br/index.php/comunicacao/noticias/149-
noticias/noticias-2020/1212-2-informativo-vspea-2020.
7. Governo de Santa Catarina: VSPEA-SC. Informativo VSPEA-SC
Dezembro/2021. Acesso em: 15 fev. 2022. Disponível em: http://
www.vigilanciasanitaria.sc.gov.br/index.php/150-noticias/noticias-
2021/1313-1-informativo-vspea-sc-2021.
 304

Skin exposure to KrCl excimer lamp

emitting at 222 nm cause tissue and
cellular alterations of concern
Tavares, Renata Spagolla Napoleão; Girassole, Alessandra; Adamoski, Douglas; Caznok, Ana Clara;
Domingues, Romênia; Leme, Adriana Paes; Carvalho, Murilo; Dias, Sandra Martha Gomes

Brazilian Biosciences National Laboratory (LNBio), Brazilian Center for Research

in Energy and Materials (CNPEM), Campinas, Sao Paulo 13083-970, Brazil.

Introduction: Ultraviolet (UV) light can inactivate
coronaviruses, but the practicality of UV light as an
engineering control in public spaces is limited by the
hazardous nature of conventional UV lamps, which
are mercury-based and emit a peak wavelength (254
nm) penetrating human skin and has carcinogenic
potential. During the SARS-CoV-2 pandemic, the
far-UVC light has emerged as a safe tool to sanitize
populated environments, once it is preconized to be
absorbed by the stratum corneum without reaching
the genetic material of living cells. Recent advances
in the development and production of KrCl excimer
lamps hold promise in this regard, as this lamp
emits a filtered shorter peak wavelength (222 nm).
The studies performed so far have evaluated the
capacity of the 222 nm light to produce cyclobutane
pyrimidine dimers and 6-4 photoproducts on the
DNA and inflammatory responses in the skin and
eyes. Objective: None of the previous studies
has evaluated molecular alterations such as ROS
formation, alterations in the protein levels and photo-
aging. Our goal is to study these phenomena on in
vitro full-thickness reconstructed human skin (RHS).
Method: We developed RHS using human primary
keratinocytes and fibroblasts embedded in a collagen
type I matrix. We added 50 µM DCF-DA on top of the
RHS to assess ROS formation. Afterwards, we exposed
the RHS to 0, 6, 25, 500 and 1500 mJ/cm2 of 222 nm
light and collected immediately the fluorescence
emission using EnSpire 2300 equipment (Perkin
Elmer). RHS were snap-frozen and the cryo-slices
analyzed for the formation of pyrimidine dimers using
immunofluorescence, immunohistochemistry and an
in-house developed Fiji script software. The RHS was
also irradiated with 500 or 1500 mJ/cm2 of 222 nm
and collected after 0 h, 24 h or 48 h of exposure. The
proteins of epidermis were extracted and digested
for MS evaluation. The MS data were analyzed in
MaxQuant and Perseus and the list of quantified
proteins were submitted to pathway enrichment.
In all experiments a traditional germicidal UVC light
at 254nm was used as a control. Results: The RHS
cultivated over eight days, as expected, developed a
fully differentiated epidermis on top of a dermis, as
evidenced by the presence of cytokeratin 14 in the
basal layer and cytokeratin 10 in the upper layers.
As already reported, even at the higher tested doses,
222 nm produced a less intense and more superficial
pyrimidine dimers staining than 254 nm, which were
more promptly removed form the skin following the
regular process of skin desquamation. However, UV at
222 nm induced ROS formation in a dose-dependent
manner, with the signal being detected with a dose
as low as 25 mJ/cm2, the preconized occupational
threshold limit value (TLV) of exposure to 222nm
in 8 hours. Moreover, the preliminary data showed
differential proteins associated with important
molecular functions and biological processes of the
epidermis. Conclusion: Our study showed for the
first time that skin exposure to filtered KrCl excimer
lamp, even within the regulatory TLV, can increase
ROS production, alterations on cellular functions and
photo-aging processes. Taken together, our results
raised important read-flags for the use of this lamp,
even within the TLV. Acknowledgments: We would
like to thank Instituto Tecnológico da Vale, Embrapii
and CNPEM for the project funding.
 305

The influence of occupational exposure

to cyanide on metahemoglobinemia
in cassava flour producers
Estumano, Saulo Braga1; Oliveira, Cláudia Simone Baltazar1,2; Araújo, Maria Eduarda
Lima1; Costa, Eliene dos Santos da Silva1; Chisté, Renan Campos2; Lameira, Christian
Neri1; Amaro, Beatriz Oliveira3; Pinheiro, Maria da Conceição Nascimento2
1
 Centro Universitário FIBRA - FIBRA; 2 Universidade Federal do
Pará - UFPA; 3 Instituto Evandro Chagas - IEC.

Introduction: Cyanide present in Manihot succulent
crantz is a highly toxic and hypoxemiant substance, as
it has properties capable of inhibiting the cytochrome
oxidase enzyme and, therefore, the chemical state of
iron. In this way, preventing the reception of oxygen
in the red cells of exposed individuals, increasing the
production of methemoglobin, for example those who
work in the production of cassava flour. Objective:
To evaluate the influence of occupational exposure
to cyanide on methemoglobin levels in cassava flour
producers. Material and Methods: Observational,
cross-sectional, analytical study developed in a
community located in the northeast region of the
state of Pará in the year 2018 to 2019. Participants
were 40 producers of both sexes between 13 and 55
years old with recent exposure and 20 residents of
the same community non-producers. Both groups
had lived in the communities for more than a year and
had not had contact with other methemoglobinizing
substances, such as tobacco, use of illicit drugs,
contact with mercury, without infectious diseases,
who had not practiced recent physical exertion and
had in the production of flour its main source of
income. By UV spectroscopy, the concentration of
free cyanide and the percentage of methemoglobin
were analyzed. Hematocrit and hemoglobin were also
measured, however using a complete blood count
device. The tests were analyzed at the Toxicology
Laboratory of the NMT/UFPA and at the University
center FIBRA. The protocol of this study was prepared
in accordance with the rules of resolution 466/2012
and approved by the Research Ethics Committee of
the Nucleus of Tropical Medicine in accordance with
ethical opinion number 2892820 of September 13,
2018. Results: The mean cyanide in blood samples
from producers with recent contact was 3.45 ± 0.8
mg/HCN, minimum of 0.7 and maximum of 5.2. In
those who declared non-producers, the average of
cyanide was 1.3 ± 0.12, minimum of 0.5 and maximum
of 2.5. A significant difference was observed according
to the Z test for two independent samples. In the
measurement of methemoglobin, the value obtained
was 4.5 ± 0.8 among producers and 1.2 ± 0.9 among
non-producers. A moderate positive correlation was
observed between free cyanide concentrations and
Methemoglobinemia r=0.45 p<0.05. In the hematocrit
measurement, the mean value was 43.6 ± 4.5 and
Hemoglobin was 13.8 ± 1.2. However, it was not
observed between hematocrit and hemoglobin levels.
Conclusion: Cyanide concentrations between groups
were statistically different, indicating the influence
of occupation on exposure to the chemical agent. The
methemoglobin values ​​show a probable participation
of cyanide in the oxidation of iron present in the red
blood cells of rural producers, although no influence
was observed on the hematocrit and hemoglobin
values. Therefore, this population needs to be
monitored more, when considering the toxicity of the
chemical compound and the risks it offers to the health
of exposed individuals. In addition, we also admit
that it is important to develop preventive measures
regarding this form of occupational exposure. Graded:
I thank my advisors and everyone who participated in
the research and for the willingness in the process of
obtaining data.
 306

Toxic metal backgrounds among waste

pickers in hair sampling: a cross-sectional
study in Brazil (Federal District)
Gonçalves, Michelly Rodrigues1; Verpaele, Steven2; Marques, Carla Pintas1; Bashash,
Morteza3,4; Cruvinel, Vanessa Resende Nogueira5; Santos, Vivian da Silva6
1
 MSc, University of Brasília, Faculty of Ceilândia, Brasília (DF), Public Health, Brasilia, Brazil.
2
 MSc, Belgian Center for Occupational Hygiene asbl, Zwijnaarde, Belgium. 3 University of
Southern California, Department of Preventive Medicine at the Keck School of Medicine,
Los Angeles, CA, USA. 4 PhD, Ryerson University, School of Occupational and Public Health,
Toronto, Canada. 5 PhD University of Brasília, Faculty of Ceilândia, Brasília (DF), Public
Health Department at Ceilândia Faculty, Brazil. 6 PhD, University of Brasília, Faculty
of Ceilândia, Brasília (DF), Pharmacy Department at Ceilândia Faculty, Brazil.

Background: This cross-sectional study was
conducted at the Brazilian open dump named as
Structural dump, also known as Jóquei dumpsite,
which was considered the second largest active dump
in the world until its closure in 2018. According to SLU,
until 2018, most of the waste pickers in the Federal
District were self-employed professionals working at
the open dump. After the open dump closure, they were
requested to organize themselves into associations
(coops) to be reallocated to the new sorting plants.
This transfer brought quality gains to work in terms
of safety, however the impact on health related to
the less ventilated environment at the sorting plants
could contribute to a greater chemical exposure and
should be monitored. Objective: the aim of this study
was to determine the metal concentration in the hair
of waste pickers who worked sorting mixed waste
including e-waste, in Brasilia, Brazil providing metal
background values to support further public policies.
Methods: There were 2112 waste pickers registered
at the SLU (urban cleaning service) in 2017. A total
of 359 waste pickers hair samples were collected.
39 samples were excluded that had been dyed or
straightened less than 3 months before collection.
All the 320 remaining samples were washed to
remove external contamination. Samples and
Certified Reference Material ERM-DB001, underwent
digestion using an ETHOS EASY and analyzed by ICP-
MS. Regarding the statistical analysis, participants
were grouped prior bivariate comparisons according
to their workplace (sorting plants or open dump),
gender, and time as waste picker using Chi-square
tests.The comparisons between age and gender
were analyzed using the non-parametric Mann-
Whitney test. Results: The results showed that the
arsenic, cadmium, barium, copper, lead, tin, especially
cobalt, manganese and molybdenum were found in
significantly higher concentrations than previously
reported. Results: Most participants are women and
have less As, Be, Cd, Co, Mn, Sn, Pb, Ba, Mo, Zn, Fe, and
Cu in their hair compared to men. Regarding working
conditions, it was not yet possible to verify significant
differences, since the migration of part of the waste
pickers from dumpsite to the sorting plants was still
in transition in 2017. Discussion/Conclusions: Waste
pickers are a vulnerable population to be exposed to
toxic metals. According to our study, most of them
are women working in this job for many years and
have less metal concentration in their hair compared
to men. In relation to working conditions, it was not
yet possible to verify significative differences, since
the migration of part of the waste pickers from
dumpsite to the sorting plants was still in transition.
Many waste pickers from the sorting centers still
were performing their occupation in the dumpsite as
extra-income in 2018. This work brings initial data
(background values) for monitoring the population
of waste pickers in the Federal District and supports
future studies already in progress. It is expected that
another sample collection carried out between 3 to
5 years after the closure of the dump, the difference
between the work environments can probably be
noticed in the metal concentrations, mainly regarding
to released particles which are better dispersed
outdoors, resulting in lower concentrations in the
workers’ breathing zones and the group 1 workers
had cooperatives that operate in indoor facilities
(sorting plants) without proper ventilation sorting
electronic materials containing Cd, Cu, and Pb. It is
very important to continue metal monitoring, as they
can have toxic effects after many years of exposure
and metal long-term poisoning might have an
insidious beginning, difficult to diagnose, which might
lead to irreversible sequelae being costly for public
healt. Acknowledgments: This research received no
external funding; however, all samples were analyzed
at no cost by the non-profit Belgium Center for
Occupational Hygiene (BeCOH).
 14 
OTHER

Attempted suicide: temporal series
and agents involved before and during
the COVID-19 pandemic in Brazil
Holanda Júnior, Wanderley Pinheiro; Magalhães, Danielle de Paula; Oliveira, Juliana Ribeiro Ibiapina Leitão
Introduction: Suicide is a behavior of self-injury with
the intention of ending one’s self-life. It is a complex,
multifaceted phenomenon which includes ideation,
attempt, and consummation of the act. It is recognized
as a serious public health concern worldwide and
it is related to several personal and social factors.
According to some studies, there was an increased
risk of suicide attempt during the Covid-19 pandemic
in Brazil. The use of substances is one of the well-
established methods in suicide attempts and leads
to exogenous intoxications. Research on the toxic
agents used motivated by suicide can contribute
to policies and strategies for case prevention and
health promotion. Objective: To delineate the toxic
agents and analyze the temporal trend of cases of
intoxication by suicide attempt (IBSA) in the period
2015 to 2019 and compare with the year 2020
regarding the pandemic by covid-19 in Brazil. Method
and Material: Descriptive study and time series of
the cases notified in the Injury Notification System
(Sinan) of the confirmed exogenous intoxications by
suicide attempt, referring to the years 2015 to 2021
by tabnet/DATA/SUS. The following parameters were
applied: notifications by year of 1st symptom(s), all
types of toxic agents and evolution (except loss to
follow-up), single and repeated acute exposures. The
following softwares were used for data tabulation,
statistical calculations: Pearson chi2 tests, prevalence
ratio (PR), 95% confidence interval (95%CI) and
verification of the temporal series: Microsoft Excel
2019®, Stata® 2016 and Joinpoint Regression Program
(version 4.9.0.1). The analysis was performed
according to stratification by sex: male (M), female
(F) and toxic agent. The crude rate of intoxication by
suicide attempt (CR-IBSA) per 100,000 inhabitants
was calculated by the following equation: (number
of notifications)/(estimated brazilian population-
year)x100,000. Population data were sourced from
308
Forensic Expertise of the state of Ceará (PEFOCE), Brazil.
the Brazilian Institute of Geography and Statistics.
Results: During the period between 2015 and 2020,
206,118 cases of IBSA were reported, the highest
number of intoxications were in 2019 (25.0%) and
72.8% (N=205,816) belonging to the female gender.
Regarding the annual temporal analysis there was
growth in CR-IBSA until the year 2019, annual percent
change (APC) = +26.4 (p<0.05) and then decrease, not
significant, in 2020 (APC = - 43.9; p=0.054). Regarding
the monthly average, in the period from 2015 to 2019
there was non-significant growth of CR-IBSA in the
second half of the year, monthy percent change (MPC)
= +4.5 (p=0.074) starting in June, in 2020 the highest
coefficients were in the first half with a decrease until
May: MPC = -15.5 (p<0.05). Medications were the most
used substances by both genders, being from 2015 to
2019: 85.5% (F); 66.0% (M), PR=1.29* (95% CI: 1.28-
1.30), in 2020: 88.3%(F); 71.5%(M), PR= 1.23*(1.21-1.25).
In comparing the period from 2015 to 2019 with the
year 2020, it was observed that among women there
was an increase in drug of abuse poisonings RP=1.93*
(1.58-2.36) and decrease in agricultural pesticide
use (2.4%; 1.4%, p<0.01). In addition, among men,
we observed an increase in drug use: (66.0%; 71.5%,
p<0.01), lower use of agricultural pesticides (8.91%;
7.01%, p<0.01) and rodenticides (12.5%; 8.84%, p<0.01)
in the corresponding periods studied. Conclusions:
There was an expressive increase in the number of
IBSA cases in the last five years before the Covid-19
pandemic (2015 to 2019) in Brazil and decrease in the
pandemic year (2020) and with greater implications
for women. Drug intoxications were predominant
in suicide attempts in both genders. A change in the
profile of the types of substances involved in suicide
attempts in the pandemic period was evidenced.
We emphasize the importance of knowing the toxic
agents used in the attempts in order to implement
specific preventive measures for vulnerable groups.

Brazil now prioritizes animal-free safety
assessment of school supplies
Research & Toxicology Department, Humane Society International, Brazil.
Introduction: Although several non-animal methods
have been accepted in Brazil since 2014, due to lack of
clarity animals are still routinely used for the safety
assessment of school supplies such as paints and
glues. According to Brazilian regulatory requirements,
all school supplies with more than 3 grams of paints,
glues, gouaches, watercolors, and powered material
per unit use must have their safety confirmed
regarding acute oral toxicity. Those susceptible to
skin contact must also have their safety confirmed for
skin irritation. Acute oral toxicity and skin irritation
could be assessed by in vivo or in vitro methods,
which means the laboratories were free to choose
between testing on living animals or using alternative
methods (cell cultures). In addition, in Brazil safety
testing has traditionally been performed on finished
products, which is expensive, time consuming and
has significant limitations. Objective: To change
the Brazilian regulatory requirements for school
supplies to allow the toxicological safety assessment
of the products based on existing information on its
ingredients, prioritizing animal-free approaches and
assuring the use of animals only as a last resort.
Methods: A series of meetings was held with national
stakeholders including the Brazilian Association of
Technical Standards (ABNT), school supplies industry
representatives and laboratories that perform safety
assessment of school supplies to discuss the proposal
309
Marigliani, Bianca
made by the Humane Society International (HSI) to
update the ABNT norm on school supplies (ABNT Norm
15236). Results: The HSI proposal for an approach
that prioritizes animal-free safety assessment was
discussed by the national stakeholders with the
support of the National Council for the Control of Animal
Experimentation (CONCEA). The ABNT committee
on school supplies have accepted HSI proposal and
published the updated norm (ABNT NBR 15236:2021)
in September 2021. This norm is referred to in the
Inmetro ordinance number 423, published in October
2021, which makes ABNT 15236:2021 the mandatory
requirement for school supplies compliance in
Brazil. Discussion: Although in vivo methods are still
allowed, the ABNT NBR 15236:2021 now prioritizes
the safety assessment of school supplies based on
existing information on its ingredients for both acute
oral toxicity and skin irritation. Another important
change is the requirement that animals are used only
as a last resort and only when technical justifications
are present. Conclusion: The changes made in the
Brazilian regulatory requirements for the safety
assessment of school supplies now prioritizes
animal-free approaches and the use of animals only
as a last resort. This is in accordance with the 3Rs
Principle, closer to regulatory harmonization with
other countries, and enables avoiding the unjustifiable
use of many animals.
 310

Early exposure to toxic metals and

child neurodevelopment: proposal
of a theoretical model based on
Bronfenbrenner’s bioecological theory
Bah, Homègnon Antonin Ferréol1,2; Santos, Nathália Ribeiro2,3; Costa, Daisy Oliveira2,3; Carvalho, Chrissie
Ferreira4; Martínez, Victor Otero; Gomes-Júnior, Erival Amorim; Menezes-Filho, José Antonio1,2,3
1
Institute of Collective Health, Federal University of Bahia, Brazil; 2 Laboratory of
Toxicology, College of Pharmacy, Federal University of Bahia, Brazil; 3 Graduate Program
in Pharmacy, College of Pharmacy, Federal University of Bahia, Brazil; 4 Department
of Psychology, Federal University of Santa Catarina, Santa Catarina, Brazil.

Background/Introduction: Early exposure to
environmental pollutants like toxic metals has been
identified as neurotoxic and interaction with social
determinants may be a mechanism of that toxicity.
The possibility of that interaction makes it necessary
to innovate in the way of approaching and studying
their effect on human population. Bronfenbrenner’s
Bioecological Theory (BBT) is a framework that
considers human development as a result of a joint
effect of its biological characteristics, immediate
setting, and the other settings of society. Objective:
To develop a conceptual framework that allows
encompassing the social and biological characteristics
of children from the gestational period to childhood,
considering exposure to toxic metals. Methods: a
literature review was conducted and the BBT besides
other concepts such as the social and structural
determinants of health, allostatic load, embodiment,
genetic and epigenetic concept were used to design
our framework. Results: Our model presents how
mechanisms based on structural determinants
(like skin color or ethnicity, socio-historical context,
power, and privilege) from the distal level of society
define the social status of a mother (and her family).
In the immediate setting, life circumstances linked to
social context define the exposure to (chronic) stress
and toxic metals, affect parents’ mental health and
their relation with the child. We argue that exposure
to heavy toxic metals and stress due to social status
may act together at a biological level to influence
the neurodevelopment of the child depending on
the quality of interaction between the growing child
and its immediate setting. Discussion/Conclusion:
Assessment of true neurotoxicity of pollutants cannot
be done separately from the ecological environment
in which they act. Although this model is based on a
specific contaminant, it opens new horizons on how
biological sciences, such as neurotoxicology, can
articulate with the theoretical models from human
sciences to have a broader approach to study human
development. Acknowledgments: We are thankful to
the National Council for Scientific and Technological
Development (CNPq), the Bahia State Research
Support Foundation (FAPESB) and Coordination of
Superior Level Staff Improvement (CAPES).
 311

Junkie – Teaching project in comic book for

the development of learning in toxicology
Bairros, André Valle1; Reginato, Fernanda Ziegler2; Ugalde, Gustavo Andrade2; Berlato,
Dener Gomes2; Pacheco, André Lucas Bezerra2; Nascimento, Marcelo Henrique Santana2;
Oliveira, Letícia Cimó2; Santos, Lara Celestina2; Chimendes, Nayomi de Andrade2; Rosa,
Victória Gomes2; Dalmazzo, André Kusser3; Maia, Affonso Enriques Montagner4
1
 Nucleus Applied to Toxicology (NAT) Federal University of Santa Maria (UFSM);
2
 NAT - UFSM; 3 Industrial Drawing Department (IDD) - UFSM; 4 IDD - UFSM.

Toxicology is a science that studies the effects of
chemical substances on organism in determinate
exposure conditions and require of knowledge from
several areas. This complexity demonstrates the
level of demand for the student during the contact
with toxicology. Alternative teaching proposal
are a good tool for fixation and improvement of
knowledge, one example is comic books. This didactic
capacity of comic books has successfully applied
in our society, from entertainment to state policy
campaigns. We developed a fictional history of José
da Silva, the Junkie, who lives in an urban center,
having drug dependence. When José da Silva uses
drugs consciously, objectively, predicting their future
biological effects, these effects are improved in high
levels. However, the protagonist still has side effects
like any normal person. In your universe, we will
approach the toxicology and adjacent themes like
abuse drugs, pesticides, industrial solvents, metals,
new psychoactive substances, medicinal plants,
sociology, law, risk and toxicity evaluation, etc.,
important to science and for the society. In this sense,
pharmacy students employed their toxicological
knowledge to develop a story on comic book based
in anti-hero journey, which it will be share with other
health area students. The scripts for the comics will be
written by NAT members as well pharmacy students
during clinical toxicology and forensic toxicology
disciplines. For NAT members, the tales will be chosen
by project coordinator while other students were
free to choose the substances that will be used on
story, explain our action mechanism, effects, and
consequences. Posteriorly, the material will be sent
for industrial drawing department for illustration
and opinions about narrative. It is estimated 2 comics
with 2 pages for each group, which 10 pharmacy
students will be involved. In the first comic book we
present José, the place where he lives, his wife and
some friends. The main drugs involved in this story
are cocaine and crack, ranging from abstinence to
use. The mechanism of these drugs is explained, also
abstinence, and illustrated to show us the effects on
the body and brain. All events bring a context to build
the story of José and his power when using drugs,
giving many possibilities to explain and explore other
drugs. José’s past is unknown, and time will tell the
truth and how he received these powers. Ten forensic
toxicology students participated in the development
of this first story. We want a popularization of
toxicology content and other pertinent subjects in
this science, which can help the learning process for
undergraduate students approaching the students
involved on comic production and other students,
including high school, via digital divulgation.
 312

The use of paracetamol as cause

for male infertility
Silva, Luíza Madureira1; Holanda, Wiron Pimentel2; Capelo, Melissa
Figueiredo2; Honório Júnior, José Eduardo Ribeiro3
1
 Academic of Nursing in the University Center Christus, Laboratory of Neurociência Translacional-
Neurocit; 2 Academic of Biomedicina in the University Center Christus, Laboratory of Neurociência
Translacional-Neurocit; 3 Doctor in Biotechnology for the RENORBIO/UFC. Professor in the University
Center Christus, Coordinator of the Laboratory of Neurociência Translacional-Neurocit.

Introduction: Paracetamol, also known as
acetaminophen, is present in a number of over-
the-counter (OTC) and prescription drugs for its
antipyretic and analgesic properties. The effects
of acetaminophen, especially in its analgesic
properties, are primarily due to activation of the
decreased serotonergic pathway. The main site of
action of paracetamol may still be the inhibition of
prostaglandins, a multifunctional substance derived
from the synthesis of polyunsaturated fatty acids.
Enzymatic reactions of molecular mechanisms may
reduce the formation of glutathione, a coenzyme
for enzymes such as prostaglandin synthase, which
inhibits prostaglandin synthesis. Many health
conditions can affect male fertility, underscoring
the need for a thorough evaluation of the patient to
identify treatable or reversible lifestyle factors or
medical conditions. The causes of male infertility vary
widely, but may be related to congenital, acquired,
or idiopathic factors that impair spermatogenesis.
Widespread use of acetaminophen at high doses has
been found to cause various forms of toxicity, such as
reproductive toxicity. The effects of paracetamol on
semen quality and sperm parameters have been found
in many published studies. Studies demonstrated
increased percentage of abnormal sperm head,
reduced sperm count, impaired motility and reduced
anogenital distance and decreased testosterone
production. Objective: To evaluate the literature
studies of the use of Paracetamol and its relation
to male infertility. Methodology: A bibliographic
search was carried out using electronic databases
(Scielo, Pubmed, and Lilacs) for articles, published
in English, Portuguese and Spanish, between 2000
and 2022. The descriptors used were infertility, male
infertility, Paracetamol, Acetaminophen. Results
and Discussion: The electronic searches identified
20 studies, of which 3 were selected that met the
criteria for inclusion. A study done by Ratnasooriya
et al., in 2000, showed that Long-term high-dose
administration of paracetamol impairs reproductive
performance in male rats. They propose that this
effect is reversible, not due to general toxicity, but
due to increased oligospermia, insufficient motility
of normal and hyperactive sperm, and decreased
sperm fertilization capacity. Another study done by
Yano and Dolder, in 2002, found that Seminiferous
tubules were altered and some degeneration, Sertoli
cell fragmentation, and sperm cell morphology were
altered in Wistar rats treated with a single dose
of acetaminophen (4.4 mmol/kg). Banihani in his
study concluded that the effects of acetaminophen
on semen quality may occur by increasing reactive
oxygen species production, inducing spermatocyte
apoptosis, reducing nitric oxide production, inhibiting
prostaglandin synthesis, and inhibit testosterone
synthesis. Conclusion: We can conclude that the
long term use of high doses of Paracetamol by men
appears to modify testosterone levels, a decrease in
the sperm parameters, specially sperm morphology,
and consequently semen quality.

INDEX OF AUTHORS
A
Abe, Flávia R. – 256
Adamoski, Douglas – 304
Aguera, Raul Gomes – 109
Aguiar, Gilberto Santos – 131
Aguiar, Larissa Muratori – 298
Aguilera, Mariana – 62
Alberton, Michele Debiasi – 195
Albino, Danielle Bibas Legat – 159
Albuquerque, Anjaina Fernandes – 206
Albuquerque, Polianna Lemos Moura Moreira – 114, 115
Alcântara, K.C. – 80
Alencar, Severino Matias – 229
Alexandre, Angela de Oliveira – 273
Alfieri, Daniela Frizon – 133, 137
Allen, David – 62
Almeida, Eduardo Alves – 176
Almeida, Elaine Renata Motta – 208
Almeida, Fernando G. – 158
Almeida, Gabriela de Oliveira – 223
Almeida, Giulia Forni – 228
Almeida, Jéssica Azarias – 42
Almeida, Mariana – 230
Almeida, Stefano Zorzal – 174
Almeida, Vivian Romero Santiago – 241
Almeida, William – 144
Alves, Carolina Esmeraldo Lima – 68
Alves, Gessé de Souza – 109
Alves, Gutemberg – 64
Alves, Jonas Alher Meira – 121, 122, 133, 137
Alves, Paula Daniela Sabino de Freitas – 219
Alves, Pedro Adolfo Pereira – 123
Alves, Rômulo Couto – 176
Amaral, Francieli Ubirajara India – 181
Amaro, Beatriz Oliveira – 305
B
Baccule, Nicole Santos – 151
Bagatin, Julia de Toledo – 27, 32
Bagio, Jéssica – 152
313
Andrade, Ana Rosa Brissant – 37, 56
Andrade, Ítalo Bertoni Lopes – 41, 165
Andrade, Ítalo B.L. – 178, 211
Andrade, Wanessa Machado – 42
André, Leiliane Coelho – 113, 125
André-Miral, Corrine – 48
Angelo, Ana Beatriz Silva – 188, 196
Antonio, Ananda da S. – 158
Antunes, Bibiana Pereira – 197
Antunes, Débora R. – 275
Antunes, Lusania Maria Greggi – 192
Antunes, Marina Venzon – 84
Antunes, Marina Vezon – 77, 102, 123, 128, 234, 301
Appelt, Patrícia – 277
Aquino, Ariana Musa – 201
Arakawa, Nilton Syogo – 269
Araki, Koiti – 271
Arantes, Ana Carolina Furiozo – 101
Araujo, Bruno Vinicios Silva – 184
Araujo, Karen Rafaela Gonçalves – 249
Araújo, Maria Eduarda Lima – 222, 305
Araujo-Souza, Patrícia Savio – 286
Arbo, Marcelo D. – 266
Arbo, Marcelo Dutra – 112, 134, 197, 200, 300
Arena, Arielle Cristina – 198, 204, 207, 210, 216
Arroteia, Kelen Fabiola – 49
Arruda, Fernanda Wolff da Silva – 129, 130
Aschner, Michael – 189
Assis, Silvia Romano – 32
Augusto, Pedro Esteves Duarte – 229
Avila, Carolina Martins – 254, 289
Ávila, Ricardo Andrez Machado – 147
Azevedo, Camilla de Marchi Sanches – 205, 212, 213
Bah, Homegnon Antonin Ferréol – 294
Bah, Homègnon Antonin Ferréol – 310
Bairros, André V. – 126, 237
Bairros, André Valle – 47, 73, 74, 75, 116, 119, 135, 236, 311 Bigão, Vitor Luiz Caleffo Piva – 94, 106
Baisch, Ana Luíza Muccillo – 175 Bigão, Vítor Luiz Caleffo Piva – 142, 226, 244
Bando, Érika – 109 Biondi, Ilka Borges – 68
Barbim, Paula Donate – 262 Birk, Letícia – 95, 239, 250
Barbisan, Luis Fernando – 201 Boff, Bruna de Souza – 242
Barbosa, Alyne Maria da Costa – 285 Boff, Everton – 299
Barbosa, Fábio de Souza – 250 Bombazar, Amanda – 147
Barbosa, Ingrid Lopes – 83 Bondan, Amanda Pacheco – 81
Barbosa Jr, Fernando – 85, 171, 192 Bonfioli, Maysa Guilherme – 248
Barbosa Junior, Fernando – 90 Borba, Maria Eduarda – 199, 215
Barbosa, Maria Clara de Oliveira – 184 Bordoni, Leonardo S. – 233, 247
Barcellos, Leonardo José Gil – 168, 181 Borges, Amanda Cecília Guimarães – 53
Barotto, Adriana Mello – 159 Borges, Cibele dos Santos – 180, 184, 187, 188, 193, 196
Barroso, Gilberto Fonseca – 173, 174 Borges, Gabriela Ramos – 78, 112, 134, 143
Barros, Silvia Berlanga de Moraes – 32 Borges, Pedro H.S. – 245
Bashash, Morteza – 306 Borgmann, Gabriela – 179, 272, 303
Bastiani, Marcos Frank – 81, 146, 167, 251 Bortoli, Stella – 302
Batista, José Márcio Machado – 114, 115 Bosquetti, Bruna – 58
Battastini, Ana Maria Oliveira – 185 Bouez, Charbel – 46
Bauermann, Lauren – 138 Braga, Ádrya Lariela Lima – 218
Bauermann, Liliane de Freitas – 194 Braga, Douglas Evangelista – 291
Baú, Morgana – 176 Braga, Jacqueline Ramos Machado – 68
Bechtold, Bruna Assunção – 40, 219 Braz, Juliana Machado – 298
Bedor, Danilo César Galindo – 56 Bresolin, Nilzete Liberato – 117
Belo, Vinícius Silva – 248 Bresolin, Tania Mari Bellé – 103
Benvenutti, Danyela Francine – 103 Brito, Maria Carolina Santos Ribeiro – 238
Benvenutti, Larissa – 38, 57, 59, 267 Broering, Milena F. – 271
Benvenutti, Régis Carlos – 299 Brohem, Carla Abdo – 58
Berlato, Dener G. – 126, 237 Brugnera, Débora Schmitz – 145
Berlato, Dener Gomes – 73, 74, 116, 119, 236, 311 Brum, Rodrigo de Lima – 175
Berlinck, Débora Zorrón – 89, 104 Brunello, Giovanna Cristina Spagnuolo – 133, 137
Berna, Gabriel da Costa – 293 Bruni, Aline Thais – 61, 67, 153, 154, 160, 161
Berta, João Antonio – 199, 215 Bruno, Vitor – 87
Berti, Fernanda Vieira – 70 Buchele, Maria Luiza Caneiro – 25
Bertoldi, Fernando – 293 Burns, Michael J. – 289
Betti, Andresa Heemann – 293 Busato, Maria Amelia de Castilhos – 84
Bhering, Cecília de A. – 158 Buzzi, Fátima C. – 267
Biazus, Inara Carbonera – 181 Buzzi, Fatima de Campos – 103

C
Cabral, Heloisi – 272 Carvalho, Deoclécio Lustosa – 37, 56
Cabral, Yngrid dos Santos – 131 Carvalho Filho, Sérgio de Morais – 51, 63
Cabrices, Oscar G. – 141, 227 Carvalho, José A.M. – 126
Cademartori, Pedro Henrique Gonzalez – 55 Carvalho, Larissa A.C. – 259
Caetano-Silva, Maria Elisa – 229 Carvalho, Larissa Anastacio da Costa – 32, 253, 257, 258
Calori-Domingues, Maria Antonia – 229 Carvalho, Larissa Anastácio da Costa – 255
Camarena, Denisse Esther Mallaupoma – 27, 32 Carvalho, Murilo – 304
Camargo João Lauro Viana – 203 Cassanego, Gabriela Buzatti – 194
Camargo, João Lauro Viana – 165 Castilhos, Maria Amélia – 81
Campos, Dyene Nascimento – 264, 265 Castilhos, Zuleica Carmen – 290
Canavez, Andrezza Di Pietro Micali – 58 Castro, Jade Simões – 61, 161
Capelo, Melissa Figueiredo – 312 Castro, Juliana Cristina – 109
Capp, Edison – 301 Catalano, Shadia M.I. – 62, 287
Cardoso, Leonardo Correa – 237 Catarino, Carolina Motter – 58
Cardoso, Leonardo Corrêa – 74, 116, 119, 135 Cattani, Shanda – 185
Cardoso, Maria Regina Alves – 166 Cavalcante, Luiz A.F. – 275
Cardoso, Marilia Santoro – 72 Cavichion, Rafael Filipe Battisti – 28, 29
Carlos Rafael – 267 Cazarin, Karen – 66
Carlson, Renato Romera – 143 Caznok, Ana Clara – 304
Carneiro, Gabriel Reis Alves – 76, 96, 100 Ceranto, Daniela de Cassia Faglioni Boleta – 199
Carriço, Murilo Ricardo Sigal – 86, 92, 172 Cesaro, Humberto Luis – 176
Carvalho, Angélica Romão – 44 Cestari, Marta Margarete – 144, 286
Carvalho, Chrissie Ferreira – 310 Cestonaro, Larissa V. – 266
Cestonaro, Larissa Vivan – 197, 200 Costa, Daisy Oliveira – 294, 310
Chaves, Pamella Eduardha Espindola – 261, 264, 265, 268 Costa, Eliene dos Santos da Silva – 222, 305
Chequer, Farah Maria Drumond – 246, 248 Costa, Eric Augusto Caravaggio – 166
Chimendes, Nayomi A. – 237 Costa, Gabriela Vanini – 158
Chimendes, Nayomi Andrade – 116, 119 Costa, Jose Luiz – 89, 98, 155, 235, 240
Chimendes, Nayomi de Andrade – 73, 74, 135, 236, 311 Costa, José Luiz – 72, 83, 101, 104, 118, 148, 150, 238, 249
Chinaglia, Kauê de Oliveira – 89, 101 Costa, Karina Oliveira – 152, 162
Chinaglia, Kauê O. – 240 Costa, Larissa Gabrielli – 40
Chisté, Renan Campos – 305 Costa, Letícia M. – 233
Choksi, Neepa – 62 Costa, Luiz Filipe – 280, 281, 283, 284
Cianci, Julio Cesar – 40 Costa, Luiz Guilherme Alonso – 201
Cisilotto, Julia – 132 Costa, Meg Cristina da Castilho – 58
Coelho, Júlia França Figueredo – 248 Costa, Nayara de Souza – 144, 190
Coelho, Maria Paula Mancini – 40 Costa, Quezia dos Santos – 133, 137
Coelho, Matheus Campelo da Costa – 76 Costa, Rony Anderson Rezende – 235
Cole, Amanda Carolina – 181 Costa-Valle, Marina Tuerlinckx – 197
Colla, Guilherme – 28, 29 Costa, Vitoria Cadore – 181
Concato, Virginia Marcia – 269 Couto, Angélica Garcia – 103
Conchon-Costa, Ivete – 269 Couto, Nilton – 230
Conte, Fernanda M. – 266 Couto, Tauer J.G. – 247
Cordeiro, Gabriela Batista Cavalcanti – 129, 130 Creczynski-Pasa,Tânia B. – 132
Cordenuzzi, Dorys Angela – 57, 59 Cruvinel, Vanessa Resende Nogueira – 306
Corralo, Vanessa da Silva – 145 Cruz, Juliana Varella – 55, 286
Corrêa, Brunna F. – 247 Cunha, Fernando – 262
Corrêa, Rogerio – 103 Cunha, Kelly F. – 98
Costa, Ana Carolina Conchon – 130, 136 Cunha, Kelly Francisco – 104, 155
Costa, Anna Carolina de Moura – 246 Cunha, L.C. – 80, 105
Costa, Brenda Natasha Souza – 166 Cunha, Ricardo Leal – 155, 232, 238
Costa, Bruno Ruiz Brandão – 94, 106, 142, 182, 224, 226, Cunha, Viviane Augusta de Medeiros Garcia – 276
244 Custódio, Flávia Beatriz – 221, 291, 292, 295
Costa, C.D.D. – 80

D
Dakic, Vanja – 40, 46 Demets, Mariana Barbieri Alvarez – 88
Dalanhol, Carolina Silveira – 78, 143 Denardin, Elton Luis Gasparotto – 86, 92, 172, 225
Dalbosco, Juliana Santos – 238 Denkena, Isadora Locilento – 155
Dal-Cheri, Beatriz Kopke de Assis – 273 Deolindo, Carolina Turnes Pasini – 97
Dallegrave, Eliane – 99, 197, 200 De Vecchi, Rodrigo – 40, 46
Dalmagro, Mariana – 199, 215 Devoz, Paula Pícoli – 192
Dalmazzo, André Kusser – 311 Devóz, Paula Pícoli – 171
Daniel, Caroline – 145 Dias, Sandra Martha Gomes – 304
Dantas, Joao Artur Diogenes – 188, 193, 196 Dias, Wanessa Amorim – 34, 69
Debiasi, Michele Alberton – 38 Diniz, Fabiana Barbosa – 291
De Carli, Diego M. – 126 Domingos, Líllian Maria Borges – 290
Delwing-Dal Magro, Débora – 272 Domingues, Romênia – 304
Delwing-De Lima, Daniela – 272 Donadel, Guilherme – 199, 215
De Martinis, Bruno Spinosa – 82, 94, 106, 108, 142, 182, 224, Dorta, Daniel Junqueira – 156, 157
226, 244

E
Eberlin, Samara – 50 Esteves, Iuri – 282
Echterhoff, Marcelo Rodrigo Franke – 38 Estumano, Saulo Braga – 222, 305
Eifler-Lima, Vera Lucia – 185 Etcheverry, Bibiana Frasson – 261, 264, 265, 268
El Haddad, Lohanna Pereira – 94, 224 Everton Boff – 145
Eller, Sarah – 78, 95, 99, 112, 134, 239, 250

F
Fabichak, Ana – 97 Facchini, Gustavo – 50
Fabris, Andre Luis – 87 Faria, Patricia Miranda – 66, 287
Fabris, André Luis – 140, 249 Farias, Beatriz Valentim – 115
Farias, Evelyn Rayani Araújo – 30, 33, 263 Flaws, Jodi A. – 201
Farias, Fabiane Moreira – 225 Flesch, Ingrid – 300
Farias, Natália Oliveira – 206 Fock, Ricardo – 262
Farsky, Sandra – 262 Foleis, Vanessa Kaplum – 276
Farsky, Sandra H.P. – 271 Fonseca, Ivana Alice Teixeira – 187, 193
Faustman, Elaine M. – 286 Fonseca, Karoline Kuhnen – 130
Fava, Luis Paulo – 40, 219 Forini, Mariana M.L. – 275
Favreto, Camila – 81 Fortuna, Milena – 168, 181
Feitosa, Emanuel Kenedy – 180, 184 França, Liana Conrado – 138
Felix, Renata Gleysiane de Sousa – 180, 184, 187, 188, 193, Freddo, Natália – 168, 181
196 Freeman, Elaine – 287
Feltrim, Fernando – 280, 281, 284 Freeman, Harold S. – 206
Fernandes, Eduarda – 124 Freire, Josiane Oliveira – 280, 281, 284
Fernandes, Fábio Henrique – 214 Freire, Livia Horrana Forte – 188, 196
Ferrazza-Heitich, Magda – 179 Freitas, Bruno Toledo – 142, 224
Ferreira, Ana Maria da Costa – 255 Freitas, Daniel Roberto Coradi – 298
Ferreira e Silva, Hendyelle Rodrigues – 114, 115 Freitas, Mariana – 81
Ferreira, Fernanda S. – 266 Freitas, Marisa – 124
Ferreira, Gabriela Kozuchovski – 272 Freitas, Vanessa M. – 27, 259
Ferreira, Julia Gabriele de Jesus – 293 Fugazza, Jonas – 274
Ferreira, Maria Augusta Drago – 114, 115 Furtuoso, Marcella Miranda Siqueira – 36, 39
Figueira, Ana Carolina Migliorini – 49 Fusco-Almeida, Ana Marisa – 44
Figueiró, Fabrício – 185 Fuzinaga, Thais – 50, 52
Figueredo, Kássia Caroline – 194 Fuzinaga, Thaís Y.T. – 256
Fitarelli, Bruna – 97

G
Gaca, Marianna – 282 Göethel, Gabriela – 185, 300
Gagosian, Viviana Costa – 55 Goetten, André L.F. – 132
Gagosian, Viviana Stephanie Costa – 286 Golçalves, A.P. – 180
Gaitani, Cristiane Masetto – 88 Goldoni, Fernanda C. – 267
Gale, Nathan – 282 Gomes, Francisca Tayná da Silva – 187, 193
Galiciolli, Maria Eduarda A. – 209 Gomes, Gabriela Cristiane Mendes – 261, 264, 265, 268
Galiciolli, Maria Eduarda de Andrade – 190 Gomes, Geovana Maria de Lima – 96, 100
Ganzerla, Melissa Dibbern – 49 Gomes-Júnior, Erival Amorim – 294, 310
Garcia, Ana Letícia Hilário – 293 Gomes, Nayna Cândida – 82, 106, 108, 226
Garcia, Cristina – 46 Gomes, S.A. – 105
Garcia, Solange – 200, 300 Gomes, Thaisângela Rodrigues Lopes e Silva – 30, 33
Garcia, Solange C. – 266 Gonçalves, Manoela Daiele – 269
Garcia, Solange Cristina – 185, 197 Gonçalves, Michelly Rodrigues – 306
Gaspar, Lorena R. – 256 Gonçalves, Odinei Hess – 276, 277
Gaspar, Lorena Rigo – 50, 52 Gonzaga, Alexsandro Pinto – 250
Gasparotto Junior, Arquimedes – 199, 215 Gonzalez, Marcelo – 64
Gastaldi, Alessandra Betina – 272, 274 Goodall, Sharon – 282
Gehlen, Günter – 293 Goulart , Christiane G.L. – 247
Gemelli, Bianca Aparecida Martins – 176 Goulart, Cristiano O.L. – 233, 247
Geraldino, Barbara Rodrigues – 131 Gouveia, Giovanna – 143
Giannini, Maria José Soares Mendes – 44 Gouveia, Giovanna Cristiano – 78, 112, 134
Gianvecchio, Daniele Muñoz – 150 Govoni, Bruna – 143
Gianvecchio, Victor Alexandre Percinio – 150 Graiczik, James Ramires Penteado – 194
Gianvecchio, Victor A.P. – 240 Grando, Ana Paula – 77, 102
Gimenes, Izabela – 64 Grando, Luciana Grazziotin Rossato – 181
Giovagnoli, Stefano – 103 Granjeiro, Jose Mauro – 273
Girassole, Alessandra – 304 Grillo, Renato – 275
Girotto, Edmarlon – 121, 122, 133, 137 Guex, Camille Gaube – 194
Gloria, Maria Beatriz A. – 292 Guidoni, Camilo Molino – 121, 122, 133, 137
Gloria, Maria Beatriz Abreu – 291 Guiloski, Izonete C. – 209
Gluzezak, Ana J.P. – 256 Guimarães, Ana Tereza Bittencourt – 169, 205, 212, 213
Gluzezak, Ana Júlia Pasuch – 52 Gündel, Augusto R. – 126
Goding, Colin – 259 Guterres, Fernanda S. – 128, 234
Godoi, Alexandre Barcia – 118 Guterres, Silvia S. – 271
Godoi, Manuella Machado – 28
H
Habe, Priscila – 62
Habib, Isabela A.3 – 126
Hahn, Roberta Zilles – 77, 81, 102, 146, 167, 251
Hardie, George – 282
Harumi, Luciana – 259
Heck, Amanda Szymansky – 194
Heghes, Crina – 254, 289
Heluany, Cíntia – 262
I
Indolfo, Nathalia de Carvalho – 49
Ineu, Rafael Porto – 276, 277
J
Jacomassi, Eliza – 199
Jager, Alessandra Vincenzi – 223
Jesus, Iracina Maura – 166
Jorge, Bárbara Campos – 198, 204, 207, 210, 216
K
Kagami, Luciano Porto – 185
Kahl, Júlia M.M. – 89
Kakuda, Priscila – 79
Kalv, Danielle Crystiane – 302
Kampmann, Micheli Gabardo – 299
Kassuya, Cândida Aparecida Leite – 204
Kawakami, Camila M. – 256
Kawakami, Camila Martins – 50, 52
Kayser, Juliana Machado – 293
Kieling, Ketelin Monique Cavalheiro – 225
Kishishita, Juliana – 37, 56
L
Lamb, Liliane Weber Bolfe – 293
Lameira, Christian Neri – 305
Lanaro, Rafael – 101, 155
Latorre, Andreia Oliveira – 62, 66, 287
Laurentino, Ana Olívia Martins – 197, 200
Leal, Leila Bastos – 37, 56
Leal, Mirna Bainy – 197, 200
Leão, Matheus C. – 271
Leimann, Fernanda Vitória – 276, 277
Leite, Jacqueline Alves – 30, 33, 263
Leme, Adriana Paes – 304
Leme, Daniela Moraes – 55
Leme, Daniela Morais – 65, 286
Lesniewski, Isabela Ramos – 212
Lima, Aliny Pereira – 30, 33
Lima, Crystiane Calado – 180
Lima, Daina – 176
M
Macarini, Leanna Camila – 169
Macedo, Larissa Matuda – 30, 33
Machado, Cibele da Silva Barbosa – 301
Herrala, Mikko – 206
Hoff, Rodrigo – 97
Holanda Júnior, Wanderley Pinheiro – 241, 243, 308
Holanda, Wiron Pimentel – 163, 218, 312
Honório Júnior, José Eduardo Ribeiro – 163, 218, 312
Hort, Mariana Appel – 48
Hoscheid, Jaqueline – 199, 215
Hueza, Isis Machado – 208
Irioda, Ana Carolina – 26, 43, 144, 190
Itinose, Ana Maria – 212
Jorge, Maria Helena Prado de Mello – 150
Jorge, Roberta Jeane Bezerra – 68
José, Gustavo Pinheiro Coelho – 162
Justulin Junior, Luís Antonio – 201
Kleemann, Cristian – 97
Kleine, Tamila – 274
Kocks, Grace – 289
Koepp, Janice – 28, 29, 70
Kohler, Cristofer José Weege – 191, 195
Kohlrausch, Ramona – 128
Kohori, Natália Akemi – 186
Krotulski, Alex J. – 98
Krüger, Nathália Ronconi Zilli – 202
Krutzmann, Maria Eduarda – 128
Lima, Daniela Delwing – 179
Lima, Geovana Cristina Ribeiro – 214
Lima, Luiza Siqueira – 144, 190
Lima, Marcelo de Oliveira – 166
Lima, Natália Cavalcante Barbosa – 68
Lima, Thania Rios Rossi – 189
Lima Thania Rossi Rios Rossi – 203
Linden, Rafael – 77, 81, 84, 102, 120, 123, 128, 146, 167, 234,
251, 301
Lini, Renata Sano – 54, 109, 151
Lizot, Lilian de Lima Feltraco – 81, 146, 167
Logan, Barry K. – 98
Lopes, Bruna Gonçalves – 38
Lourenço, Emerson Luiz Botelho – 199, 215
Lovatel, Ivy Bauer – 75
Lucinda-Silva, Ruth Meri – 57, 59
Luz, Heloisa Peres – 93
Machado, Isabel Daufenback – 38, 191
Machado, Michel Mansur – 261, 264, 265, 268
Machado, Sérgio de Paula – 76
Machado, Simone Caetani – 280, 284 Medeiros, Elvis A. – 240
Machinski Junior, Miguel – 109 Meirelles, Gabriela de Paula – 91
Maffi, Victoria Costa – 168 Mello, Raissa – 143
Magalhaes, Danielle de Paula – 243 Melo, Paolo Oliveira – 180, 184
Magalhães, Danielle de Paula – 241, 308 Melo, Saulo Nascimento – 248
Magalhães, Karla do Nascimento – 115 Mendanha, Sebastião Antônio – 60
Magalhães, Washington – 55 Mendes, Michele Polyana Rocha – 113
Magosso, Natália – 201 Mendes, Rosivaldo de A. – 297
Maia, Affonso Enriques Montagner – 311 Mendonça, Izadora Caroline Furtado – 31, 51, 60
Maia, Thayná Patachini – 272 Menegasso, Aloma Santin – 168
Maldaner, Adriano Otávio – 232 Menegatti, R. – 105
Maluf, Ana Cristhina Sampaio – 148 Menezes-Filho, José Antonio – 294, 310
Manfio, Cleofas Sates – 197 Menezes, Francisco Paz – 250
Manoel, Beatriz de Matos – 198, 204, 207, 216 Messias, Nayara Casagrande – 117, 159
Marchioni, Camila – 93, 107, 109, 138, 151, 159, 162, 242 Migliato, Ketylin Fernanda – 44
Marciano, Luiz Paulo de Aguiar – 280, 281, 283, 284 Milanez, Guilherme Paier – 83
Marco, Mariana – 293 Miranda, Antônio M.M. – 297
Marcon, Scheila – 145 Miranda, Raul Ghiraldelli – 156, 157
Marek, Carla Brugin – 212, 213 Miranda-Sapla, Milena Menegazzo – 269
Maria-Engler, Silvya S. – 256, 259 Molina, Higor Severo – 86, 172
Maria-Engler, Silvya Stuchi – 27, 32, 52, 253, 255, 257, 258 Monteiro, Fabíola Branco Filippin – 25
Mariani, Noemia Aparecida Partelli – 214 Monteiro, Silvia Gonzalez – 74, 236, 237
Marigliani, Bianca – 279, 309 Moraes Junior, Manoel Oliveira – 255
Marinho, Pablo Alves – 224, 226 Moraes, Liliana S. – 126
Mariotto, Lívia Salviano – 154, 160 Moraes, Natália Valadares – 88
Marize Campos Valadares – 53 Moraes, Niely Galeão da Rosa – 170
Marques, Ana Maria – 263 Morais, Déborah Araújo – 85, 90
Marques, Carla Pintas – 306 Morales, Daniel Alexandre – 206
Martinez, Victor Otero – 294 Mora, Tamara Dal – 25
Martínez, Victor Otero – 310 Moreira, Beatriz – 124
Martins, Aline Franco – 83 Moreira, Camila Francisco – 113
Martins, Aline R. – 275 Moreira, Camila Queiroz – 298
Martins, André Bittencourt – 147 Moreira, Rhubens Levy Rodrigues – 114
Martins, Isarita – 131, 280, 281, 284, 285 Moreira, Suyane da Silva – 204, 216
Martins Jr., Airton Cunha – 189 Moretti, Gabriela – 223
Martins, Mayane Emanuela Melo Lopes – 243 Mori, Matheus P. – 259
Martins, Mayane Emanuella Melo Lopes – 241 Mossini, Simone Aparecida Galerani – 54, 109, 151
Massucato, Lucas Eduardo – 54 Motomura, Larissa Tiemi Akamine – 54
Matias, William Gerson – 274 Moura, Kézia – 50
Matos, Ricardo Luiz Nascimento – 269 Moura, Maria Joana Nogueira – 180, 184, 187, 188, 193, 196
Matsui, Andresa – 219 Mozzato, Mateus Timbola – 168, 181
Mattos, Cristiane Bastos – 293 Muccillo-Baisch, Ana Luiza – 48
Mattos, Guilherme – 46 Müller, Gabrielle do Amaral e Silva – 176
Mazete, Fernanda Pine S. – 127 Muñoz, Rodrigo A.A. – 245
McEwan, Michael – 282 Murata, Rosana Zoriki Hosomi – 62

N
Nagaoka, Lívia Trippe – 198, 207 Nogueira, Janer Alves – 138
Nardi, Jessica – 181, 300 Nogueira, Jéssica Bueno – 204, 210, 216
Nascentes, Clésia C. – 233 Nolasco, Daniela – 113
Nascimento, Marcelo Henrique Santana – 47, 73, 74, 116, Nolasco, Daniela M. – 125
119, 236, 311 Nold, Juliana C. Lago – 27
Nascimento, Marcelo H.S. – 237 Noma, Isabella Harumi Yonehara – 257
Nascimento, Sabrina – 300 Noma, Isabella H.Y. – 259
Nazari, Evelise Maria – 202 Nonino, Elisa de Castro Wille – 65
Nerilo, Samuel Botião – 109 Nossol, Edson – 245
Ness, Sandro Luis Ribeiro – 301 Nunes, Cleia Justino – 255
Neves, Gustavo Machado – 185 Nunes, Rafaella Ferreira Nascimento – 131, 283
Neves Júnior, Luiz F. – 249 Nunes, Viviane Abreu – 70
Nogueira, Caroline Lacerda – 86, 172, 225
O
Ogasawara, Maryanne – 62 Oliveira, Lara Luiza Freitas – 246
Oliveira, Adriana Sousa – 158 Oliveira, Leandro Leal Rocha – 30, 33
Oliveira, Aline Lima – 232 Oliveira, Letícia Cimó – 311
Oliveira, Allan Santos – 166 Oliveira, Marcos Antônio Fernandes – 201
Oliveira, Ana Paula – 181 Oliveira Neto, J.R. – 80
Oliveira, Antonio Anax F. – 254, 289 Oliveira, N.R.L. – 80
Oliveira, Arthur Lima – 156, 157 Oliveira, Sarah Carobini Werner de Souza Eller Franco – 116
Oliveira, Celinalva da Silva Lima – 232 Oliveira, Sarah C.W.S.E.F. – 126
Oliveira, Claudete C. – 72 Oliveira, Sidney Julio Vieira – 222
Oliveira, Cláudia S. – 209 Oliveira, Silvana Ruella – 85, 90
Oliveira, Cláudia Simone Baltazar – 222, 305 Oliveira, Therezinha Maria Novais – 274
Oliveira, Cláudia Sirlene – 26, 43, 144, 190 Oliveira, Tiago F. – 126
Oliveira, Danielle P. – 256 Oliveira, Tiago Franco – 78, 95, 99, 112, 116, 134, 239, 250
Oliveira, Danielle Palma – 55 Olympio, Kelly Polido Kaneshiro – 166
Oliveira, Érica Aparecida – 258 Osaki, Luciana Harumi – 27
Oliveira, Franciele Aparecida Mendes – 190 Ossanes, Daniela Souza – 239
Oliveira, Jordana Meirelles – 121, 122 Otero, Ubirani Barros – 131
Oliveira, José Miguel P. Ferreira – 124 Ott, Isabela Ritter – 234
Oliveira, Juliana Ribeiro Ibiapina Leitão – 241, 243, 308

P
Pacassa, Pâmela – 38 Pereira, Lilian Cristina – 165
Pacheco, André L.B. – 237 Pereira,, Lilian Cristina – 186
Pacheco, André Lucas Bezerra – 73, 74, 116, 119, 135, 236, Pereira, Lílian Cristina – 41, 183, 189, 217
311 Pereira, Meire Ellen – 144
Padua, Amanay Sousa – 40 Pereira, Pedro Afonso de Paula – 232
Pais, Mariana Castello Novo – 62, 287 Pericolo, Suellen – 162
Paiva, Maria José Nunes – 113, 125 Perini, Camila Maria – 191, 195
Palmeira, Carlos Manuel Marques – 186 Perjessy, Gisele – 62
Pappis, Lauren – 194 Peruzzi, Caroline Portela – 185, 300
Papsun, Donna M. – 98 Pestana, Cynthia Bomfim – 65
Parabocz, Gisele Chibinski – 162 Petry, Adriana Ubirajara Silva – 250
Paschoalini, Beatriz Rizzo – 204, 210, 216 Petry, Andrea – 129
Paula, Eliza Bianchini – 107, 138 Pimenta, Camila de Almeida Perez – 37
Paula, Favero R. – 103 Pinc, Mariana Moraes – 199, 215
Paula, Fávero Reisdorfer – 47 Pinelli, Juliana Junqueira – 221
Paulo, Breno F. Pereira – 149 Pinheiro, Adriano da Silva – 50
Paulucci, Leticia Trevisan – 111 Pinheiro, Ana L.T.A. – 50
Pavanelli, Wander Rogério – 269 Pinheiro, Dávylla Rennia Saldanha – 218
Pavlak, Jaíne Luana – 205 Pinheiro, Fabriciano – 228, 288
Paz, Maria Elizabeth Gomes – 86, 92 Pinheiro, Jacqueline – 202
Pechansky, Flavio – 143 Pinheiro, Maria da Conceição Nascimento – 305
Pedralli, Bruna Cristiane Oliveira – 35, 36 Pinto, Nadja C. Souza – 259
Pedroso, Bruno – 302 Pires, Janaina Aparecida Cardoso – 62
Pego, Ana Miguel Fonseca – 99 Pires, Sumaia Araújo – 125
Peixoto, Paloma Vitória Lima – 41, 165 Piton, Yasmin V. – 266
Peixoto, Paloma V.L. – 178, 211 Plautz, Katherine – 179, 272, 274, 303
Pepato, Ana Claudia de Andrade – 66 Poça, Kátia Soares – 131
Perdoná, Gleici da Silva Castro – 142 Pohlmann, Adriana R. – 271
Pereira, Alessandra de Oliveira – 74, 236, 237 Polido, Lucas Roberto Ferreira – 183
Pereira, Artemia Kelly Holanda – 180, 184, 187, 188, 193, 196 Pompermeier, Aline – 181
Pereira, Edimar Cristiano – 208 Ponce, Júlio de Carvalho – 249
Pereira, Eduardo Manoel – 272 Pontes, Montcharles S. – 275
Pereira, Elizeu Chiodi – 166 Ponting, David J. – 289
Pereira e Silva, Jefferson – 91 Ponting, David. J. – 254
Pereira, Henrique Marcelo Gualberto – 76, 96, 100 Presgrave, Octavio – 64
Pereira, I.B. – 105 Priedols, Gustavo Abud – 121, 122
Pereira, João Paulo Goes – 166 Priedous, Gustavo Abud – 133, 137
Pereira, Leonardo da Cunha Boldrini – 273 Proença, Carina – 124
Pereira, Lílian C. – 178, 211 Pupo, André Sampaio – 207
Pereira Lilian Cristina – 203
Q
Queijo, Rodrigo Gonçalves – 253 Quintão, Nara L.M. – 267
Queiroz, Maria Eugênia Costa – 79 Quintas, Luís E.M. – 263
Queiroz, Thais Karolina Lisboa – 166 Quiozini, Nathaly de Matos – 205, 212, 213
Quintão, Nara Lins Meira – 38

R
Räisänen, Riikka – 206 Rios, Nathália Vieira – 261, 265, 268
Ramadan, Debora R. – 111, 127 Rizzi, Joyce Santana – 183, 217
Ramborger, Bruna Piaia – 92 Rizzo, Elizete – 169
Ramos, Adriano T.1 – 132 Rocha, Anna Carolina Furaer – 59
Ramos, David L.O. – 245 Rocha, Cássia C.S. – 297
Ramos, Silvia Aparecida – 162 Rocha, Cecília Cristina de Souza – 171, 192
Ramos, Thalita da Silva – 285 Rocha, Daniela Barbosa – 42
Ramos, Vitor Serrão – 295 Rocha, Danilo Galvão – 68
Rangel, Ana Lúcia Carrinho Ayroza – 205 Rocha, Raquel G. – 245
Rangel, Lara Luiza Pimenta – 173 Rocha, Thiago L. – 297
Rath, Susanne – 285 Rocha, Vanessa Aguiar – 201
Reck, Carolina – 132 Rodrigues, Caio Henrique Pinke – 61, 67, 154, 161
Reginato, Fernanda Z. – 237 Rodrigues, Gabriela Zimmermann Prado – 293
Reginato, Fernanda Ziegler – 73, 74, 75, 116, 119, 135, 194, Rodrigues, Leonardo Costalonga – 89
236, 311 Rodrigues, Marina Diaz – 86, 92, 172
Reis, Ana Carolina Casali – 198, 204, 207, 210, 216 Rodrigues, Taís B. – 240
Reis, Emily Marques – 28, 29, 70 Rodrigues, Vanessa Fernandes – 182
Reis, Karsonn B. – 245 Roehrs, Miguel – 75
Reis, Luciane Ayres Castro – 173, 174 Roehrs, Rafael – 86, 92, 172, 225
Remor, Aline Pertille – 176 Romero, Alessandra de Cássia – 229
Resener, Marisete Canello – 129, 130, 136, 159 Romoli, Jéssica Cristina Zoratto – 109
Rezin, Kéttulin Zomer – 242 Rosa, Victória Gomes – 73, 74, 116, 119, 135, 236, 237, 311
Ribeiro, Daniela – 124 Rossato-Grando, Luciana Grazziotin – 168
Ribeiro, Milena Mariano – 26, 43 Rossi, Bruna Franzon – 276, 277
Ricci, Maurizio – 103 Rudolf, Carline – 59
Richter, Eduardo M. – 245 Rufino, Ana T. – 124
Rigaudeau, Anne-Sophie – 46 Rysä, Jaana – 206
Rios, Nathalia Vieira – 264

S
Saboya, Andrea Luiza Rocha – 241
Saboya, Andréa Luiza Rocha – 243
Sá, Clodoaldo Antônio – 145
Sakakibara, Isarita Martins – 283
Saldanha, Geovane A. – 126
Saleh, Najla Adel – 25
Sales, Bianca Camargo Penteado – 41, 165
Sales, Thais Lorenna Souza – 246
Salles, Fernanda Junqueira – 166
Salles, Gabriela Pereira – 114, 115
Salomón, Janaína – 200
Salvadori, Daisy Maria Fávero – 214
Sampaio, Amanda Mello Kasper Vaz – 75
Sanches, Alex O. – 275
Sanches, Cristina – 246
Sandri, Silvana – 262, 271
Santana, Davi Pereira – 37, 56
Santana, Thatiane Nunes – 40, 219
Santiago, Etenaldo F. – 275
Santiago, Mayla Andra de Andrade – 222
Santiago, Vivian Romero – 243
Santin, José Roberto – 38, 57, 59, 103, 267
Santos, Bruno Pereira – 78, 112, 134, 143
Santos, Caio Cesar Araújo – 180, 184, 187, 188, 193, 196
Santos, Carlos Eduardo Matos – 103
Santos, Christiano – 153
Santos, Claudia Regina – 93, 107, 117, 129, 138, 159
Santos, Fabiana Pereira – 87
Santos, Jeniffer Farias – 70
Santos, Jordana Andrade – 34, 53, 69
Santos Junior, Wilson José Ramos – 106
Santos Júnior, Wilson José Ramos – 244
Santos, Lara C. – 237
Santos, Lara Celestina – 73, 74, 116, 119, 135, 236, 311
Santos, Nathália Ribeiro – 294, 310
Santos, Nícolas Guimarães – 197
Santos, Rachel – 74, 116, 119, 126, 135, 237
Santos, Rafaela Knak – 120
Santos, Raquel – 124
Santos, Sérgio Alexandre Alcantara – 201
Santos, Thaís Rosa Marques – 53
Santos, Thiago Santana – 298
Santos, Vanessa Farelo – 76, 96, 100
Santos, Vivian da Silva – 306
Saraiva, Illyushin Zaak – 176
Saraiva, Thalia Emmanoella Sebulsqui – 293
Sarpa, Márcia Sarpa de Campos – 131 Silva, Luíza Madureira – 163, 218, 312
Sartori, Alan Giovanini de Oliveira – 229 Silva, Maria Isabel Gonçalves – 145
Sartori, Daniela Carlos – 182 Silva, Mariana Cristina – 118
Scanferla, Deborah Thais Palma – 109, 151 Silva, Mateus Limerio Carlos – 188, 196
Scarano, Wellerson Rodrigo – 201 Silva, Priscilla Muniz Ribeiro – 40
Scavone, Cristoforo – 263 Silva, Rafael Araújo – 283
Schamne, Tatiane – 302 Silva, Rivia Regina Lopes – 263
Scharf, Pablo – 262, 271 Silva, Samanta de Matos – 44
Scherer, Juliana – 78 Silva, Stephanie Soares – 117, 159
Scherer, Juliana N. – 143 Silva, Victória da Costa – 114, 115
Schimith, Lucia Emanueli – 48 Silveira Filho, Jair – 242
Schmitt, Maria Laura Videiro – 172 Silvério, Alessandra Cristina Pupin – 280, 281, 284
Schmitz, Felipe – 266 Silvério, Kérolyn Aparecida – 288
Schneider, Ayda Henriques – 262 Simioni, Carmen – 202
Schroeder, Samilla Driessen – 242 Simon, Jaqueline – 205, 212, 213
Schuck, Desiree Cigaran – 58 Singulani, Junya de Lacorte – 44
Schwengber, Heloysa Talia – 212 Siqueira, Gabriella Ferreira – 54
Sebben, Viviane – 200 Siqueira, Janas – 131
Sebben, Viviane Cristina – 112, 134 Siqueira, Lisiane – 181
Seloto Danielle Gabriel – 203 Smalley, Keiran S.M. – 259
Seloto, Danielle Gabriel – 217 Smidt, Mariana – 251
Sérgio de Morais – 63 Soares, Daniel – 287
Siena, Ádamo D.D. – 259 Soares, Marcela de Oliveira – 111, 127
Silva, Adny Henrique – 25 Soares, Suelen Mendonça – 168, 181
Silva, Agnes Soares – 166 Sobjak, Thaís Maylin – 169
Silva, Alan Andrew dos Santos – 214 Sotelo, Êmily Clori – 261, 268
Silva, Aline Gabrielle Gomes – 180, 184, 187, 188, 193, 196 Sousa Junior, Wellington Tavares – 90
Silva, Ana Cléia Cardoso – 26, 43 Sousa Júnior, Wellington Tavares – 85
Silva, Ana Lucelha dos Santos – 188, 196 Sousa, Roberto Cesar Santos – 292
Silva, Artur Christian Garcia – 31, 34, 45, 51, 53, 58, 60, 63 Souza, Asley Thalia Medeiros – 37, 56
Silva, Bruna Espíndola – 93, 242 Souza, Daniela Cristina – 277
Silva, Carla Brigagão Pacheco – 182 Souza, Douglas – 293
Silva, Claudia Larissa Viana – 32 Souza, Isisdoris Rodrigues – 286
Silva, Denise Bousfield – 117 Souza, Israel Donizeti – 79
Silva, Erick José Ramo – 214 Souza, João Pedro Silveira – 185
Silva, Fabiano – 230 Souza-Kaneshima, Alice Maria – 54
Silva, Fernanda Coleraus – 205, 212, 213 Souza, Karla Aparecida de Oliveira – 150
Silva, Gerlane Modesto – 180 Souza, Luiz Claudio Cindra – 173
Silva, Graciele Machado – 111 Souza, Mirna Maciel D'Auriol – 125
Silva, Gustavo Henrique – 50 Souza, Natacha Medeiros – 263
Silva, Jonas Joaquim Mangabeira – 226 Souza, Patrick Vieira – 201
Silva, José Wellithom Viturino – 56 Souza, Tainá Brumate – 113, 125
Silva-Jr, Wilson A. – 259 Souza, Wanderson – 273
Silva, Juliana F. – 209 Stainki, Daniel Roulim – 237
Silva, Julia Rezende – 258 Steiner, Bethina Trevisol – 147
Silva Júnior, Clovis Reis – 223 Stein, Julia – 198, 204, 207, 210, 216
Silva Júnior, Flavio Manoel Rodrigues – 170, 175 Stival, Ana Clara Silva – 45
Silva, Laura Cé – 77, 102, 123, 301 Sugawara, Eduardo Kinio – 111, 127
Silva, Luciana Stein – 301 Summy, Maria Julia – 176

T
Tadeu, Vitória Costa – 225 Tennant, Rachael E. – 254, 289
Tamagno, Wagner – 181 Teodoro, João Soeiro – 186
Tanamachi, Amanda Rodrigues – 214 Thá, Emanoela Lundgren – 58, 286
Taruhn, Lillian Freitas – 136 Tonietto, Bruna Ducatti – 197, 200
Tavares, Kvetta Pinheiro Teixeira – 36, 39 Tostes, Rita C. – 182
Tavares, Renata S.N. – 256 Trajano, Christian Farias – 96, 100
Tavares, Renata Spagolla Napoleão – 52, 304 Tufik, Sergio – 111, 127
Tegner, Mariane – 234 Tumas, Vitor – 79
Tenfen, Adrielli – 303
U
Uchiyama, Mayara K. – 271 Uhera, Adriana H. – 259
Ugalde, Gustavo A. – 237 Umbuzeiro, Gisela de Aragão – 206
Ugalde, Gustavo Andrade – 73, 74, 75, 116, 119, 135, 236, 311

V
Valadares, Marize Campos – 30, 31, 33, 34, 35, 36, 39, 45, Veiverberg, Andriele – 293
51, 58, 60, 63, 69 Vellosa, Jose Carlos Rebuglio – 302
Valente, Letícia Cardoso – 198, 207 Verpaele, Steven – 306
Vasconcelos, Mailton – 143 Viana, Roberta Rodrigues – 99
Vasconcelos, Mayrla Emilia Dantas – 88 Vicente, Eduardo – 50
Vasconcelos Neto, Milton Cabral – 295 Vieira, Bruna Todeschini – 213
Vaz, Milena Menegazzo – 267 Viola, Patrícia Pacheco – 143
Vecina, Juliana Falcato – 40, 219 Viriato, Cristina – 41, 178, 211
Veiga, Ângela – 132 Vivani, Riccardo – 103

W
Waechter, Fernanda – 289 Willett, Catherine – 279
Wagner, Eduardo José – 191 Winter, Evelyn – 132
Wagner, Theodoro – 103 Wurzler, Gleicielle T. – 158
Walton, Sara E. – 98 Wyse, Angela T.S. – 266
Werner, Anne-Laure D. – 254

Y
Yli-Öyrä, Johanna – 206 Yonamine, Mauricio – 87, 91, 140, 249

Z
Zardeto, Giuliana – 199, 215 Zimmath, Michel – 303
Zauli, Danielle Alves Gomes Breno – 149 Zoghaib, I.V.J. – 105
Zazula, Matheus Felipe – 169 Zuravski, Luísa – 261, 264, 265, 268
Zebele, Patricia – 282
Zimermann, Francielli C. – 132
 PATROCINADOR OURO:

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ALTERNATIVE TOXICOLOGY

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