Guideline No: H G P T 4 1

Technical Guideline on Irritation, Sensitization and Hemolysis Study for Chemical Drugs
(Second draft) March 1st , 2004 Table of Contents
1 Introduction..................................................................................... ........................................3 2 Overall principles......................................................................................... ............................3 3 Irritation test.................................................................................................. ............................4 3.1 Definition.................................................................................................................................. 4 3.2 Objective................................................................................................................................... 4 3.3 Considerations in irritation test design ........................................................................................ 4 3.4 Skin irritation test ....................................................................................................................... 5 3.5 Irritation test of injection site...................................................................................................... 5 3.6 Ocular irritation test.................................................................................................................... 5 3.7 Irritation test for other routes at the administration site .............................................................. 5 4 Sensitization test ................................................................................................. ......................5 4.1 Definition.................................................................................................................................. 5 4.2 Objective................................................................................................................................... 6 4.3 Considerations in sensitization test design................................................................................... 6 4.4 Sensitization test of transdermal administration .......................................................................... 6

4.5 Sensitization test of injection administration ............................................................................... 6 4.6 Sensitization test of other administration routes.......................................................................... 7 4.7 Test method ...............................................................................................................................7 5 Hemolysis test ................................................................................................. .........................7 5.1 Definition.................................................................................................................................. 7 5.2 Objective................................................................................................................................... 7 5.3 Scope......................................................................................................................................... 7 5.4 Considerations in hemolysis test design ...................................................................................... 7 5.5 Test method ...............................................................................................................................8 6 Test report.............................................................................................. ..................................8 7 Evaluation of results............................................................................................. ....................8 8 Some considerations ................................................................................. ................................8 9 References....................................................................................... .........................................8 10 Appendix.......................................................................................... ........................................9 10.1 Method for irritation test .......................................................................................................... 9 10.1.1 Skin irritation test........................................................................................................... 9 10.1.2 Irritation test to injection site ............................................................................................... 11 10.1.3 Eye irritation test.................................................................................................................. 11 10.2 Method for sensitization test................................................................................................... 14

10.2.1 Passive cutaneous anaphylaxis test(PCA)...................................................................... 14 10.2.2 Active systemic anaphylaxis test (ASA)......................................................................... 14 10.2.3 Guinea pig maximization testGPMTand Buehler testBT .............................. 15 10.2.4 Skin phototoxicity test.................................................................................................. 17 10.2.5 Skin photoallergy test ................................................................................................... 18 10.3 Method for hemolysis test....................................................................................................... 19 10.3.1 general in vitro tube method (observation with eyes).................................................... 19 10.3.2 Improved in vitro hemolysis test method(spectrophotometer) ..................................... 20 11 Drafting notes .............................................................................................. ........................... 21 12 The author ............................................................................................. ................................. 23

1 Introduction
1.1 Definition and Objective The irritation, sensitization and hemolysis of chemicals is defined as the production of toxicity at the local site of administration, eg, irritation and sensitization, etc, and/or the systemic toxicity, eg, sensitization and hemolysis following the unoral application of a chemical drug products to eye, ear, nose, mouth, respiratory tracts, joint cavity, derma, recta, vagina, vein, artery, muscle, undermic, vein besides, theca, and so on. It is a part of preclinical safety evaluations. The drugs active substances and its metabolites, excipientsome relative components and physical-chemical properties (PH, osmotic Pressure, etc) are all lead to irritation and/or sensitization/or hemolysis, and serious toxicity may affect safety and efficacy. As a result, it is necessary to study the local and /or systemic toxicity of the drug product after being applied, to suggest the possible toxicity, target organ of toxicity,

safety range and parameters of clinical monitoring, further, give reference to clinical detoxifcation or rescuing methods and guarantee safety and efficacy of the drug in clinic. 1.2 Scope The Guideline is applied to the study of irritation, sensitization and hemolysis of chemicals. 1.3 The main content The Guidelines main content includes the overall/ general principles and commonly used test methods of study on irritation, sensitization and hemolysis of chemicals.

2 Overall principles
The study of irritation, sensitization and hemolysis must comply with GLP Nonclinical Study Good Practices The experiment design should adhere to randomized, control, repetitive principles. The study of irritation, sensitization and hemolysis should take account of toxicity mechanism, influent factors, clinical significance, indication and administration method. Selection of relevant animal species should be based on proposed experimental model and observational parameters. Gender, age, and physiological state should meet the experiment requirements. The experimental drug should be identical with those applied to human body. The drug delivery, including dose, route and treatment regimen, is determined by experiment model and proposed clinical practice. Applying place is expected to be the observational place, and peripheral tissue or exposure place should also be considered to be observed at the same time. If the test substance that will be investigated in clinic is identical or analogous with other non-clinical safety study, eg, long-term toxicity, and the findings can reflect one of the toxicity among irritation, sensitization and hemolysis, the corresponding toxicity study may be omitted. To study the irritation, sensitization and hemolysis of the chemical, it is best to make clear of components of the test substanceincluding the names and

amount of principal drug excipientimpurity. If marketed drug product which have the same route of administration with the test substance indicate certain toxicity in the research of irritation and/or sensitization/or hemolysis, it is necessary to conduct compare study, and the toxicity should be equivalent or less compared with the existing ones. The sample size is determined by basic statistics theory. Due to the limits of animal experiments, irritation, sensitization and hemolysis of the chemical shall be integrally evaluated with pharmacy, pharmacology, toxicology and clinical useto provide the possibility of clinical use of the chemical and considerations when used in clinic.

3 Irritation test
3.1 Definition Irritation test means reversible changes at administration site after non-oral drug product delivery, irreversible tissue damage is named as corrosion. 3.2 Objective Observation on irritation after animal locally exposure to the test substance, suggestion on possible inflammation reaction, denaturalization and necrosis at the administration site when clinical use. 3.3 Considerations in irritation test design 3.3.1 Selection of animal The selection of animal should be based on the observational parameters and the planned animal model, in general, the estimation of each experiment is expected to select one species of animal. 3.3.2 Frequency and duration of administration The frequency and duration of administration should be deicide according to proposed clinical use. Repeated dose test is conducted everyday, duration is less than 4 weeks. The test substance which is administrated casually can be studied by single-dose test. 3.3.3 Reversible observation To investigate the recover after toxicity reaction, convalescence observation after stopping

administration is recommendedReactions in local and relative places should be included in reversible evaluation of the toxicity reaction. 3.3.4 Test substance The test substance should be consistent with that of human body. 3.3.5 Control group The negative control is adopted menstruum and/or excipient. Active control or marketed drug preparation shall be added if necessary. 3.3.6 Dose Dose design shall mainly consider concentration and total dose of the test substance. In general, one of the concentration should be consistent with that in clinic, and dose can be modified through changing the frequency of administration. However, it is not feasible to increase plaster thickness or extend plaster area to increase dose in skin irritation study. Irritation tests of different concentrations should be conducted where necessary. 3.3.7 Manner of delivery 3.3.7.1 The treatment regimen should be as close as possible to that for clinical use. 3.3.7.2 The route and site of administration should be consistent with clinical use. 3.3.7.3 Anatomical and physiological characters at the administration site should be taken into account when design volume, velocity and frequency of administration. 3.3.8 Irritation tests of different administration routes can be performed in the same animal as long as no interaction among different routes and the systemic tolerance permitted. The control test may also be conducted at opposite side. 3.3.9 Data Statistic Appropriate statistical methods should be selected on the basis of models and experimental methods. 3.4 Skin irritation test 3.4.1 Skin irritation test should be performed in terms of transdermally applied drug and nonoral drug product to which skin possibily expose. 3.4.2 When skin irritation has been evaluated in acute toxicity test, long term toxicity test or

skin sensitization test, and the preparation of the test substance is identical or comparable with that of the proposed clinical study, a separate skin irritation test may not perform. 3.4.3 Test method See the commonly used methods and related literature for irritation test in appendix. 3.5 Irritation test of injection site 3.5.1 For injection, irritation test at injection site should be considered. 3.5.2 When irritation at injection site has been evaluated in acute toxicity test, long term toxicity test, and the formulation of the test drug is identical or comparable with that of the proposed clinical study, a separate skin irritation test at injection site may not be performed. 3.5.3 Test method See the commonly used methods and related paper for irritation test in appendix. 3.6 Ocular irritation test 3.6.1 Ocular drugs and the drugs to which eyes are possible exposed should be considered to perform ocular irritation test. 3.6.2 When ocular irritation test has been evaluated in acute toxicity test, long term toxicity test, and the formulation of the test substance is identical or comparable with that of the proposed clinical study, there is no need to perform a separate ocular irritation test. 3.6.3 Experimental method See the commonly used methods and related paper for irritation test in appendix. 3.7 Irritation test for other routes at the administration site This sector shall refer to the irritation test on skin, injection site and ocular, under the guidance presented in overall principals and irritation test design considerations.

4 Sensitization test
4.1 Definition Sensitization in this Guideline is synonymous with allergy or hypersensitivity, refers to increased reactivity to an antigen to which a person (or animal) has been previously exposed, with the specific immunoreaction presenting tissue damage and or

physiological disfunction, . It is a kind of abnormal and pathological immunoreaction, for which the most common categories are Type I (immediate or rapid) allergy, which is mediated by IgE antibodies, represent urticaria allergic shock, bronchial asthma , allergic coryza, allergy of gastraenteric tract; Type II (cytotoxic or cytolytic ), which is mediated by IgG antibodies , represent haemolytic anemia with positive in Coombstest, agranulocytosis and hematoblastic purpura; Type III(immune complex initiated hypersensitivity or vasculitis ), is mediated by IgG, IgM, represent limited pneumonia, vasculitis, lupus-like response, glomerular nephritisetc; Type IV (delayed hypersensitivity or tuberculin hypersensitivity) is mediated by T lymphocytes, represent contact dermatitis. Photosensitivity includes phototoxicity(light irritation)and photoallergy. Phototoxicity is some kind of non-immunologic response of the skin to sunlight activated by light, drug plays a role in the response directly or indirectly. Photosensitivity is an acquired, immune-mediated response of the skin to sunlight, and photosensitive substances transdermal absorbed or through circulation reach skin, and response with absorbed light at epidermal cells layer. It is a special type of the type IV hypersensitivity. 4.2 Objective To observe systemic and local hypersensitivity after the animal is exposed to the test substance. 4.3 Considerations in sensitization test design 4.3.1 The problem that which kind of sensitization study should be performed should be identified in conjunct with the self characters, eg. Chemical structure and so on, pharmacological and toxicological findings, especially the long term toxicity test result, indication and the delivery method. 4.3.2 The selection of the specified test method is on the basis of the administration route and the sensitization mechanism, along with the interpretation of

rationality. For instance, transdermal administration should consider conduct the guinea pig maximization test(GPMT), Buehler test(BT); For injection administration ,the active systemic anaphylaxis(ASA) and the passive cutaneous anaphylaxis test(PCA) should be taken into account; as to inhale administration, the guinea pig inhale sensitization test and irritation test should be considered. 4.3.3 Dose should be designed rationally, and the quality-efficacy relation is also concerned. Active control and passive control is needed. 4.3.4 If it has been demonstrated the type of hypersensitivity in other tests, eg, long term toxicity test, and the test substance is identical or comparable with that of the proposed clinical study, it need not perform the sensitization test separately. 4.3.5 Due to limitation of animal model, egthere is no appropriate animal model of type II and type III, no clear clinical significance of photosensitive model, flexible patterns may be applied to preclinical sensitization evaluation for some drugs with adequate arguments, and the potential toxicity should be interpreted in detail in doctor brochure and labeling, as well as the responding study in clinic. 4.3.6 Analysis of data: Appropriate statistical methods should be selected on the basis of models and test methods. 4.4 Sensitization test of transdermal administration Skin sensitization reaction should be investigated, and it is commonly used BT, GPMT or other rational methods. If drugs chemical structure is identical or similar with that of other reported compounds which lead to hypersensitivity, it is appreciated to perform other suitable methods to investigate whether the drug can cause other hypersensitivity, eg. systemic hypersensitivity. 4.5 Sensitization test of injection administration ASA, PCA or other rational methods with the interpretation of rationality are commonly used to investigate systemic hypersensitivity. If the chemical

structure of the drug is identical or similar with that of a compound reported by literatures that lead to other hypersensitivity, the suitable methods should be considered to investigate whether the drug can cause other hypersensitivity, eg. skin sensitization reaction, etc. 4.6 Sensitization test of other administration routes Inhale administration drug should conduct guinea pig inhale sensitization test and irritation test. Sensitization test of mucous membrane administration drug should consider own characters and be reference to transdermal administration test method. If the chemical structure of the drug is identical or similar with that of a compound reported by literatures that lead to other hypersensitivity, the suitable methods should be considered to investigate whether the drug can cause other hypersensitivity, eg. photosensitization reaction, etc. 4.7 Test method See the commonly used methods and related papers for sensitization test in appendix.

5 Hemolysis test
5.1 Definition Hemolysis means inner or outer blood vessels hemolysis and hemagglutination caused by drug products. Hemolysis consists of immunological hemolysis and nonimmunological hemolysis as well. Immunological hemolysis is defined as the hemolysis produced by specific antibody through immune response, and it belongs to type II and type III hypersensitivity; non-immunological hemolysis includes drug induced oxidationcaused hemolysis and hemolysis and hemagglutination resulted from changes of blood stable state by drug product. 5.2 Objective To investigate if drug can lead to hemolysis in inner or outer blood vessels and hemagglutination reaction. 5.3 Scope All the injection and other drug products which can lead to immunological hemolysis and

non-immunological hemolysis should be conducted hemolysis test. 5.4 Considerations in hemolysis test design 5.4.1 The hemolysis mechanism presents complicated, and there is no standard preclinical test in vivo to comprehensively assess hemolysis reaction of drug product. As a result, Hemolysis observation of the drug product, to which no marked drug has the same administration route, is strongly recommended in long term toxicity study. The relative parameters and body sign, (eg. reticulocyteerythrocyte number, bilirubin, urine protein, nephritis and spleen gore and so on) if hemolysis occurs, study should be furthered. 5.4.2 Injection to which the marketed drug has the same route can be evaluated hemolysis through general in vitro tube method. If gained a positive result, the comparative research is recommended between the test drug product and the marketed product, and animal experiments will be employed if necessary. 5.5 Test method See the common methods and related literatures for hemolysis test in appendix. 6 Test report The test report should be in compliance with GLP (Good Laboratory Practice), covering: test method, test substance, test animal, test procedure, parameter, result and result evaluation. 7 Evaluation of results To explain the methodology, test substance and rational choice of animals and analyse the possible impacts on the test result. To correctly analyse the test results, well understand the significance, justify toxicity and target organ of toxicity. To analyse the integrity of toxicity test and estimate whether the toxicity has been fully demonstrated by the toxicity study after administration, and the possible reasons elicited toxicity. To identify whether the drug can be clinically used and the considerations in clinical use, systemically evaluation should be carried out in terms of pharmacy, pharmacology, oxicology and clinical

practice. 8 Some considerations Regarding the irritation, sensitization and hemolysis studies, considerations should be put in the following aspects. 8.1 Integrated and comprehensive principal Depending upon the drugs characters, irritation, sensitization, hemolysis studies and evaluations should be conducted together with pharmacy, pharmacology and toxicology and clinical practice. 8.2 Analyses case by case At present, there are limitations in existing test methods, so new approaches can be adopted to provide more valuable results, consequently improve the sensitivity and veracity in the studies of irritation, sensitization and hemolysis. However, rationality should be interpreted for the other approaches, and specific methods and operational flow should also be explained.

9 References
1FDA Guideline for Industry Skin irritation and sensitization testing of generic transdermal drug products 2FDA Guideline for Industry Photosafety testing 3FDA Guideline for Industry Immunotoxicology evaluation of investigation new drug 4EPA Health effects test guidelines OPPTS 870.7800 Immunotoxicity 5EPA Health effects test guidelines OPPTS 870.2500 Acute dermal irritation 6EPA Health effects test guidelines OPPTS 870.2600 Skin sensitization 7EPA Health effects test guidelines OPPTS 870.2400 Acute eye irritation 8EMEA Non-clinical local tolerance testing of medicinal products 9ISO 10993-10:2002(E) Biological evaluation of medical devicesPart 10- Tests for irritation and delayed-type hypersensitivity 10Office of Pharmacy Policy of Ministry of Health Peoples Republic of China Collection of Guideline of clinical and preclinical study for new drug (Western medicine)

11Xu Shuyun, Bian rulian, Chen xiu. Methods of pharmacological experimentation. 12Chen Qi. Methods of pharmacological Study on Chinese Traditional Medicine.

10 Appendix
The test methods collected in appendix only provide references, the researcher should adopt scientific and rational methods accepted by domestic and overseas based on the own characters of the test drug, and dont limit in the test methods collected in appendix. 10.1 Method for irritation test 10.1.1 Skin irritation test 10.1.1.1 Definition Skin irritation test means the reversible inflammatory changes on the skin induced by transdermal administration, inreversible tissue damage is named as skin causticity. 10.1.1.2 Objective To observe of irritation after skin of animal has been exposed to the test substance and provide possible ADR such as inflammation reaction, denaturalization and necrosis of skin 10.1.1.3 Test method 10.1.1.3.1 AnimalAlbino rabbit is preferred, 4 to 8 animals, half females and half males. Excipient control is designed. Right and left sides of each animal serves as its own control. Fur should be removed from the test area 24 h before the test, (generally the dorsal area of the trunk of the animals, by clipping or shaving or suitable depilatory). The area of removing fur is approximately 3cm3cm each side. Skin should not be abraded. Only animals with healthy intact skin should be used. But if the irritation of abraded skin is like to be observed, using sandpaper to abrade or lacerate # until blooding. 10.1.1.3.2 Application of the test substance: The test substance 0.5ml should be applied to the area with no fur, and two layers of gauze patch 2.5 cm2.5cmand one piece of cellophane paper or analog should be covered on the test area, unstimulating adhesive plaster and bandage should be fixed; Excipient serves as the control

at the other side. The recommended exposure duration is 4 hours at least. At the end of the exposure period, residual test substance should generally be removed, where practicable, using water or an appropriate solvent. Skin irritation test of repeated dose should be performed at the identical area with the same time, and the exposure period often less than 4 weeks. 10.1.1.3.3 Animal observation: Dermal irritation should be observed in the natural light or holospectrum light. Dermal irritation should be scored according to the grades in the following Table. 1. As for the single dose skin irritation test, exposure site should be examined and recorded with eyes for signs of erythema and edema at 3060 min, 24, 48, and 72 hours after removing test drug. For permanent damage, it is necessary to prolong observation period to determine the recovery and its duration. But the postponement should not exceed 14 days. Histopathology examination should be conducted for animals with moderate or severe skin irritation after observation period. Regarding the repeated dose irritation test, each time after 1 hour of removing the test substance and prior to the next exposure ,animals should be examined and recorded for signs of erythema , edema, pigmentation, hemorrhage, coarse or thin skin and their beginning time and ending time. Erythema, edema should also be scored. Animals should be examined for signs of erythema and edema at 3060 min, 24, 48, and 72 hours after removing the test drug. For permanent damage, it is necessary to extend observation period to determine the recovery and its duration. But the postponement should not exceed 14 days. Histopathology examination should be conducted for animals with moderate or severe skin irritation after observation period. 10.1.1.3.4 Evaluation of results: As for the single dose skin irritation, the mean of skin

response scores of each test and excipient group at every observation point should be calculated. The mean of the excipient response scores subtract that of skin gets primary irritation index. Degree of irritation should be evaluated as table 2. The repeated dose skin irritation, at first, the mean of primary irritation scores of each group at every observation point should be calculated. Then the irritation integral mean score of each individual animal (namely, accumulative irritation score) in the observation duration should be calculated. Irritation degree evaluation shall abide by table 2. Table 1.Evaluation of Skin Irritation Response Irritation Response Score Erythema formation No erythema 0 Mild erythema (barely perceptible) 1 Moderate erythema( Well-defined). 2 Severe erythema 3 Beet redness erythema to slight eschar formation (injuries in depth) 4 Edema Formation No edema 0 Mild edema (barely perceptible) 1 Moderate edema (definite raised edges) 2 severe edema (raised approximately 1 mm) 3 Severe edema (raised more than 1 mm and extending beyond area of exposure 4 Maximum total score 8 Table 2.Evaluation Criteria of Skin Irritation Degree Value Evaluatin 0-0.49 No irritation 0.5-2.99 Mild irritation 3.0-5.99 Moderate irritation 6.0-8.0 Severe irritation 10.1.1.4 Test report Test report should be in compliance with GLP, covering: test substances name, physical-chemical properties, formulation and dose; animals species, strain, sex, age, weight, source (marked by certificate code and animal grade); description of

animal raising conditions (source of feed, ambient temperature, humidity, photoperiod, and certificate code of facility of test animal); specific operational procedure; skin responses scores for each individual animal at each observation point; narrative description of other toxicity; test conclusion, etc. 10.1.1.5 Explanation to test results The test system is more sensitive than the human skin irritation test, so the negative result can be justified as no irritation and false active response should be excluded. Mild irritation can be ignorant except that test substance has been used for a long time or with a broad area. 10.1.2 Irritation test to injection site 10.1.2.1 Definition Irritation test of injection site means the reversible tissue changes in the injection sites after injecting test substance, but for irreversible tissue damages, called Corrosion. 10.1.2.2 Objective Determination of the irritant and/or corrosive effects at the injection place is useful to refer possible inflammation reaction, denaturalization and necrosis in clinical practice. 10.1.2.3 Test method 10.1.2.3.1 Test animal: Albino rabbit is r preferred, at least 3 animals should be used. Saline control is needed. Right and left sides of each animal may serve as its own control. Injection site shall basis on administration route, e.g. ear edge veinear centra artery other animanls can use fore/hand limb vein and femoral artery, etc.femoral and dorsal muscles hypodermis of lateral chest wallvein side tissue, etc. . 10.1.2.3.2 Method of administration: test substance is administered depending on the clinical use, and drug volume and velocity can be modified according to animal condition. Administration duration is determined by proposed clinical practice, and repeated dose administration cannot surpass 7 days generally.

10.1.2.3.3 Animal observation: in light of single dose irritation test, injection sites and animals should be examined with eyes; and for repeated dose irritation test, injection sites and animals should be examined with eyes prior to administration each day and after 48-96 hours of the last administration. At the end of observation period, histopathology examination of some animals should be conducted on the administration site. The rest of animals should be further observed in the following 14-21 days depending on the characters of test substance and the process of irritative response, then conduct histopathology examination to learn the irritation reversibility. 10.1.2.3.4 Evaluation of results: safety evaluation of test substance is based on the results of observation by eyes and pathological examination. 10.1.2.4 Test report Test report should be in compliance with GLP, covering: test substances name, physical-chemical properties, formulation and dose; animals species, strain, sex, age, weight, source (marking certificate code and animal grade); description of animal raising conditions (source of feed, ambient temperature, humidity, photoperiod, and certificate code of facility of test animal); specific operational procedure; skin responses scores for each individual animal at each observation point; narrative description of other toxicity; test conclusion, etc. 10.1.3 Eye irritation test 10.1.3.1 Definition Eye irritation is the production of reversible tissue changes in the eye following the application of a test substance to the anterior surface of the eye, corrosion is irreversible tissue damage in the eye following application of a test substance to the anterior surface of eye. 10.1.3.2 Objective Observation of the irritant effects on the eye delivered substances may give reference to possible inflammation reaction after clinical usage, denaturalization

and necrosis in the eyes. 10.1.3.3 Test method 10.1.3.3.1 Test animal: Albino rabbit is recommended as the preferred species, At least three animals should be used. Saline control should be designed, and right and left sides of each animal serves as its own control. Both eyes of each test animal should be examined within 24 h before testing starts, including Nafluoresecin examination. Animals with eye irritation, corneal defects, or conjunctiva injury should not be used. 10.1.3.3.2 Method of administration: A dose of 0.1 ml or 0.1g test substance should be placed or daubed respectively in one eye of each animal. The lids are then gently held together for about 10 seconds. The eyes of the test animals need not be washed out. The administration period is determined by the proposed clinical treatment plan. Normally, for the repeated dose drug, the period may not exceed 4 weeks. 10.1.3.3.3 Observation on eyes: for single dose eye irritation test, the eyes should be examined at 1, 24, 48, and 72 h after drug delivery. For repeated dose eye irritation test, the eyes should be examined ahead of the administration and at 1, 24, 48, and 72 h after last administration. If there is no evidence of irritation at 72h, the study may be ended. Extended observation (normally need not exceed 21 days) may be necessary if there is a permanent damage. Examination of reactions can be facilitated by use of a binocular loupe, hand slit-lamp, biomicroscope, or other suitable device. After recorded observation at 24 hours, fluorescein dyeing examination may be further conducted. The grades of ocular reaction using the table 3 should be recorded at each examination. In addition to the observations of the cornea, iris and conjunctivae, any other lesions which are noted should be recorded and reported. 10.1.3.3.4 Evaluation of results: As the requirement of table 3, scores of cornea, iris and

conjunctivae of each group at every observation point should be summarized to get the total scores. Summation of each group is divided by the number of animal, then the final score is gotten. Irritation degree is justified using table 4. Table 3.Evaluation of eye Reaction Eye irritation reaction value Cornea No opacity 0 Scattered or diffuse areas of opacity, details of iris clearly visible 1 Easily discernible translucent area, details of iris slightly obscured 2 Nacrous area, no details or iris visible, size of pupil barely discernible 3 Opaque cornea, iris not discernible through the opacity 4 Iris Normal 0 Markedly deepened rugae, congestion, swelling moderate circumcorneal hyperemia, or injection, iris still reacting to light 1 Hemorrhage / necrosis visible with eyes / no reaction to light (any or all of these) 2 Conjunctivae Redness (refers to palpebral and bulbar conjunctivae) Blood vessels normal 0 Blood vessels hyperemic, cardinal red 1 Blood vessels hyperemic, crimson color, not easily discernible 2 Diffuse beefy red 3 Chemosis (refers to lids and/or nictitating membranes) No swelling 0 Slightly swelling (includes lids) 1 Obvious swelling with partial eversion of lids 2

Swelling with lids about half closed 3 Swelling with lids more than half-closed 4 Secretion No secretion 1 A little secretion 2 Lids and eyelashes humid or cohesive by secretion 3 ` The whole eyes area humid or cohesive by secretion 4 Maximum score 16 Table 4.Evaluation of Skin Reaction Degree Value Evaluatin 0-3 No irritation 4-8 Mild irritation 9-12 Moderate irritation 13-16 Severe irritation 10.1.3.4 Test report Test report should be in compliance with GLP laboratory administrant criterion, the following specific information should be reported: name , physicalchemical properties, formulation and dose; Species, strain, sex, age ,weight, source(marked by identification code and animal grade) of test animal; Description of caging conditions including source of feed, ambient temperature, humidity, photoperiod, and identification code of facility of test animal; Specific operational procedure; Eyes response scores for each individual animal at each observation time point; Narrative description of other toxicity; test conclusion, etc. 10.1.3.5 Explanation of results The test system is more sensitive than the eye irritation test of human, so the negative result can be justified no irritation, but false active response should be excluded when got active results. Mild irritation can be ignorant except that test substance has been used for a long time or in a broad area. 10.2 Method for sensitization test 10.2.1 Passive cutaneous anaphylaxis test(PCA) 10.2.1.1 Elementary principle Normal animal is given a intraderma injection of IgE-rich serum from an allergen-sensitized animal. IgE binds to Fc3 acceptors of mast cells in the skin, the latter is sensitized passively.

Local mast cells release allergied mediators, followed by increased local vascular permeability . The injected dye can leak from wheal, making a bule. Sensitization degree is justified according to the range of blue or spectrophotometer measurement. 10.2.1.2 Test animal: Rats are the commonly used animal for PCA reaction, and mice are also employed. Albino rabbit is used to meet the test requirements. Because PCA reactions of all the animals are mediated by IgE. 10.2.1.3 Groups of test: groups should be designed as negative group, active group and test substance group in different dose. Negative control group should be administered same volume vehicle, and positive control group is given ovalbumin or allergen sensitized drug. 4 animals in each group are available at least. 10.2.1.4 Route and manner of sensitization: The route of administration is in compliance with that of in proposed clinical practice. Sensitization is employed every other day, 5 times in total. Blood is collected at 10 days approximately after the last sensitization, After centrifugation (2000 rpm) for 10 min, and serum is separated and stored at -20 until used within 2 weeks. 10.2.1.5 Excitation: The above anti-serum is commonly diluted in saline to 1:2, 1:8, 1:32. fur should be removed from the dorsal area of the trunk of the animals for 3cm4cm2. After 24 or 48 hours, each group is injected iv with excitation antigen which is an equal amount with the sensitization dose and mixed with 0.5-1% evans blue dye, the total amount is 1 ml. 10.2.1.6 Test and evaluation of results: After 30 min, all animals are sacrificed by anaesthesia, skin of the dorsal area of the trunk are clipped .A reaction was scored as positive if the spot of the skin was more than 0.5 cm in diameter. 10.2.2 Active systemic anaphylaxis test (ASA) 10.2.2.1 Elementary principle A venous injection of antigen is given to allergen-sensitized animal to observe systemic

anaphylaxis resulted from degranulation of mast cell and basophil, active medium release after antigen binding with IgE. 10.2.2.2 Objective To observe anaphylaxis induced by systemic administration of the test substance. 10.2.2.3 Test method 10.2.2.3.1 Test animal Male Guinea pig of Hartley strain, 300-400g of weight, is commonly used 10.2.2.3.2 Dose Group Negative group, active group, high dose group and low dose group. At least 4 animals in each group. 10.2.2.3.3 Sensitization 10.2.2.3.3.1 Route of sensitization: follow the route of clinical administration. 10.2.2.3.3.2 Times of sensitization: every other day, 5 times in total. 10.2.2.3.3.3 Dose of sensitization Negative control group: Vehicle diluting drug is administrated with the equal volume. Active control group: 1-5mg BSA or valbumin /per animal or allergen sensitized substance Low dose group: clinically maximum dose/kg or m2 High dose group: Several multiple amount of low dose group 10.2.2.3.4 Excitation 10.2.2.3.4.1 Route of excitation: A venous injection commonly. 10.2.2.3.4.2 Time of excitation: single excitation at the 10-14 days after last injection. 10.2.2.3.4.3 Dose of excitation: generally 2-5 times of sensitization dose. 10.2.2.3.5 Observation variable 10.2.2.3.5.1 Sensitization period: Symptoms of each animal is observed everyday. Weight of individual animal in each group should be weighed at the initial and terminal sensitization days and excitation day. 10.2.2.3.5.2 Excitation: Animal responses and appear /disappear time of symptoms should be closely observed according to the table 5 from right after injection through 30 min after injection. It should persist 3 hours at most. Table 5 symptoms of anaphylaxis 0 normal 7 increased inspiratory rate 14 unsteady gait

1 dysphoria 8 emiction 15 jump 2 pilar erecti 9 defecation 16 wheeze 3 tremor 10 watering 17 convulsion 4 scratch around the nose 11 labored repiratory 18 turn 5 sneeze 12 rale 19 wavy respiration 6 tussis 13 purpura 20 death Table 6 evaluation of systemic sensitization 0 negative anaphylaxis 1-4 symptom + weak positive 1-10 symptom ++ positive 1-19 symptom +++ strong positive 20 ++++ highly strong positive 10.2.3 Guinea pig maximization testGPMTand Buehler testBT 10.2.3.1 Definition The test animals are initially exposed to a sensitization dose through intradermal and/or epidermal application. After that, a period of 10 to 14 days induction period develops an immune response. Then the animals are exposed to an excitation dose to observe whether anaphylaxis occurs. The response and degree of skin reaction to the sensitization and excitation exposure should be compared, as well as compared with sham treatment group. 10.2.3.3 Test method 10.2.3.3.1 Test animal 10.2.3.3.1.1 Species and strain: it is better the young adult guinea pig and commonly used strains in laboratory. 10.2.3.3.1.2 Housing and feeding. The temperature should be 203 with the relative humidity 3070 percent. Where artificial light, the sequence should be alternate 12 h light/12 h dark. Conventional laboratory diets may be used without limitation of water. Guinea pigs should receive an adequate amount of ascorbic acid. 10.2.3.3.1.3 Number and sex The number and sex depends on the choice of method, either male or female. If females are used, they should be nulliparous and no pregnant. The Buehler test recommends a minimum of 20 animals in the treatment group and no less than10 in control treatment. For GPMT, no less than 10 animals in

the treatment group and 5 in the control group should be used. If the test result fails to suggest the test substance is a sensitizer, the test should be continued, for which no less than 20 test animals in treatment group and 10 animals in control group. 10.2.3.3.1.4 Control animals 10.2.3.3.1.4.1 Sensitivity and reliability of the method should be examined by known mild-to-moderate positive substances every 6 months. Mildmoderate sensitizers with adjuvant shall at least response by 30%, without adjuvant by 15%. Recommended positive substances are mercaptobenzothiazole , benzocaine , dinitro-chlorobenzene ,or DER 331 epoxy resin. Other sensitizers substances may also be used. 10.2.3.3.1.4.2 A separate sham-treated group which only receive vehicle should be designed to ensure the excitation response arising from sensitization rather than irritation. The solvent should not interfere or change the judgement of results. 10.2.3.3.1.5 Dose Dose depends on the test method. In the Buehler test, the sensitization dose should be high enough to cause mild irritation, and the excitation dose is the highest non-irritating dose. In the GPMT, the sensitization dose should be well tolerated systemically, and should be high enough to cause mild-to-moderate skin irritation; the GPMT excitation dose is the highest non-irritating dose. 10.2.3.3.1.6 Observation to animals 10.2.3.3.1.6 .1 Skin reactions should be graded and recorded after the excitation exposures at the time specified in the methodology, usually at 24 and 48 hours. Time adjustment should be made as necessary to describe abnormal responses. 10.2.3.3.1.6.2 Body weights at initial and terminal should be recorded 10.2.3.3.1.7 Test procedure 10.2.3.3.1.7.1 The Buehler test uses topical administration via a closed patch on days 0, 68, and 1315 for sensitization, with topical excitation of the untreated flank for 6 hours on day 2728. Readings are made approximately 24 hours and 48 hours

after removing the patch. If the results are equivocal, the animals may be re-excitated one week later, using either the original control group or a new control group. 10.2.3.3.1.7.2 The GPMT uses intradermal injection to topically sensitize for 58 days, with and without adjuvant. After that, topical excitation will be given for 24 hours on day 2022. Readings are made approximately 24 hours and 48 hours after removing the excitation dose. As same as the Buehler test, if the results are equivocal, the animals may be reexcitationd 1 week later. If only 10 animals were used initially and lead to equivocal results, 10 treatment animals and 5 control animals shall be added. 10.2.3.3.1.7.3 Blind access to both test and control animals 10.2.3.3.1.7.4 Removal of the test material by water or an appropriate solvent, without altering epidermis existing response and integrity. 10.2.3.3.1.7.5 Hair is removed from the site of application by clipping, shaving, or depilating, depending on the test method. 10.2.3.4 Test report Test report should comply with GLP, covering: test substances name, physical-chemical properties, formulation and dose; animals species, strain, sex, age, weight, source (marking certificate code and animal grade); description of animal raising conditions (source of feed, ambient temperature, humidity, photoperiod, and certificate code of facility of test animal); specific operational procedure; the skin response results of each individual animal subjected to sensitization exposure at 1 and 24 hours, and the excitation exposure at 24 hours and 48 hours. As a minimum, the record should include erythema and edema grad, any abnormal finding, the proportion of sensitization in each group and the extent (slight, moderate, severe) of the sensitization reaction in each individual animal, as well as test conclusion, etc. 10.2.3.5 Explanation of results The test system is more sensitive than human sensitization test, so the strong active response

on Guinea pig possibly means sensitization on human body, and week active response on Guinea pig may means week or non-sensitization on human body. . 10.2.4 Skin phototoxicity test 10.2.4.1 Definition Phototoxicity reaction means the release of ultraviolet energy embodied in drug to skin results in skin damage. Namely, a skin toxicity reaction generated when skin or whole body first exposure to chemical, and subsequently to ultraviolet radiation. It is a most common adverse reaction, and has dose dependency. The clinical symptoms are similar with sunburnshowing erythema , edema, prutitis, pigmentation, and local necrosis, canker or desquamation of epidermis in severe condition. Once occur, symptoms develop very quickly, usually around 24 hours light irradiation or even shorter 10.2.4.2 Test method 10.2.4.2.1: Test animals: Albino or hairless Guinea pig, healthy, 300500g of weight, 14 animals at least, half females and half males. 10.2.4.2.2 Test groups:, Guinea pig are randomizly divided into 2 groups depending upon weight and sex, 4 in control group, 10 in treatment group, half females and half males. The control group should not expose to light after administration of test substance, but active control drug expose to light. 10.2.4.2.3 Drug and dose: 8- methoxypsoralen is preferred as an active control substance; the dose of test substance is determined by deliquescence, required concentration, chemical/ physical properties, local/systemic tolerance of animals and their rationalities. Two concentrations are commonly selected; the low concentration is same as the clinically used concentration. 10.2.4.2.4 Method of administration: the back should be removed hair and divided into four parts. 0.025mlg/cm2 drug delivered to each part. The application as following: Group Site Treatment Irradiation

Control group 1 solvent or excipient no 2 test substance concentration 1 no 3 test substance concentration 2 no 4 active control no Treatment group 1 solvent or excipient yes 2 test substance concentration 1 yes 3 test substance concentration 2 yes 4 active control yes After administration, the animals are placed in the given detents to limit activity. Before exposure to light, the test substance and control substance should have enough time(60 min is preferred) to permeate into cuticleand then interact with epidermal cells. At the proper time after administration, generally 0.5-4 hours, residual test substance and excipient at the test site of control group should be removed, using water or an appropriate solvent, the animal are then placed back into cages. The treatment group does as the control group did after irradiation. 10.2.4.2.5 Method of irradiation: wave band should be sensitive to the test substance. Irradiation dose, light intensity, irradiation period and distance should be carefully determined through Pre-test. During the experiment, the animals are fixed at the irradiation desks with the heads covered, and irradiated for appropriate time, using proper light source, eg, water-cooled xenon arc lamps. It is generally considered that wavelengh should cover 280-450nm, and irradiation period is within 0.5-2 hours, and irradiation intensity is proper, which should be measured before and after irradiation. 10.2.4.2.6 Test items: Individual animal in each group should be weighed when initiate and terminate the test. Skin responses should be observed and recorded by photos at 24 and 48 hours. Skin responses should be scored according to the grades in the table 7. Table 7.Evaluation of Skin Responses Degree Skin response value No erythema 0 Mild erythema 1

Moderate erythema 2 Severe erythemawith or without edema 3 10.2.4.2.7 Judgement and evaluation of results: compared with control group, if score of individual animal in the control group is less than 1, those in treatment group whose score is equal with or more than 1 is regarded as positive; if score of individual animal in the control group is equal with or more than 1, those in treatment group whose score is more than the highest in the control group is justified as positive. Positive ratio can be further obtained. 10.2.5 Skin photoallergy test 10.2.5.1 Definition The drug becomes active state after absorbing light energy, and binds to protein in the skin, in a manner of hapten, to become drug-protein complexcomplete atigenwhich is transferred to immune active cells by Langerhans cells in epidermis, inducing supersensitive reaction. Photoallergy belongs to type IV bradyanaphylaxis. It undergoes latent period. The reaction occurs after a relatively long time. In general, continued administration for 5-10 days and irradiation can induce immune system response. When the drug is applied again photoallergy happens within 24-48 hours after drug and irradiation treatment. 10.2.5.2 Method of test 10.2.5.2.1 Preparation: removing hairs of animals ( 24cm2 area). Freunds complete adjuvant is injected to the four corners of the hairless area, 0.1ml respectively. 10.2.5.2.2 Sensitization: 20% Sodium dodecyl sulfate is applied to the hairless area, followed by the test substance. The residual drug should be removed after some time. The positive drug can chose salicylamide bromide. 10.2.5.2.3 Irradiation: Wave band should be sensitive to the test substance. Irradiation dose, light intensity, irradiation period and distance should be carefully determined through the pilot test to select wave and the site of application for UV irradiation. The evident eythema is

taken as the selection standard. The operational procedure of application of 20% Sodium dodecyl sulfate and then the test substance which is followed by irradiation should be repeated for 5 times. 10.2.5.2.4 Excitation: two weeks after the last sensitization, drug is applied to the prepared skin area, for which the concentration lower than that of sensitization to prevent irritation and phototoxicity. The test substance and control substance should have enough time(60 min is preferred) to permeate into cuticleand then to interact with epidermal cells. UV irradiation is charactered by sub-erythema (1/2-2/3 of minimum amount of erythem) which doesnt elicit erythema reaction. 10.2.5.2.5 Observation of results: Skin responses should be observed and presented by photoes within 1-72 hours after irradiation. The test substance which is exposed repeatedly and subject to UV irradiation, and then lead to skin response even systemic response is regarded to cause photoallergy. 10.3 Method for hemolysis test 10.3.1 general in vitro tube method (observation with eyes) 10.3.1.1 Objective: Whether the state of erythrocyte is affected by test substance. 10.3.1.2 Test method 10.3.1.2.1 Preparation of blood cell suspensionput rabbit (or sheep) blood of milliliters (about 20 ml ) into a triangular conical flask with a beading for vibration 10 minutes, or stirring blood by a Glass rod, to remove away fibrinogen, then become decellulose blood. 10-fold amount of 0.9% Nacl solution is added, shake, and centrifugation (2000 rpm) for 10 min. The supernatant is removed, and precipitated red cells should be washed for 2-3 times until the supernatant shows no red color in the 0.9% Nacl solution according to the method described above. 2% mixed suspension is prepared with RBC dissolved in 0.9% Nacl solution, which is prepared for test. 10.3.1.2.2 Preparation of test substance: Except additional

requirements, for non blood vessel injection, the clinical use concentration in drug labeling should be diluted as 1:3 by 0.9% saline solution to be test solution; in terms of in blood vessel injection, the test solution is made in the concentration recommended in drug labeling. 10.3.1.2.3 Test method: 7 clean test tubes are numbered, number 15 are the tubes for test substance. Number 6 is for negative control, number 7 for positive control. 2% RBC suspension, 0.9% NaCl solution or distilled water are added in sequence as the following table, after mixed, immediately incubated at 370.5 in incubator. Observation is conducted every other 15min at the beginning, one hour later, observing every other one hour. The total observation period lasts 3 hours. Various solutions are added in the following sequence:. tube number 1 2 3 4 5 6 7... 2RBC suspension (ml) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Nacl(ml) 2.0 2.1 2.2 2.3 2.4 2.5 Distilled waterml 2.5 Test substanceml 0.5 0.4 0.3 0.2 0.1 10.3.1.2.4 Observation of results: When the solution is red and there is no cell residual or few RBC residual in the tube bottom, it demonstrates hemolysis occurred; When RBC are all completely precipitated, and the supernatant is colorless and pellucid, it demonstrates no hymolysis occurred. When there is red brown or brown red floccule which does not disperse when shaked, it means RBC have agglutinated. If there is a agglutination phenomenon, distinguishing true agglutination from the false should be further justified as this method: if the agglutinated substance is dispersed uniformly after shaking, or put on the microscope slide. Two drops of 0.9% Nacl is added at the verge of coverslips to observe under the microscope. If the agglutinated RBC is dispersed, false agglutination can be concluded, conversely, true agglutination.

10.3.1.2.5 Judgement of results: When there are hemolysis and agglutination in the positive control tube but no in the negative control tube, and there are/is no hemolysis and/or agglutination happening in solution in the test substance tube within 3 hours, the test substance can be used as injection, otherwise, it can not be used as injection. 10.3.1.3 Test report Test report should be in compliance with GLP, covering: name, physical-chemical properties, formulation and dose of the test substance; test method; specific operational procedure; observation results of individual tube at each observation point; test conclusion and so on. 10.3.2 Improved in vitro hemolysis test method(spectrophotometer) 10.3.2.1 Objective: observation on whether the test substance affects erythrocyte state 10.3.2.2 Test method 10.3.2.2.1 Preparation of blood cell suspensionput rabbit (or sheep) blood of milliliters (about 20 ml ) into a triangular conical flask with a beading for vibration 10 minutes, or stirring blood by a Glass rod, to remove away fibrinogen, then become decellulose blood. 10-fold amount of 0.9% Nacl solution is added, shake, and centrifugation (2000 rpm) for 10 min. The supernatant is removed, and precipitated red cells should be washed for 2-3 times until the supernatant shows no red color in the 0.9% Nacl solution according to the method described above. 2% mixed suspension is prepared with RBC dissolved in 0.9% Nacl solution, which is prepared for test. 10.3.2.2.2 Preparation of test substance: Except additional requirements, for non blood vessel injection, the clinical use concentration in drug labeling should be diluted as 1:3 by 0.9% saline solution to be test solution; in terms of in blood vessel injection, the test solution is made in the concentration recommended in drug labeling. 10.3.2.2.3 Test method: 7 clean test tubes are numbered, number 1-

5 are the tubes for test substance. No. 6 is for negative control, no. 7 for positive control. 2% RBC suspension, 0.9% NaCl solution or distilled water are added in sequence as the following table, after mixed, immediately incubated at 370.5 in incubator. Observation is conducted every other 15min at the beginning, one hour later, observing every other one hour. The total observation period lasts 3 hours. Various solutions are added in the following sequence: tube number 1 2 3 4 5 6 7 2RBC suspension (ml) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 0.9% Nacl(ml) 2.0 2.1 2.2 2.3 2.4 2.5 Distilled waterml 2.5 Test substanceml 0.5 0.4 0.3 0.2 0.1... 10.3.2.2.4 Observation of results: Solution in individual tube is put into dried centrifugation tube for centrifugation, and supernatant fluid is obtained. At 545nm of spectrophotometer, OD value is read base on distill water is taken as the blank. 10.3.2.2.5 Judgement of result: hemolysis ratio(%) can be calculated using the following formula: Hemolysis ratioODtODnc/(ODpcODnc) X 100 In the formula: ODtAbsorbency of the tube for test substance; ODncAbsorbency of negative control tube ODpcAbsorbency of positive control tube. When the hemolysis ratio is more than 5% (>5), it demonstrates there is hemolysis happening, and the test substance should not be used as injection. 10.3.2.3 Test report Test report should be in compliance with GLP, the following specific information should be reported: name, physical-chemical properties, formulation and dose of the test substance; test method; Specific operational procedure; observation results of individual tube at each observation time point; test conclusion and so on.

11 Drafting notes
Based on the original New Drug Review Regulation, skin irritation, sensitization of dermal administration, eye irritation, drops irritation and inhalational product irritation are applied

to the study of irritation of rectal and vaginal products. In terms of the study and evaluation on sensitization, hemolysis of vein administration, there is a principle requirement. With the development of science and technology, the understanding of mechanism, influent factors and clinical meaning of irritation, sensitization and hemolysis is deepen, so is the position in the nonclinical safety evaluation. We revise technical Guideline principles about irritation, sensitization and hemolysis of chemicals according to Drug Registration Regulation, referring to relative technical Guideline principles of each country of three sides under the ICH framework and the research development in domestic and foreign countries on irritation, sensitization and hemolysis of nonoral administration drug, incorporating in general rule of nonclinical safety evaluation for drugs and the working practice in drug research technic. It is intended to provide technical reference for study and estimation and approval of irritation, sensitization and hemolysis of chemicals. The technical Guideline principle only reflects the present understanding, viewpoint and advice on irritation, sensitization and hemolysis of chemicals, and will be revised and improved with the development of science and technology. The revised guidline is the inheriting and development of the original one. Compared with the printed Guideline, it pays much attention on expounding how to design the study of irritation, sensitization and hemolysis of chemicals scientifically and rationally. The test samples are the proposed clinical drug products. It includes the local and/or systemic toxicity after administration of various routes covering eyes, ears, noses, mouth, respiratory tracts, joint cavity, skin, recta, vagina, vein, artery, muscle, derma, vein besides, theca, and so on. It emphasizes the study of irritation, sensitization and hemolysis of chemicals should execute the Quality Administrative Criterion of Drug Nonclinical Study. It

also stresses that the test design should be based on mechanism of toxicity and clinical pertinence, combined with pharmacy eg, Structure-activity relationships and/or physicalchemical properties pharmacology and toxicology eg acute toxicity, log term toxicity, pharmacokinetics and Pharmacodynamics, and clinical use to justify how to study and evaluate irritation, sensitization and hemolysis of chemicals. However, it should be noted that specific issue should be and represented integrated, compositive principle. Considering the close relationship between drug products and drug toxicity, whereas new drug research in our country is mainly imitated, and drug preclinical animal safety evaluation is usually lack of systematization and integrity, therefore, to guarantee its safety and efficacy in clinical use, in the revised technical Guideline, it suggests marketed drug products should compare with those of the marketed products which have the same administration route, when the former showed positive results in toxicity study. The study on the toxicity in the revised technic Guideline principle has improved a lot comparing with the printed one, eg, sensitization includes systemic sensitization, skin sensitization and photoallergy; hemolysis covers immunological and nonimmunological hemolysis. In addition to evaluate the irritation and skin sensitization of transdermal drug products, if it is possible to have other toxicity, eg systemic sensitization, hemolysis and so on, appropriate test method should be performed to fully investigate its possible related toxicity during clinical use. In terms of injection drug products, it also should be considered to adopt suitable methods to study the responding toxicity if the drug products maybe cause other toxicity, eg, skin sensitization, in spite of irritation systemic sensitization, hemolysis. Irritation and/or sensitization and/or hemolysis of the drug products with other routes of administration should be investigated in conjunction with their own

characters. The study on the toxicity in the revised technic Guideline principle also has big difference with the printed one. For instance, the investigation of skin sensitization commonly uses BT or GPMT; Systemic sensitization is performed using ASA and PCA. Irritation and/or sensitization and/or hemolysis can be considered when other toxicity tests eg, long term toxicity test are being performed, but the test substance must be identical or comparable with the proposed clinical drug products, and the test result should completely show the possible irritation and/or sensitization and/or hemolysis which maybe occur in clinical use. In the appendix of the Guideline, commonly used test methods are embodied. But they are only viewed as a reference. Owing to the differences of chemical structure and drug products, clinical use, mechanism, character, intensity of toxicity response, the researcher should select recognized method to perform the test according to the characters of the drug. In this Guideline, irritation is defined as the production of reversible inflammatory changes at the administration site, whereas the production of irreversible tissue damage is defined as corrosion, which are referred to the Methods of pharmacological Experimentation, EPT, OECD and the Guideline for skin and eye irritation response to cosmetic. In the present, the sensitivity and veracity of nonclinical safety evaluation of irritation, sensitization, hemolysis are not highly predicted, it is thought to be related to: 1. The tests arent designed with the consideration of mechanism of toxicity, effected factors, clinical meaning and indication symptoms in clinical practice and administration, eg, in the skin sensitization test, active skin anaphylaxis is often used, but the method is designed based on the mechanism of type I hypersensitivity, the test results only can predict the possible systemic anaphylaxis, to the transdermal administration, skin

sensitization response is more important to the safety and efficacy of administration. Therefore, the sensitization test should be designed with the consideration of pathogenesis, eg, BT or GPMT is recommended.
in the test design, the test substance, dose, route, period, times of administration and so on, can not reflect the condition in clinical use. 2. there are limits in the present methods, eg, there are not ideal animal models for II and III type hypersensitivity at present; there is not definite clinical meaning about the model of photoallergy; the reliability of measuring small molecular substances using PCA and ASA is not certain. 3. The observation of some toxicity reactions is neglected in the test design. eg, with regard to the hemolysis test, only routine test tube method in vitro is adopted, but this test can not predict immunological hemolysis and oxidation-caused hemolyis induced by drug as an inducer; for vein injection, blood vessel irritation is taken into account merely, and it is a pity to ignore the irritation response to muscle when the drug leaks to the out of blood vessel. 4. Sensitivity of the test method is not high enough, eg, when the hemolysis is observed using the routine test tube method, the hemolysis is often justified with eyes, thus subjective error is bigger. It is also affected by the drugs color and obscuration facors. 5. The evaluation methods and standards is not perfect, eg, there are not semi-quantitative and/or quantitative evaluation tests. Due to the unceasing progress and development of scientific research, other methods which provide more valuable results and enhance the prediction on irritation, sensitization and hemolysis are encouraged. But when other methods are used, their rationale and specific methods and operational flow should be interpreted.

12 Author
The project research group of Technical Guideline on Irritation, Sensitization and Hemolysis Study for Chemical Drugs