FINAL STUDY REPORT
‘STUDY TITLE
Germicidal and Detergent Sanitizing Action of Disinfectants
Test Organisms:
‘Staphylococcus aureus (ATCC 6538)
Escherichia coli (ATCC 11229)
Salmonella typhi (ATCC 6539)
Methicilin Resistant Staphylococous aureus ~ MRSA (ATCC 33592)
PRODUCT IDENTITY
Enagic Super 501 Strong Acidic Water 2.5 pH
Machine 1 (Serial #. 87100333) Lot #1
and Machine 2 (Serial #: 87100339) Lot # 2 with pre-fiter C-1000
DATA REQUIREMENTS
U.S. EPA 40 CFR Part 158
"Data Requirements for Registration”
Pesticide Assessment Guidelines - Subdivision G, 91-2 (k)
AUTHOR
Anne Stemper, B.S.
Study Director
STUDY COMPLETION DATE
June 22, 2010
PERFORMING LABORATORY,
ATS Labs
1285 Corporate Center Drive, Suite 110
Eagan, MN 55121
SPONSOR
YNR Marketing
Box 7735
Laguna Niguel, CA 92607
PROJECT NUMBER:
‘09385
Page 1 of 47
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Protocol Number: YNRO10401 10,6DST Page 2 of 47
STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS
No claim of confidentiality is made for any information contained in this study on the basis of its falling
within the scope of FIFRA Section 10 (d) (1) (A), (B), or (C).
‘Company: YNR Marketing
‘Company Agent
Title
Date:
Signature
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Protocol Number: YNRO1040110.GDST Page 3 of 47
GOOD LABORATORY PRACTICE STATEMENT
‘The study referenced in this report was conducted in compliance with U.S. Environmental Protection
‘Agency Good Laboratory Practice (GLP) regulations set forth in 40 CFR Part 160.
‘The studies not performed by or under the direction of ATS Labs are exempt from this Good Laboratory
Practice Statement and include: characterization and stability of the compounds,
‘Submitter, Date:
Sponsor. Date:
Study Director, Date_b- 2.2/2
‘Anne Stemper, B.S.
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Protocol Number: YNRO1040110,GDST Page 4 0f 47
QUALITY ASSURANCE UNIT SUMMARY
Study: Germicidal and Detergent Sanitizing Action of Disinfectants
The objective of the Quality Assurance Unit is to monitor the conduct and reporting of nonclinical
laboratory studies. These studies have been performed under Good Laboratory Practice regulations (40
CFR Part 160) and in accordance to standard operating procedures and standard protocols. The Quality
Assurance Unit maintains copies of study protocols and standard operating procedures and has
inspected this study on the dates listed below. Studies are inspected at time intervals to assure the
integrity of the study.
2 Date of Phase Date Reported to | Date Reported to
Shase inspecies Inspection ‘Study Director | Management
Critical Phase Audit April 30, 2010 April 30, 2010
May 11, 2010
Draft Report May 6, 2010. May 7, 2010
Final Report dune 18,2010 June 18, 2010 ‘June 22,2010 |
‘The findings of these inspections have been reported to management and the Study Director.
uty Assurance Autor fet = Date ase
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Protocol Number: YNRO1040110.GDST Page 50f 47
TABLE OF CONTENTS
Title Page aren ite 1
Statement of No Data Confidentiality Claims 2
Good Laboratory Practice Statement 3
Quality Assurance Unit Summary... oe so
Table of Contents. a6:
Study Personnel 6
General Study Information. 7
Test Substance Identity... a Et
Study Dates i
Objective 7
Summary Of RESUS... snnnrene 8
Study Meteriels 9
Test Method... 10
Study CONHOIS essen Senate titrant : Eee ed
Study Acceptance Criteria 4
Protocol Changes 4
Data Analysis... 15
Study Retention 6
References. 6
Resuts 16
Analysis. 7
Conclusion 18
Table 1: Control Results. 19
Table 2: Neutralization Confirmation Control RESUIS....n.uenmnnnninneuninenennincnnen 20
Table 3: Test Results - Staphylococcus aureus (ATCC 6538) 2
Table 4: Test Results - Escherichia coll (ATCC 11229) : 2
Table 5: Test Results - Salmonella typhl (ATCC 6639) ...n.nne 23
Table 6: Test Results - Methicilin Resistant Stephylococcus aureus - MRSA (ATCC 33592) 24
Table 7: Verification of Antibiotic Resistance az 25
TeSt PTOt000l oe nnnninennsnnenenn : 26
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STUDY PERSONNEL,
STUDY DIRECTOR: Anne Stemper, B.S.
Professional personnel involved:
Amy S. Jeske, B.S. = Manager, Microbiology Operations
Scott R. Steinagel, B.S. - Manager, Microbiology Laboratory Operations
Joshua Luedtke, M.S. - Research Scientist |
‘Adam W. Pitt, B'S ~ Research Assistant I!
Peter Toll, B.S. - Research Assistant Il
Megan Polos, B.S. - Research Assistant |
Jessica Underwood, B.S. ~ Research Assistant |
Christine Chan, B.S. - Research Assistant |
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‘STUDY REPORT
GENERAL STUDY INFORI
Study Title: Germicidal and Detergent Sanitizing Action of Disinfectants
Project Number: 09385
Protocol Number: =YNR01040110.GDST
‘Sponsor: YNR Marketing
Box 7735
Laguna Niguel, CA 92607
Test Facility: ATS Labs
41285 Corporate Center Drive, Suite 110
Eagan, MN 55121
‘TEST SUBSTANCE IDENTITY
Test Substance Name: Enagic Super 501 Strong Acidic Water 2.5 pH
LotBatch(: Machine 1 (Serial #: 87100333) Lot # 1 and Machine 2 (Serial #: 87100339)
Lot # 2 with pre-filter C-1000
A OOOO eee eee
Test substance characterization as to content, stability, etc., (40 CFR, Part 160, Subpart F [160.105],
is the responsibility of the Sponsor.
‘STUDY DATES
Date Machines Receives April 7, 2008
Date Pre-filter C-1000 Receive March 24, 2010
Date of Test Substance Preparation: April 27, 2010
Study Initiation Date: April 19, 2010
Experimental Start Date: April 28, 2010
Experimental End Date: April 30, 2010
Study Completion Dat June 22, 2010
OBJECTIVE
The objective of this assay was to determine the minimum concentration of chemical that can be used in
sanitizing precleaned, nonporous food contact surfaces using the AOAC Germicidal and Detergent
Sanitizing Action of Disinfectants method.
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Protocol Number: YNRO1040110.6DST Page 8 of 47
SUMMARY OF RESULTS
Test Substance: Enagic Super 501 Strong Acidic Water 2.5 pH, Machine 1 (Serial #
87100333) Lot # 1 and Machine 2 (Serial #: 87100339) Lot # 2 with pre-filter
c-1000
Test Organisms: Staphylococcus aureus (ATCC 6538)
Escherichia coli (ATCC 11228)
Salmonella typhi (ATCC 6539)
Methicillin Resistant Staphylococcus aureus - MRSA (ATCC 33592)
Exposure Time: 30 seconds
Exposure Temperature: Room temperature (20.0°C)
Organie Soil Load: 5% fetal bovine serum
Efficacy Result: Enagic Super 501 Strong Acidic Water 2.5 pH demonstrated efficacy of two
lots against Staphylococcus aureus, and therefore, meets the requirements
set forth by the U.S. EPA for sanitizer label claims following a 30 second
exposure time at room temperature (20.0°C) in the presence of a 5% fetal
bovine serum organic soil load.
Enagic Super 501 Strong Acidic Water 2.5 pH demonstrated efficacy of two
lots against Escherichia coli, and therefore, meets the reauirements set forth
by the U.S. EPA for sanitizer label claims following a 30 second exposure
time at room temperature (20.0°C) in the presence of a 5% fetal bovine
‘serunorgani¢-soil load z
Enagic Super 501 Strong Acidic Water 2.5 pH demonstrated efficacy of two
lots against Salmonella typhi, and therefore, meets the requirements set
forth by the U.S. EPA for sanitizer label claims following a 30 second
exposure time at room temperature (20.0°C) in the presence of a 5% fetal
bovine serum organic soil load
Enagic Super 501 Strong Acidic Water 2.5 pHi demonstrated efficacy of two
lots against Methicilin Resistant Staphylococcus aureus - MRSA, and
therefore, meets the requirements set forth by the U.S. EPA for sanitizer
label claims following a 30 second exposure time at room temperature
(20.0°C) in the presence of a 5% fetal bovine serum organic soil load,
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Page 9 of 47
STUDY MATERIALS
Test System/Growth Media
Incubation
__Test Organism | Atco # : cane
‘Staphylococcus aureus 6538 Nutrient Agar A & B 35-37°C, aerobic
Escherichia coll 11229 Nutrient Agar A & B 35-37°C, aerobic
Salmonella typhi 6539 Nutrient Agar A & B 35-37°C, aerobic
Methicillin Resistant aay 35-37°C, aerobic
Staphylococcus aureus - MRSA
Nutrient Agar A & B
The microorganisms used in this study were obtained from the American Type Culture Collection
(ATCC), Manassas, Virginia.
Recovery Media:
Neutralizer: Modified Fluid Thioglycollate Medium
‘Subeulture Medium:
Reagents
Organic Soil Load Description:
‘A 0.40 mL aliquot of fetal bovine serum was added to 7.6 mL of each broth culture to yield a 5% fetal
bovine serum soil load.
Tryptone Glucose Extract Agar (TGEA)
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TEST METHOD.
Preparation of Test Substance
‘The Sponsor supplied two machine units, Machine 1 (Serial #: 87100333) and Machine 2 (Serial #
87100339) with pre-filter C-1000, to generate two lots of Strong Acidic Water 2.5 pH to be used in
testing. Each machine was also connected to a corresponding pre-fter C-1000 unit. Lot # 1 was
prepared using Machine 1 and pre-fiter C-1000 # 1 and Lot # 2 was prepared using Machine 2 and pre-
filter C-1000 #2, Each lot of test substance was prepared as follows on April 27, 2010.
The corporation cock supplied by the Sponsor was installed in an appropriate manner to tap water faucet
# 2 in the ATS Labs Microbiology Laboratory. Each main machine unit was installed following the
operations manual supplied by the Sponsor. Each machine unit was primed prior to use in testing
following the Sponsor provided instructions. The priming procedure was performed on April 23, 2008
Each machine was appropriately connected to be used in combination with the appropriate pre-fiter C-
1000 unit supplied by the Sponsor.
Each machine was set up to produce the strong acidic water. Prior to producing the strong acidic water,
the temperature of the tap water running through each machine was recorded. The temperature of the
tap water used to generate Lot # 1 was 18.0°C and the temperature of the tap water used to generate Lot
# 2was 17.0°C.
The flow rate of the tap water was set to approximately 0.4 gallons/minute and the flow rate was adjusted
as needed per the message on the machine display.
The ionizing accelerator (sodium chloride) was prepared for each machine following the instructions in
the operations manual supplied by the Sponsor. A 300 g sample of sodium chloride, supplied by ATS
Labs, was measured using the measuring cup supplied by the Sponsor. The sample was diluted as
‘specified in the operations manual and added to the built-in tank located on the back of the machine unit.
This procedure was performed for each machine,
Each machine was turned on, the water supply was tured on, then the “Strong Acid” button located on
the operation panel was pressed. The appropriate volume of Lot # 1 and Lot # 2 was collected from the
respective machine and the “Stop” button located on the operation panel was pressed when the
generation of strong acidic water was no longer required.
ATS Labs performed a pH reading for each lot of strong acidic water generated. The pH was determined
using the Fisher Scientific accument® pH meter. The pH of Lot # 1 was 2.68 and the pH of Lot # 2 was
2.73. The temperature of each lot of strong acidic water was also taken. The temperature of Lot # 1 was
18.0°C and Lot #2 was 17.0°C.
Following preparation, each lot of strong acidic water was stored in a sterile glass container, wrapped in
aluminum foil and stored at room temperature for less than 24 hours prior to use in testing
Following the use of each machine, the built-in sodium chloride tank was detached from the machine unit
and rinsed well with tap water. While each unit was stopped, the air discharge pipe located at the upper
part of the unit was cleaned by feeding approximately 20-50 oc of tap water through the hose using the
‘Sponsor provided wash bottle
Each prepared test substance was homogenous as determined by visual observation,
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Preparation of Test Organism
For Staphylococcus aureus, the test organism was transferred daily on Nutrient agar A slants. Three to
thirty consecutive daily transfers were performed prior to the inoculation of agar bottles. The bacterial
growth was washed from a 24 + 2 hour Nutrient Agar A slant of the test organism using 5.0 mL
phosphate buffer dilution water (PBDW). This growth suspension was aspirated and added to 99.0 mL
PBDW. A 2.0 mL aliquot of this suspension was inoculated onto eight French square bottles containing
Nutrient agar B. The bottles were titted back and forth to distribute the inoculum. The excess inoculum
was aspirated off. The bottles were incubated for 18-24 hours at 35-37°C (agar side down). After
incubation, the culture was harvested from the bottles using PBDW and approximately 15-20 sterile glass
beads to target 1x 10'° CFU/ mL. A 1.5 ml. aliquot of PBDW, per bottle, was used to harvest the test
organism. The suspension was removed from the bottles, filtered through sterile Whatman No. 2
Paper pre-wetted with approximately 1.00 mL sterile of PBDW, and collected into a sterile vessel.
The test organism culture suspension was placed in a spectrophotometer and the absorbance value was
recorded at 620 nm. The absorbance value for the Staphylococcus aureus culture suspension was
2.487 and no adjustments were necessary.
For Escherichia cof, the test organism was transferred daily on Nutrient agar A slants. Three to thirty
consecutive daily transfers were performed prior to the inoculation of agar bottles. The bacterial growth
was washed from a 24 + 2 hour Nutrient Agar A slant of the test organism using 5.0 mL phosphate buffer
dilution water (PBDW). This growth suspension was aspirated and added to 99.0 mL PBDW. A 2.0 mL
aliquot of this suspension was inoculated onto four French square bottles containing Nutrient agar B.
The bottles were tilted back and forth to distribute the inoculum. The excess inoculum was aspirated off.
The bottles were incubated for 18-24 hours at 35-37°C (agar side down). After incubation, the culture
was harvested from the bottles using PBDW and approximately 16-20 sterile glass beads to target 1 x
10° CFU/ml. A3.0 mL of PBDW, per bottle, was used to harvest the test organism. The suspension
was removed from the bottles, filtered through sterlle Whatman No. 2 paper pre-wetted with
approximately 1.00 mL sterile of PBDW, and collected into a sterile vessel,
‘The test organism culture suspension was placed in a spectrophotometer and the absorbance value was
recorded at 620 nm. The absorbance value for the Escherichia coli culture suspension was 2.030 and a
5.0 mL aliquot of PBOW was added to the culture suspension. The absorbance reading was taken again
and was 1.785. No additional adjustments to the culture suspension were necessary.
Salmonella typhi was transferred daily on Nutrient agar A agar slants. Three to thirty consecutive daily
transfers were performed prior to the inoculation of agar bottles. The bacterial growth was washed from
a 24 + 2 hour slant of the test organism using 5.0 mL phosphate buffer dilution water (PBDW). This
growth suspension was aspirated and added to 99.0 mL PBDW. A 2.0 mL aliquot of this suspension was
inoculated onto 12 French square bottles containing Nutrient agar B. The bottles were tilled back and
forth to distribute the inoculum. The excess inoculum was aspirated off. The bottles were incubated for
18-24 hours at 35-37°C (agar side down). After incubation, each suspension was harvested from the
bottles using PBDW and 15-20 sterile glass beads to target 1 x 10" CFU / mL. A 1.00 mL aliquot of
PBDW was used, per bottle, to harvest this organism. The suspension was removed from the botties,
filtered through sterile Whatman No. 2 paper pre-wetted with approximately 1.00 mL sterile of PBDW,
and collected into a sterile vessel.
‘The test organism culture suspension was placed in a spectrophotometer and the absorbance value was
recorded at 620 nm. The absorbance value for the Salmonella typhi culture suspension was 2.019 and a
2.00 mL. aliquot of PBDW was added to the culture suspension. The absorbance reading was taken
again and was 1.958. No additional adjustments to the culture suspension were necessary.
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Methicilin Resistant Staphylococcus aureus was transferred daily on Nutrient agar A agar slants. Three
to thirty consecutive daily transfers were performed prior to the inoculation of agar bottles. The bacterial
growth was washed from a 24 + 2 hour slant ofthe test organism using 5.0 mL phosphate buffer dilution
water (PBDW). This growth suspension was aspirated and added to $9.0 mL PBDW. A 2.0 mL. aliquot
of this suspension was inoculated onto five French square bottles containing Nutrient agar B. The bottles
were tilted back and forth to distribute the inoculum. The excess inoculum was aspirated off. The bottles
were incubated for 18-24 hours at 35-37°C (agar side down). After incubation, each suspension was
harvested from the bottles using PBDW and 16-20 sterile glass beads to target 1 x 10'°CFU/mL. A3.0
mL aliquot of PBDW was used, per botile, to harvest this organism. The suspension was removed from
‘the bottles, filtered through sterile Whatman No. 2 paper pre-wetted with approximately 1.00 mL sterile of
PBDW, and collected into a sterile vessel.
The test organism culture suspension was placed in a spectrophotometer and the absorbance value was
recorded at 620 nm. The absorbance value for the Methicillin Resistant Staphylococcus aureus culture
suspension was 2.185 and a 3.0 mL aliquot of PBDW was added to the culture suspension, The
absorbance reading was taken again and was 2,078, No additional adjustments to the culture
suspension were necessary.
Antimicrobial susceptibility testing was performed utilizing a representative culture from the day of
testing to verify the antimicrobial resistance pattem stated,
Methicilin Resistant Staphylococcus aureus (ATCC 33592) was purchased from the American Type
Culture Collection (ATCC) by ATS Labs. ATS Labs verified that the organism was resistant by
performing a Kirby Bauer Susceptibility assay on the day of testing. The organism was subcultured
‘onto a Tryptic Soy + 5% Sheep Blood agar plate and was incubated for 24 hours at 35-37°C.
Following incubation, a suspension of the test organism equal to a 0.5 MoFarland Standard was made
in 0.85% sterile saline. The suspension was streaked onto Mueller Hinton agar plate in three planes.
An oxacillin disc was placed in the center of the inoculated Mueller Hinton agar plate, The plate was
inverted and incubated for 224 hours at 36-37°C. Following incubation, the zone of inhibition was
measured using a calibrated caliper. A control organism, Staphylococcus aureus (ATCC 25923), was
Tun concurrently with the test organism to confirm the validity of the assay. The interpretation of the
zone of inhibition is based on established performance standards of the Clinical and Laboratory
Standards Institute (CLSI). See Table 4 for results.
‘An organic soil load was added to the test culture per the Sponsor's request.
Inoculation of Flasks.
Duplicate 250 - 300 mL Erlenmeyer flasks containing 99.0 mL of the test substance at the concentration
to be tested were placed into a room temperature (20.0°C) waterbath and was allowed to equilibrate for
220 minutes.
Flasks containing 99.0 mL sterile PBDW were prepared to be used for “initial numbers" control. The
PBDW was placed in the room temperature (20.0°C) waterbath and allowed to equilibrate 220 minutes.
A volume of 1.00 mL of culture suspension was added to each flask as follows:
a) The flask was whirled in a circular motion, and stopped just before the suspension was added, which
created enough residual motion of liquid to prevent pooling of the suspension at the point of contact
with the test substance.
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») The test organism suspension was added midway between the center and the edge of the surface
with the tip of the pipette slightly immersed in test solution. Touching of the pipette to the neck or
side of the flask during addition of the test organism suspension was avoided
Neutralization/Subcutture
At the specified exposure time point, one (1.00) mL of the inoculated test substance was transferred to 9
mL ofneutralizer, The neutralizer was vortex mixed. Four 1.0 mL and 0.1 ml aliquots were transferred
to individual sterile Petri dishes. A sufficient volume (approximately 15-25 mL) of tryptone glucose extract
agar was transferred to each Petri dish and each dish was allowed to solidify.
Incubation and Observation
The subculture plates were incubated for approximately 48 hours at 35-37°C before enumeration of
survivors Following incubation, the plates were visually examined for growth.
STUDY CONTROLS
Purity Control
‘A“streak plate for isolation” was performed on the organism culture and following incubation examined in
order to confirm the presence of a pure culture. The acceptance criterion for this study control is a pure
culture demonstrating colony morphology typical ofthe test organism.
Neutralizer Sterility Control
A representative sample of uninoculated neutralizer (1.00 mL) was transferred to a sterile Petri dish and
pour plated using TGEA, incubated, and visually examined, The acceptance criterion for this study
control is lack of growth.
Organic Soil Sterility Control
The serum used for soil load was cultured, incubated, and observed for lack of growth. The acceptance
criterion for this study control is lack of growth.
PBDW Sterility Control
A representative sample of PBDW (1,00 mL) was transferred to a sterile plate and pour plated using
GEA. The plate was incubated and visually examined. The acceptance criterion for this study control is
lack of growth.
‘Subculture Agar Sterility Control
‘One sample of the subculture agar was pour plated at the end of testing, incubated with the test and
visually examined. The acceptance criterion for this study control is lack of growth.
Viability Control
‘An aliquot of the test organism (0.10 mL) was transferred to a plate and the subculture agar added and
allowed to harden. The plates were incubated and visually examined. ‘The acceptance criterion for this
study control is growth,
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Numbers Control
Prior to addition of the organism, the PBDW was placed in the room temperature (20.0°C) waterbath
and allowed to equilibrate for 220 minutes. For each organism on the day of testing, one (1.00) mL of
the initial suspension was added to 99.0 mL of PBDW representing the “Test Substance’. A volume
of 1.00 mL of this suspension was added to 99.0 mL PBDW (Dilution 1, representing the 10” dilution).
After mixing thoroughly, 1.00 mL of Dilution 1 was transferred to 99.0 mL PBDW (Dilution 2,
representing the 10° dilution). third dilution was made using 1.00 mL Dilution 2 into 99.0 mL PBDW
(Dilution 3, representing the 10° dilution). Four 1.0 mL and four 0.1 mL. aliquots from Dilution 3 were
transferred to individual Petri dishes. Approximately 15-25 mL of TGEA was added to each plate.
The plate was swirled to mix and the agar was cooled to solidify. The plates were inverted and
incubated with the test. The acceptance criterion for this study control is a calculated result between
75 and 125 x 10° CFU/mL of “test substance"
Neutralization Confirmation Control
Aliquots of 1.0 mL and 0.1 mL of the neutralization control samples (a 9 mL neutralizer tube inoculated
with 1.0 mL of the test substance) were transferred to individual sterile Petri dishes in duplicate. A 1.0
mL aliquot of a diluted suspension of the test organism (serially diluted to target approximately 100 CFU)
was added to each dish. Approximately 15-25 mL of TGEA was added to each Petri dish, swirled to mix
and allowed to harden. A volume of 1.0 ml of the diluted test organism suspension was also plated as a
numbers control to verify the amount of organism inoculated. This control was performed with multiple
replicates representing different dilutions of the test organism. The control result is reported using data
from the most appropriate dilution. The acceptance criterion for this study control is growth within 1.0
logo of the neutralization confirmation numbers control.
STUDY ACCEPTANCE CRITERIA
Test Substance Performance Criteria
‘The EPA efficacy performance requirements for label claims state that a sanitizer must show a
'99,999% reduction of the test organism within 30 seconds.
Control Acceptance Criteria
The study controls must perform according to the criteria detailed in the study controls description
section.
PROTOCOL CHANGES
Protocol Amendments:
No protocol amendments were required for this study.
Protocol Deviations:
No protocol deviations occurred during this study.
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DAT: \LYSI
Calculations
CFUmL = (Average number of survivors on duplicate plate counts) x (Test dilution)
(Volume plated)
Test dilution = 10 (Test)
10° (Numbers Control)
Percent Reduction = 1 — (average of survivors/average numbers control) X 100
Five digits were used when caloulating Percent Reduction values.
Statistical Analysis
None used.
STUDY RETENTION
Record Retention
All of the original raw data developed exclusively for this study shall be archived at ATS Labs, 1285,
Corporate Center Drive, Suite 110, Eagan, MN 55121. These original data include, but are not limited
to, the following:
1. All handwritten raw data for control and test substances including, but not limited to, notebooks,
data forms and calculations.
2. Any protocol amendments/deviation notifications.
3. All measured data used in formulating the final report.
4. Memoranda, specifications, and other study specific correspondence relating to interpretation
and evaluation of data, other than those documents contained in the final study report.
§. Original signed protocol.
6. Certified copy of final study report.
7. Study-specific SOP deviations made during the study.
Test Substance Retention
The test substance will be discarded following study completion per Sponsor approved protocol. Itis the
responsiblity of the Sponsor to retain a sample of the test material.
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REFERENCES
1. Official Methods of Analysis of the AOAC, Seventeenth Edition, 2000. Chapter 6-Disinfectants,
960.09. Germicidal and Detergent Sanitizing Action of Disinfectants with modifications as described
in the experimental design.
2. U.S. Environmental Protection Agency, Registration Division, Office of Pesticide Programs, 1979.
Efficacy Data Requirements, Sanitizing rinses (for previously cleaned food-contact surfaces),
DISiTss-4.
3. U.S. Environmental Protection Agency, Registration Division, Office of Pesticide Programs,
1979. Efficacy Data Requirements, Supplemental Recommendations, DIS/TSS-2.
RESULTS
For Control and Neutralization Results, see Tables 4, 2 and 7.
All data measurements/controls including the culture purity, viability, organic soil sterility, diluent
sterility, PBDW sterility, subculture agar sterility, neutralizer sterility, numbers control, and
neutralization confirmation controls were within acceptance criteria. Furthermore, antibiotic resistance
Verification results met established standard acceptance criteria.
For Numbers Control and Test Results, see Table 3-6.
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ANALYSIS
Enagic Super 501 Strong Acidic Water 2.5 pH, Machine 1 (Serial # 87100333) Lot # 1 and Machine 2
(Serial #: 87100339) Lot # 2 with pre-fter C-1000, demonstrated a >99.999 percent reduction of
‘Staphylococcus aureus following a 30 second exposure time at room temperature (20.0°C) in the
presence of a 5% fetal bovine serum organic soil load
Enagic Super 501 Strong Acidic Water 2.5 pH, Machine 1 (Serial #. 87100333) Lot # 1 and Machine 2
(Serial #. 87100339) Lot # 2 with pre-fiter C-1000, demonstrated a >9.999 percent reduction of
Escherichia coli, following a 30 second exposure time at room temperature (20.0°C) in the presence of a
5% fetal bovine serum organic soll load.
Enagic Super 501 Strong Acidic Water 2.5 pH, Machine 1 (Serial #. 87100333) Lot # 1 and Machine 2
(Serial # 87100339) Lot # 2 with pre-fiter C-1000, demonstrated a >99.999 percent reduction of
Salmonella typhi, following a 30 second exposure time at room temperature (20.0°C) in the presence of
a 5% fetal bovine serum organic soil load.
Enagic Super 501 Strong Acidic Water 2.5 pH, Machine 1 (Serial #: 87100333) Lot # 1 and Machine 2
(Serial # 87100339) Lot # 2 with pre-fiter C-1000, demonstrated a >99,999 percent reduction of
Methicillin Resistant Staphylococcus aureus - MRSA, following a 30 second exposure time at room
temperature (20.0°C) in the presence of a 5% fetal bovine serum organic soil load.
1286 Corporate Center Ove, Sle 170 + Eagan, MV 55171 + 677:267.6978 » Fax 651,79.5540 + ww asbs.con Propet Wo, A386 wees /ANTS&LABS
Protocol Number: YNRO10401 10.GDST Page 18 of 47
CONCLUSION
Under the conditions of this assay, Enagic Super 501 Strong Acidic Water 2.5 pH, Machine 1
(Serial #: 87100333) Lot # 1 and Machine 2 (Serial #: 87100339) Lot # 2 with pre-filter C-1000,
demonstrated efficacy against Staphylococcus aureus for precleaned, nonporous food contact
surfaces following a 30 second exposure time at room temperature (20.0°C) in the presence of
a 5% fetal bovine serum organic soil load as required by the U.S. EPA for sanitizer label claims
Under the conditions of this assay, Enagic Super 501 Strong Acidic Water 2.5 pH, Machine 1
(Serial #: 87100333) Lot # 1 and Machine 2 (Serial #: 87100339) Lot # 2 with pre-filter C-1000,
demonstrated efficacy against Escherichia coli for precleaned, nonporous food contact
surfaces following a 30 second exposure time at room temperature (20.0°C) in the presence of
a 5% fetal bovine serum organic soil load as required by the U.S. EPA for sanitizer label claims
Under the conditions of this assay, Enagic Super 501 Strong Acidic Water 2.6 pH, Machine 1
(Serial #: 87100333) Lot # 1 and Machine 2 (Serial #: 87100339) Lot # 2 with pre-filter C-1000, did
demonstrated efficacy against Salmonella typhi for precleaned, nonporous food contact
surfaces following a 30 second exposure time at room temperature (20.0°C) in the presence of
5% fetal bovine serum organic soil load as required by the U.S. EPA for sanitizer label claims
Under the conditions of this assay, Enagic Super 01 Strong Acidic Water 2.5 pH, Machine 1
(Serial #: 87100333) Lot # 1 and Machine 2 (Serial #: 87100339) Lot # 2 with pre-filter C-1000,
demonstrated efficacy against Methicillin Resistant Staphylococcus aureus - MRSA for
precleaned, nonporous food contact surfaces after a 30 second exposure time at room
temperature (20.0°C) in the presence of a 5% fetal bovine serum organic soil load as required
by the U.S. EPA for sanitizer label claims
In the opinion of the Study Director, there were no circumstances that may have adversely affected the
quality or integrity of the data,
The use of the ATS Labs name, logo or any other representation of ATS Labs without the
written approval of ATS Labs is prohibited. In addition, ATS Labs may not be referred to in
any form of promotional materials, press releases, advertising or similar materials (whether
by print, broadcast, communication or electronic means) without the express written
permission of ATS Labs.
1285 Corporate Center Ove ise 110 + Eagan, MN SS121 + 677-267 8R7B « Fax 651 STR.SEA9 « wn. atesabe com Project No. A09385
Protocol Number: YNRO10401 10.GDST
YNR Marketing
Page 19 of 47
ATS#&LABS
TABLE 1: CONTROL RESULTS
The following results from controls confirmed study validity:
Type of Control Results
Neutralizer Sterility Control No Growth
Purity Control Staphylococcus aureus (ATCC 6538) Pure
Purity Control Escherichia coli (ATCC 11228) Pure
Purity Control Salmonella typhi (ATCC 6539) Pure
Purity Control Methicilin Resistant Staphylococcus aureus — Pure
MRSA (ATCC 33592)
Viability Control Staphylococcus aureus (ATCC 6538) Growth
Viability Control Escherichia coli (ATCC 11229) Growth
Viability Control Salmonella typhi (ATCC 6539) Growth
Viability Control Methicilin Resistant Staphylococcus aureus — aan
MRSA (ATCC 33592) _ te att
Subculture Agar Sterility Control (before testing) No Growth
Organic Soil Load Sterility No Growth
PBDW Sterility Control No Growth
1285 Corporate Center Ove, Suite 110 + Eagan, MI 85121 « B77 287.8078 « Fax 681 3T9.AGEG « wawatsiabs com Project No. A09386
Protocol Number: YNRO1040110.GDST
YNR Marketing
Page 20 of 47
ATS#LABS
TABLE 2: NEUTRALIZATION CONFIRMATION CONTROL RESULTS
Volume of
Numbers | Neutralized
ee 5 | jContel | tnocutatea bog Passi
‘Test Substance Test Organism | & | (rom) - reuse ore eae
: 6 SEY Logi.
cru forme) FP | me
‘Staphylococous
‘aureus | 13,18 | 12.17 | 11,8 | 015 | Pass
(ATCC 8638)
Enagic Super 501 Escherichia coli
srong nase waer2s | "asrhie col 104 | 610 | 18) 02 | Pass
gta ‘Salmonella typhi
(Serial #: 87100333) ] 6s | w2sfom| o2 |p
Lot # 1 with pre-fiter (ATCC 6539) : 7 ee
1000 Methicillin Resistant
‘Staphylococcus
ee unen 17,7 | 166] 11,9 | 008 | Pass
(ATCC 33692) : |
‘Staphyiooecus | "© |
‘aureus 13,15 | 12,20] 815 | 007 | Pass
(ATCC 6538)
Enagle Super 501 Escherichia col
Strong Acidic Water2.5 | (ATCC 11229) Ora estte eerie eedeeara tel ee
pH, Machine 2
(Serai#: 67100330) | Somonete yen! 68 | 11,18 | 10,10] 02 | Pass
Lot #2 with pre-fiter )
¢-1000 Methicillin Resistant
Staphylococcus
penlureon 17,7 | 13,20] 14,13) 007 | Pass
(ATCC 33592)
CFU = Colony forming unit
1285 Corporate Conor Driv, Suto 40 » Eager, MN 55121 « €77.287,8378 « Fax 851.370.5549 + wnw.ateisbacom Projet No ADE885 vnruaistrs ANT Se
Protocol Number: YNRO1040110.6DST Page 21 of 47
TABLE 3: TEST RESULTS - Staphylococcus aureus (ATCC 6538)
Raw Data for Staphylococcus aureus (ATCC 6538)
"Microbes Initially Present
= | Numbers | Numbers
Amb of 10" (in Control | Controf
= neutralizer) 0.4 mL of 10% | 4.0 mL of 10%
Enagic Super 504
Strong Aciaic Water Runt
2.6 pH,
Machine 4
(Serial #: 87100333)
Lot with pre-fiter Run2
©1000 30
Enagic Super 501 | Seconds
Strong Acidic Water
25 pH,
Machine 2
{Serial #: 87100339)
Lot #2 with presfiter Run2
¢-1000
10,6,9,14 | 75, 80, 74,77
Run 1
Avvalue of <1 was used in place of zero for calculation purposes only.
Calculated Results for Staphylococcus aureus (ATCC 6538) by Lot, Exposure, and
Corresponding % Reduction
: 5 Wicrobes Initially :
rectSubeinane: | Exposure | AYPEeae Number Present, Porcent
os | Time erORAEY Numbers Control Reduction.
‘ ) (CEUImL)
Enagic Super 501 Strong
Acidic Water 2.5 pH,
Machine 1 99,909
(Serial #: 87100333) Lot # 4
with pre-fiter C-1000
Enagic Super 501 Strong
Acidic Water 2.5 pH,
Machine 2 99,909
(Serial #: 87100339) Lot # 2
with pre-fiter C1000
CFU = Colony Forming Unit
30 seconds 77X10"
1286 Corporat Center Dive, Suite #10 + Eagan, MIN S5421 + 677.287.8978 « Fax 651.378.5549 + wownatelabs.com Project No, 08005 wawatein ANT S&LABS
Protocol Number: YNRO1040110.GDST Page 22 of 47
TABLE 4: TEST RESULTS — Escherichia coli (ATCC 11229)
Raw Data for Escherichia coli (ATCC 11229)
Duplicate Plate Counts (CFUIplate)
{@ sets of 4 plates for each volume)
Tost Substance Estee Run#[___ Number Surviving s
: Test Test Numbers | Numbers
| OAmLof10" | 4.0mLof10" | Control Control
(in neutralizer) | (in neutralizer) | 0.1 mL of 10° | 1.0 mL of 10°
Enagic Super 504
Strong Acidic Water Run
2.5 pH,
Machine +
(Serial #: 87100333)
Lot # 1 with preefiter Run 2
1000
Enagic Super 501
‘Strong Acidic Water Run 1 0,0,0,0 0,0,0,0
2.5 DH,
Machine 2
(Serial #: 87100339)
Lot # 2 with ore-fiter Run2] 0,0,0,0 0,0,0,0
c-1000
30 seconds
11,16, 13,6 | 92,95, 93, 88
‘A value of <1 was used in place of zero for calculation purposes only.
Calculated Results for Escherichia coli (ATCC 11228) by Lot, Exposure, and Corresponding %
Reduction
: fvarago number | M"Biesent | percent
_ Test Substance Sunnving: ‘Numbers Control_| Reduction
2 (CFUimL) Teun)
Enagic Super 601 Strong
‘Acidic Water 2.6 pH,
Machine 4 99.999
(Serial #: 87100333) Lot # 4
with pre-fter C-1000
Enagic Super 501 Strong
Acidic Water 2.5 pH,
Machine 2 99.999
(Serial #: 87100339) Lot #2
with pre-fiter C-1000
30 seconds 9.2% 107
1285 Carports Carter, Sute 110 + Eagan, MN SS121 « B77.287.8978 + Fac 661.2795549 « wwe ats om Project No. Ao6s85 wirwersies ANT S#LABS
Protocol Number: YNR01040110.GDST Page 23 of 47
TABLE 5: TEST RESULTS ~ Salmonella typhi (ATCC 6539)
Raw Data for Salmonella typhi (ATCC 6539)
Duplicate Plate Counts (CFUiplate)
: (2 sets of 4 plates for each volume)
Test Substance | EPOSUS | pang ‘Number Surviving ‘Microbes initially Present
ime, Test Test ‘Numbers
O.tmLof 107 | 4,0mLof 10" | Control
{in neutralizer) | {in neutralizer) | 0.1 mt of 10° | 1.0 mL of 10*
Enagic Super 601
Strong Acidic Water Runt] 0,0,0,0 0,0,0,0
2.5 pH,
Machine 1
(Serial # 87100333)
Lot #1 with pre-fiter Run2] 0,0,0,0 0,0,0,0
¢-1000
Enagic Super 501
Strong Acidic Water
2.5 pH,
Machine 2
(Serial #: 87100339)
Lot # 2 with pre-fiter Run2] 0,0,0,0 0,0,0,0
€-1000
111, 109, 91,
113
8, 15, 10, 17
seconds
0,0,0,0
0,0,0,0
A value of <1 was used in place of zero for calculation purposes only.
Calculated Results for Salmonella typhi (ATCC 6539) by Lot, Exposure, and Corresponding %
Reduction
Wicrobes initially
Prosent Percent
Reduction
‘Average Number
Surviving
(CFUImL)
Exposure
Test Substance
Enagic Super 501 Strong
‘Acidic Water 2.5 pH,
Machine 1 <1x10"
(Serial #: 87100333) Lot # 1
with pre-fiter C-1000
Enagic Super 50 Strong
Acidic Water 2.5 pH,
Machine 2
seers ines oe
; ;
1288 Corporate Center Ove, Sule 110 + Eagan, MA SS121 + 877-287.8978 « Fax 681 79.5549 + wn aelabs com Project No, Aas wanes /XTS&LABS
Protocol Number: YNR01040110.GDST Page 39 0f 47
Hd de YWROLOWCI/A, C-US4
D-
PHP pe ;
Name of each parts
+285 Cororoe Center Drive, Sule 110 + Eagan, MN G6121 » 877.287.8878 « Fax 651 9795549 « wer atsabs com Pret, A086 wanes ANTS&LABS
Protocol Number: YNRO1040110.GDST Page 40 of 47
0ST
[brbmnt bo yar oft lle
pry S41
4 ca comae
"205 Corporate Center Dive, Sie £10 + Epan, MN 65421 « 677.287.8878 « Fax: 651.979.5540 « wn aesabacem Project No. A00395 winners ACT S&LABS
Protocol Number: YNRO1040110.GDST Page 41 of 47
Mhbmt & YNRelcYo vo. C0sT
be a Ir 7 EXACT py
nS 9
Displays and Description eb Adve
on Operation Panel
Operation Panel within Cover
+288 Cerprat Center Dive, Suite 110 + Eagan, NAW 86121 « 877.287.8578 + Fax: 651.970.5549 « wnatslads.com Project No, 00085 wrvercio ANT SLABS
Protocol Number: YNRO1040110,GDST Page 42 of 47
Mirckinnd fo YARol Ch HD. @ OST
Vem ph 4-tes? 7
adam S00
nate
| -2 Installing the Corporation Cock = 1° & a.
(© incase ofa general type of faucet and tho ming faucet
Acta fs foasbe by using the standard acogssoné)
rer me noagns ap tom
vena ge anh oe a
do tosinch
om
tray bm coca
cerpeaion ‘on th war aucet, oun to water
oo faucet pipe on the cock. At this time,
eo
opp,
OM AN pg
© Pross the [Strong Acisic Wate] button locatod on the top
‘operation panel Song Acidic Watr willbe dscharged
‘tom the left ex pio (orango). (Strong KANGEN Wester
‘willbe discharged from the water discharge hose (gray).)
re tha [Sep baton on op poratin pane.
QGG88 One ee ay
‘lpe (orange) and Strong KANGEN Water from the water \
eakge hos (rey) nse te dopa” :
peoe of }
?
|insgecne” Use pH test poper (included). /
Acidic Water. (© carey oe at paper to mao i
+ Book pH tect paper ‘out immediately. f :
(atached} nfbeasea Sb
@ shake the pH test paper genty to remove y
coma
SS
"
© creck tne ctr o th wot orton othe paper
‘gaat andard calor cha
"Cid conparion and tamination
Sons
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