Final report GLP-SP-240 - Biosenta Inc.Southwest Research Institute Project 01.20792 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal

Southwest Research Institute Project 01.20792

Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240

Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 1 of 17

FINAL REPORT

STUDY TITLE

Germicidal Spray Test of ZeroMold Plus for Additional Bacteria

SOUTHWEST RESEARCH INSTITUTE PROJECT NUMBERS

01.20792.01.001 (Notebook#14-0207-036)

TESTING FACILITY Southwest Research Institute (SwRI

®)

Chemistry and Chemical Engineering Division

Applied Physics Division

Applied Power Division

6220 Culebra Road

San Antonio, TX 78238-5166, USA

Tel: 210-522-6354, Fax: 210-522-4565

STUDY SPONSOR BIOSENTA Inc.

1120 Finch Ave West, Suite 503

Toronto, Ontario, M3J 3H7, CANADA, Tel: 416-410-2019

TEST GUIDELINE OCSPP 810.2200

TEST ORGANISMS: Methicillin-Resistant Staphylococcus aureus (MRSA)

(ATCC 33592)

TEST PRODUCT IDENTITY

ZeroMold PlusTM

(48538, BI# 0019-6, Lot #3, >60 days)

ZeroMold PlusTM

(48539, BI# 0020-1, Lot #4, >60 days)

STUDY DATES

Date Sample Received: 02/17/2014

Study Initiation Date: 11/07/2014

Experimental Start Date: 11/14/2014

Experimental End Date: 12/13/2014

Study Completion Date: 12/19/2014




Table of Contents

TABL OF C

STUDY TITLE ............................................................................................................................... 1

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS ................................................... 2

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT......................................... 3

QUALITY ASSURANCE UNIT STATEMENT OF COMPLIANCE.......................................... 4

TABL OF C .................................................................................................................................... 5

1. OBJECTIVE ............................................................................................................................ 6

2. TEST SUBSTANCE, MATERIALS, EQUIPMENT & PERSONNEL ................................. 6

Test Substance ............................................................................................................................ 6

Materials and Equipment ............................................................................................................ 7

Personnel ..................................................................................................................................... 9

3. PROCEDURES ..................................................................................................................... 10

4. PROTOCOL CHANGE AND STUDY DEVIATION ......................................................... 10

Protocol Amendments ............................................................................................................... 10

Study Deviations ....................................................................................................................... 10

5. STUDY DESIGN .................................................................................................................. 10

6. CONTROLS AND ACCEPTANCE CRITERIA .................................................................. 12

7. STUDY ACCEPTANCE CRITERIA ................................................................................... 13

8. CALCULATION AND STATISTICAL ANALYSIS .......................................................... 13

9. RESULTS .............................................................................................................................. 14

Methicillin-Resistant Staphylococcus aureus (MRSA) ............................................................ 14

10. STUDY CONCLUSION ....................................................................................................... 16

11. RECORD AND SAMPLE RETENTION ............................................................................. 16

REFERENCES ............................................................................................................................. 16

APPENDICES .............................................................................................................................. 17




1. OBJECTIVE The purpose of this study was to determine the effectiveness of the Sponsor’s product: ZeroMold

Plus disinfectant, for hard surface disinfection following the Association of Analytical

Communities (AOAC) Germicidal Spray Method in accordance with the Environmental

Protection Agency (EPA) 40 CFR 160 Federal Insecticide, Fungicide, and Rodenticide Act

(FIFRA) Good Laboratory Practice (GLP) Standards. This method is in compliance with the

requirements of EPA (OCSPP 810.2200) and Health Canada Therapeutic Products Directorate

(TPD). AOAC Official Method 961.02 (2012) is applicable for testing spray and pressurized

spray disinfectants to determine effectiveness as disinfectant for contaminated hard, nonporous,

inanimate environmental surfaces. Additionally, the Clinical and Laboratory Standards Institute

Performance Standards (CLSI) for Antimicrobial Disk Susceptibility Tests (M02-A11) was

followed for the antibiotic resistance confirmation requirement when using antibiotic resistant

test organisms. The test conducted under these protocols included:

• GLP AOAC Germicidal Spray Test (MRSA) for “Additional Bacteria” claim

2. TEST SUBSTANCE, MATERIALS, EQUIPMENT & PERSONNEL

Test Substance Test substance information was shown below and active ingredient concentration was shown in

Table 1. All ZeroMold Plus tested were previously titrated and diluted according to

manufacturer’s instructions to ensure compliance with EPA guidance on LCL (SwRI GLP-SP-

210 study).

Name: ZeroMold Plus

Active ingredient: NaClO

Lower certified limit (LCL): 0.43%

Acceptable active ingredient concentration for efficacy testing: 0.43%-0.44 % (2% above

LCL)

Storage Conditions: Ambient Temperature, cap closed (all lots tested)

Test substance spray condition is as follows:

Distance = 6 - 8"

Number of pumps = 4

Contact Time = 10 minutes

Exposure Temperature = 18 - 24°C (room temperature)

Table 1. Test substance active ingredient concentration (LCL to up to 2% above LCL).

ZeroMold Plus

Test batch/lot information

Active ingredient

(NaClO)

concentration

Concentration/Dilution Tested

BI# 0019-6, Lot #3, >60 days 0.44% Ready-to-use, Trigger Spray

BI# 0020-1, Lot #4, >60 days 0.44%




Materials and Equipment The materials and major equipment used in the study are indicated in Table 2 and 3.

Table 2. Materials used in this study. COA/Calibration documents archived in QA department.

Materials CAS# Batch/Lot # Supplier Grade Expiry

10 mL

Serological Pipet N/A 3266820 Becton Dickinson N/A 9/30/2018

25 mL

Serological Pipet N/A 3324836 Corning N/A 11/30/2018

5 mL Serological

Pipet N/A 3276848 Becton Dickinson N/A 10/31/2018

Cefoxitin N/A 1505808 Oxoid/Fisher

Scientific 30 mcg/disc June-17

CHROMagar

MRSA II N/A 4281612 Becton Dickinson

Individual

plates 12/25/2014

Coagulase Cryo N/A 14224 Hardy Diagnostics N/A 4/9/2015

Cotton applicators N/A 20130926 Fisher Scientific N/A 9/26/2015

Ethanol 64-17-5 136936 Fisher Scientific Anhydrous,

Histology Oct-16

Glycerol 56-81-5 B0514622 Fisher/Acros Spectrophot

ometric October-18

Gram Stain Kit

Multiple,

(see

CoA)

3330114,33

30126,

3291128,

3330116

Becton Dickinson N/A 4/30/2015

Hydrochloric acid 7647-01-

0

SHBD5057

V Sigma-Aldrich

36.5-38%,

Bio Reagent Oct-15

HyPure Water N/A AZA181036 HyClone

Molecular

Biology

Grade

Jan-16

Inoculation loops N/A 140108607,

130805607 Fisher Scientific N/A N/A

Letheen Broth N/A 4069265 Becton Dickinson N/A 2/29/2016

McFarland

Turbidity

Standard

N/A 536267 Remel/Fisher

Scientific 0.5 11/18/2015

Mueller Hinton

Agar N/A 14274 Hardy Diagnostics

Individual

plates 12/30/2014

Nutrient Agar N/A 4233924 Becton Dickinson Individual

plates 12/17/2014

Nutrient Broth N/A 3254131 Becton Dickinson N/A 2/29/2016




pH Buffer Kit

Multiple,

(see

CoA)

136183 Fisher Scientific N/A Feb-15

Phosphate Buffer N/A 542965 Remel/Fisher

Scientific Butterfield 8/13/2015

Saline 7647-14-

5 544813

Remel/Fisher

Scientific 0.85% 8/18/2015

Sodium

Hydroxide

1310-73-

2

SLBH8376

V Sigma-Aldrich

Reagent

grade,

≥98%

pellets

Oct-15

Sodium

Thiosulfate

7772-98-

7

BCBM3765

V Sigma-Aldrich Anhydrous 9/15/2024

Staphylococcus

aureus N/A 3601741

Hardy

Diagnostics/Micro

biologics/ATCC

#25923 Aug-15

Staphylococcus

aureus (MRSA) N/A 59363660 ATCC #33592 N/A

Staphylococcus

epidermidis N/A 3711113

Hardy

Diagnostics/Micro

biologics/ATCC

#12228 Nov-15

Trypticase Soy

Agar N/A 4239613 Becton Dickinson

Individual

plates 12/25/2014

Trypticase Soy

Broth N/A 1194164 Becton Dickinson N/A 5/31/2015

Table 3. Equipment used in this study. Calibration/Verification documentation archived in QA

department.

Equipment Supplier/Model# Serial Number Expiry if

applicable Location

Autoclave Tuttnauer, 3870M 2304553 N/A B244, L1.115

Check weights Class N/S 20364 2/13/2015 B244, L1.115

Data Logging

System N/A N/A N/A B244, L1.115

Incubator Lab-Line, Imperial III,

305 09045401 N/A B244, L1.115

Incubator Lab-Line, Imperial III,

305 04033589 N/A B244, L1.115

pH Meter Oakton pH/CON, 510

series 181287 N/A B244, L1.115

Pipette Eppendorf Research

Plus, P200 284481A 2/26/2015 B244, L1.115





Plus, P200 115108Z 2/26/2015 B244, L1.115


Plus, P1000 361974Z 2/26/2015 B244, L1.115

Pipette Socorex, Acura 815,

P1000 18061341 2/26/2015 B244, L1.115

Refrigerator Sanyo Medicool, MPR-

411F 00909013 N/A B244, L1.115

Refrigerator/Freezer Fisher Scientific,

Isotemp, 13986106A 15210900000000 N/A B244, L1.111

Scale Sartorius, ED2202S 21951005 9/2/2015 B244, L1.115

Scale Mettler Toledo,

AB104-S 1125311520 9/2/2015 B244, L1.115

Temp./RH Probe Vaisala J3040001 9/5/2015 B244, L1.115

Thermocouple Omega, type K, -5 to

+105°C 18096 9/29/2015 B244, L1.115

Thermocouple Duro-Sense Corp., type

K, -80 to +180°C 336 2/24/2015 B244, L1.115

Thermometer Data

Logger Omega, HH306 050501588 9/29/2015 B244, L1.115

Timer Fisher

Scientific/S40799-2 61773758 2/24/2015 B244, L1.115

Ultra Low -80°C

Freezer Sanyo, MDF-C8V1 12010009 N/A B244, L1.111

Personnel

The following persons contributed to the study:

Xingguo Cheng, Ph.D., Senior Research Scientist, Study Director

Amy De los Santos, Research Scientist




3. PROCEDURES

The following Test and Analytical Procedures (TAPs) were utilized in this study and are shown

in Table 4.

Table 4. TAPs used in this project and their applications (TAP 01-0207-037 was attached in

Appendix 1, all TAPs were archived by QA)

TAP

Number

Name Application

01-0207-030 Autoclave Operation and Validation for use in the

Microbiology Laboratory

Sterilization

01-0207-028 Use of the Oakton® pH/CON Meter in the

Microbiology Laboratory

pH measurement

01-0207-033 Verification and Use of Automatic Pipettes in the

Microbiology Lab

Pipetting

01-0207-029 Calibration and Verification of Analytical Balances Weighing

01-0207-031 Calibration of Thermocouples and Relative Humidity

Sensors

Temperature

01-0207-037 Germicidal Spray Product Test for Additional

Bacteria: Disinfectant Testing Procedure.

Disinfectant testing

4. PROTOCOL CHANGE AND STUDY DEVIATION

The GLP study protocol was attached in Appendix 2.

Protocol Amendments No protocol amendments were required for this study.

Study Deviations No study deviation occurred during this study.

5. STUDY DESIGN

The brief study design is shown in Figure 1 below. The detailed test procedure is shown in TAP

01-0207-037 in appendix 1. Brief test parameters are shown below:

Carrier Type: Glass slides (25 mm×25 mm)

Contact Time: 10 Minutes

Test Temperature: 18-24 °C (room temperature)

Growth Medium: Nutrient Media

Sub-culture Medium: (Modified) Letheen Broth

Diluent Type: N/A




Organic Soil Load: None

Carrier Dry Time: 30 minutes

Carrier Dry Temperature: 36 ± 2°C

Incubation Temperature: 36 ± 2°C

Incubation Time: 48 ± 2 hours

Figure 1. Schematic diagram showing the study design of this GLP study.

Test substance received at SwRI

Preparation of Carriers and Test organisms

Inoculation of Carriers (control and test)

Exposure (Spray) of test substance onto carriers

Test System Recovery after contact time

Incubation and Observation for growth

Study controls confirmation




6. CONTROLS AND ACCEPTANCE CRITERIA

Purity Control A ‘streak plate for isolation’ was performed on each 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 of the test organism.

Carrier Sterility Control On the day of testing, a sterile, un-inoculated carrier was placed into a tube of neutralizing

subculture media. This subculture tube was incubated as said ‘test’ carrier tubes and examined

for growth. The acceptance criterion for this study control was lack of growth to confirm the

sterility of the carriers prior to test inoculation.

Viability Control On the day of testing, two dried inoculated carriers were placed into individual tubes of

neutralizing subculture media. These subculture tubes were incubated as said ‘test’ carrier tubes

and examined for growth. The acceptance criterion for this study control was growth which

confirms exposure to and viability of the test organism.

Neutralizing Subculture Medium Sterility Control A representative sample of un-inoculated neutralizing subculture medium was incubated with

said ‘test’ carrier tubes and other controls and examined. The acceptance criterion for this study

control was lack of growth which confirms sterility of prepared neutralizing subculture media.

Neutralization Confirmation Effective neutralization of the test substance was confirmed by exposing sterile carriers to the

test substance and transferring them to subculture tubes containing the neutralizing media. The

subculture tubes were inoculated with 10-100 CFU of the test organism, incubated under test

conditions and visually examined for the presence of growth. This control was performed with

multiple replicates using different dilutions of the test organism and performed for each test

organism the test substance was tested against. A standardized spread plate was run concurrently

in order to enumerate the number of CFU actually added. The acceptance criterion for this study

control was growth in the subculture tube, minimally, following inoculation with ≤ 100 CFU.

Antibiotic Resistance Confirmation Confirmation of the antibiotic resistance of the test organism was conducted to include numerical

values for the antibiotic tested and the method used to obtain the results. Per the Clinical and

Laboratory Standards Institute (CLSI), Performance Standards for Antimicrobial Disk

Susceptibility Tests, Cefoxitin was tested as a surrogate for oxacillin. As oxacillin is not reliable

for testing and is the replacement antimicrobial agent for methicillin, which is no longer

commercially available in the United States. Table 5 contains the CLSI zone diameter

interpretive criteria for Staphylococcus aureus against the antimicrobial agent oxacillin.

Additionally, the test organism was characterized according to the following: the source and

identity, the transfer history, the method used to confirm the identity, and the method of

preservation/storage.




Table 5. Zone Diameter Standards for Staphylococcus aureus extracted from CLSI M100-

S24 Tables 2C and 4A.

Antimicrobial Agent

Oxacillin

Zone Diameter

(nearest whole mm)

Susceptible Resistant

Staphylococcus aureus ≥ 22 ≤ 21

Staphylococcus aureus ATCC 25923 23-29 N/A

Carrier population control (Enumeration of Viable Bacteria from Carriers) Three inoculated carriers were individually placed into a tube containing the subculture media.

Each carrier tube was vortexed to remove the bacteria from the carrier surface and then a series

of 10-fold dilutions was generated using phosphate-buffered dilution water. A sub-set of the

dilution series was plated, incubated, and the resulting colonies enumerated to determine the

CFU per carrier. The log10 density (LD) for each carrier was determined and used to calculate the

average LD for the test. The mean LD acceptance criteria for this study control are organism

dependent as shown in Table 6.

Table 6. Acceptable values of mean density, required per organism, when performing the

Germicidal Spray Product test.

Organism Average LD Corresponding Mean Density

MRSA ≥4.0 ≥1.0 x 104

7. STUDY ACCEPTANCE CRITERIA

Test Substance Performance Criteria

The U.S. EPA efficacy performance requirements for ‘Additional Bacteria’ claimed on the label

in addition to the base broad-spectrum claim, requires 10 out of 10 carriers are negative for

growth of the test organism.

Control Acceptance Criteria The study controls must perform according to the criteria detailed in the study controls

description section above. If any of the control acceptance criteria are not met, the test may be

repeated under the current protocol number.

8. CALCULATION AND STATISTICAL ANALYSIS

Neutralization Control Inoculum Enumeration:

[(Plate Count 1 + Plate Count 2/2) / (dilution factor x plated volume)] = Neutralization control

inoculum (CFU) Enumeration

Carrier Population Calculation:




CFU/Carrier =

rvolumeneutralizectordilutionfaxedvolumeplat

dilutionplateofcoloniesaverage×

)()(

)@/#(

Average Log10 Carrier Population Control = N

XNLogXLogXLog 10210110 ++

X= CFU/carrier

N= number of control carriers

9. RESULTS

Methicillin-Resistant Staphylococcus aureus (MRSA)

Control and Neutralization Results Control and neutralization results (Table 7) were in compliance with the aforementioned study

acceptance criteria. The neutralization control tube for MRSA was inoculated with 61.5 CFU,

which met the acceptance criteria.

Table 7. Various controls, neutralization, and carrier population results ZeroMold

Plus

Purity

Control

Carrier

Sterility

control

Viability

Control

Neutralizing

Subculture

Medium

Sterility

Control

Neutralization

Confirmation

Carrier

population

control

SwRI

test #

BI# 0019-6,

Lot #3

Acceptable No

growth

Growth No growth Growth 3.98 x 106 1

BI# 0020-1,

Lot #4

Acceptable No

growth

Growth No growth Growth 3.98 x 106 2

Test Carrier Results ZeroMold Plus demonstrated the following results as shown in Table 8. All test carriers were

negative following 10 min exposure.

Table 8. Summary of testing results ZeroMold

Plus at

LCL

BioSenta

Batch/Lot

number

Test organism Contact

time

Soil

load

Number

of carriers

tested

Carriers

positive

Carrier

negative

>60 days

old

BI# 0019-6,

Lot #3 Staphylococcus

aureus (MRSA)

#33592

10 min.

None

10 0 10

>60 days

old

BI# 0020-1,

Lot #4 10 min. 10 0 10

Antibiotic Resistance and Culture Identification Confirmation

On the day of testing, the antibiotic resistance (CLSI disk diffusion test) and identity (e.g.

biochemical test, Gram stain, morphology) of the test organism utilized was confirmed per EPA

OCSPP 810.2000 (e) (3). A summary of these test results are stated in Table 9.




Table 9. Results of test and quality control strains for identification and antibiotic

resistance tests performed.

Test

Staphylococcus aureus

MRSA

ATCC 33592a

Staphylococcus aureus

ATCC 25923b

Staphylococcus

epidermidis

ATCC 12228b

Gram stain +, non-spore coccus,

singly, pairs and clusters N/A N/A

Colony morphology:

Nutrient agar/TSA

Entire, glistening, circular,

low convex, yellow-white,

opaque, and smooth


low convex, yellow-white,

opaque, and smooth


convex, opaque, and

smooth

Coagulase + + ̶

CHROMagar MRSA II Mauve colony color No growth N/A

CLSI Disk diffusion test

zone diameter

30 µg Cefoxitinc

Resistant

0 mm

Susceptible

24-25 mm N/A

a Test organism, b Quality control strain, c Cefoxitin was tested as a surrogate for oxacillin. Oxacillin is not reliable for

testing and is the replacement antimicrobial agent for methicillin, which is no longer commercially available in the United

States.

Per OCSPP 810.2000 (e)(3)(i) through (e)(3)(iv), the following information should be included

when using an antibiotic resistant test organism.

Source and Identity: American Type Culture Collection, Methicillin-resistant Staphylococcus

aureus; subsp. aureus #33592.

Transfer History/Storage: Prior to testing, the organism was rehydrated and propagated per the

ATCC product sheet in nutrient broth, to include a QC culture purity plate and media only

controls. After 24 hours of growth, the liquid culture was vortexed, aliquoted 1:1 in glycerol,

vortexed again and stored at -80°C until a single-use vial was thawed for testing. This

represented one passage from the ATCC freeze-dried vial prior to test culture initiation.

Culture Identification: Gram stain, colony morphology, coagulase test, and CHROMagar

MRSA II along with the appropriate controls, confirmed the test culture identity of methicillin-

resistant S. aureus (see Table 9).

Scientific Method for Antibiotic Resistance Confirmation: The Clinical and Laboratory

Standards Institute Performance Standards for Antimicrobial Disk Susceptibility Tests;

Approved Standard-Eleventh Edition (M02-A11) and the Performance Standards for

Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement (M100-S24)

were used to perform the disk diffusion test by the direct colony suspension method.




10. STUDY CONCLUSION

Under the conditions of the GLP testing, ZeroMold Plus disinfectant at LCL, was effective

(PASSED) against Methicillin-Resistant Staphylococcus aureus (MRSA) (ATCC 33592) per

EPA performance test guidelines.

11. RECORD AND SAMPLE RETENTION The study for a research or marketing permit approved by EPA shall be maintained for five years

from the period during which the sponsor holds that research or marketing permit to which the study

is pertinent unless all originals are provided to the client. For samples remaining at the conclusion of

the study SwRI will either return samples to the sponsor or dispose the sample after which the

sponsor holds that research or marketing permit to which the study is pertinent unless all originals are

provided to the client (EPA). Materials that degrade will not be maintained.

REFERENCES http://www.epa.gov/pesticides/methods/atmpmethods/MB-06-05.pdf

U.S. Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention,

Product Performance Test Guidelines, OCSPP 810.2000: General Considerations for Public

Health Uses of Antimicrobial Agents, March 12, 2012.

EPA Product Performance Test Guidelines; OCSPP 810.2200: Disinfectants for Use on Hard

Surfaces-Efficacy Data Recommendations, September 4, 2012.

http://www.epa.gov/oppad001/dis_tss_docs/dis-01.htm

http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series810.htm

AOAC Official Method 961.02 Germicidal Spray Products as Disinfectants (2012).

US Environmental Protection Agency, Office of Pesticide Programs, Lower Certified Limit

Testing Guidance, http://www.epa.gov/oppad001/lower-cert-limit-test-guidance.pdf

Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Disk

Susceptibility Tests; Approved Standard-Eleventh Edition, M02-A11, January 2012.

Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Disk

Susceptibility Tests; Twenty-Fourth Informational Supplement, M100-S24, January 2014.

http://www.cdc.gov/MRSA/lab/index.html

http://www.atcc.org/~/media/PDFs/mrsa09.ashx




APPENDICES Appendix 1. TAP 01-0207-037 (Rev. 0): Germicidal Spray Product Test for Additional Bacteria:

Disinfectant Testing Procedure used in this study.

Appendix 2. GLP Study Protocol (GLP-SP-240).

UNCONTROLLED COPY IF PRINTED

SOUTHWEST RESEARCH INSTITUTE®

Chemistry and Chemical Engineering Division Test/Analytical Procedure (TAP)

Group/Section TAP ID No. Revised By: Microbiology Laboratory

(B244, Labs 1.115 and 1.115C) TAP-01-0207-037 Xingguo Cheng

Title: Germicidal Spray Product Test for Additional Bacteria:

Disinfectant Testing Procedure

Effective Date: Revision Page

November 2014 0 1 of 18

Southwest Research Institute Proprietary

1.0 SCOPE

The purpose of this procedure is to serve as a detailed description to the performance of a Germicidal Spray Product Test that is repeatable and meets the Environmental Protections Agency’s specifications and criteria in accordance with EPA 40 CFR 160 Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Good Laboratory Practice (GLP) Standards.

2.0 PURPOSE

2.1 Identification of the Test Methods

AOAC Official Method 961.02 Germicidal Spray Products as Disinfectants US EPA Office of Pesticide Programs, Standard Operating Procedure for Germicidal Spray Products as Disinfectants: Testing of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica US EPA Office of Pesticide Programs, Standard Operating Procedure for Neutralization Confirmation Procedure for Products Evaluated with the AOAC Use Dilution Method and the AOAC Germicidal Spray products as Disinfectants Test

2.2 Applicable Test Matrix/Matrices

Additional bacteria disinfectant/hard, non-porous surfaces; Bacteria claimed on the label in addition to the base broad-spectrum claim

2.3 Identification of Test Organisms and Testing Parameters

Efficacy Claim Test Organism No. of

Batches/Carriers Evaluation of

Success

Additional bacteria disinfectant/hard non-

porous surfaces

Methicillin-Resistant Staphylococcus aureus (MRSA) (ATCC 33592)

Two batches at LCL, 10 carriers/batch

10/10 carriers are negative for growth in

≤ ten minutes

Table 1. Summary of testing for efficacy claims per EPA OCSPP 810.2200 for Germicidal Spray Product Test.











3.0 SAFETY 3.1 Safety is performed in accordance with requirements of the Microbiology

Hygiene Plan (B 244), 01-ECP-001, Biohazard Exposure Control Plan, and B244/Lab 1.115C Addendum to CHP-008, Chemical Hygiene Plan for Chemistry and Chemical Engineering Division.

3.2 The following PPE (Personal Protection Equipment) is required when performing

the Germicidal Spray Products test:

3.2.1 Laboratory coat

3.2.2 Nitrile gloves

3.2.3 Safety glasses 3.3 All culture inoculations/manipulations (stock tubes, test culture, carriers, sub-

culture tubes) shall be performed under the safety of the biological safety cabinet (BSC).

4.0 RESPOSIBILITIES

4.1 It is the responsibility of the Study Director (project manager) to assure

that all steps described in this procedure are performed.

4.2 It is the responsibility of the personnel performing the analysis to follow the procedure thus ensuring accurate results.

5.0 ABBREVIATIONS

5.1 AOAC- Association of Official Analytical Chemists

5.2 ATCC – American Type Culture Collection 5.3 BSC- Biological Safety Cabinet

5.4 CLSI- Clinical Laboratory Standards Institute


http://nt01a.chem.swri.edu/sops/CHP/008/CHP-008/CHP-008.htm�










5.5 CFU- Colony Forming Unit

5.6 DI- Deionized (water)

5.7 EPA- Environmental Protection Agency

5.8 MHA- Mueller-Hinton agar 5.9 MRSA- Methicillin-Resistant Staphylococcus aureus 5.10 PBDW – Phosphate Buffered Dilution Water

5.11 pH- potential of hydrogen, a measure of the acidity or basicity of a solution

in terms of activity of hydrogen ions [H+] 5.12 LCL- lower certified limit

5.13 LD – log10 density

5.14 TSA or TSB- Tryptic Soy Agar or Broth

6.0 REFERENCES

6.1 http://www.epa.gov/pesticides/methods/atmpmethods/MB-06-05.pdf

6.2 U.S. Environmental Protection Agency, Office of Chemical Safety and Pollution

Prevention, Product Performance Test Guidelines, OCSPP 810.2000: General Considerations for Public Health Uses of Antimicrobial Agents, March 12, 2012.

6.3 EPA Product Performance Test Guidelines; OCSPP 810.2200: Disinfectants for

Use on Hard Surfaces-Efficacy Data Recommendations

6.4 http://www.epa.gov/oppad001/dis_tss_docs/dis-01.htm

6.5 http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series810.htm

6.6 AOAC Official Method 961.02 Germicidal Spray Products as Disinfectants (2012)


http://www.epa.gov/pesticides/methods/atmpmethods/MB-06-05.pdf�

http://www.epa.gov/oppad001/dis_tss_docs/dis-01.htm�

http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series810.htm�










6.7 US Environmental Protection Agency, Office of Pesticide Programs, Lower Certified Limit Testing Guidance, http://www.epa.gov/oppad001/lower-cert-limit-test-guidance.pdf

6.8 CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests;

Approved Standard-Eleventh Edition, M02-A11, January 2012. 6.9 CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests;

Twenty-Fourth Informational Supplement, M100-S24, January 2014. 6.10 http://www.cdc.gov/MRSA/lab/index.html

6.11 http://www.atcc.org/~/media/PDFs/mrsa09.ashx

7.0 PROCEDURE

Test Organism ATCC # Growth Medium

Sub-Culture Medium

Incubation Parameters

MRSA 33592 Nutrient media Modified

Letheen Broth 36°C, aerobic

Table 2: Summary of efficacy test organism and associated growth conditions.

7.1 Reagent Preparation 7.1.1 Nutrient Broth

7.1.1.1 Weigh out 8 grams of Nutrient media (TAP-01-0207-029) and

suspend in 1L of reagent grade water. For smaller volumes of broth, calculate required grams of media based on the above ratio per liter.

7.1.1.2 Mix thoroughly.

7.1.1.3 Heat with frequent agitation and boil for 1 minute to completely

dissolve the powder.


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7.1.1.4 Check the final pH and adjust, if needed, to 6.8 ± 0.2 (TAP-01-0207-028).

7.1.1.5 Autoclave at 121°C for 1 hour (TAP-01-0207-030).

7.1.1.6 After the sterilization cycle, let the media cool under the BSC until

it can be capped tightly and store at room temperature for 1 month.

7.1.1.7 For test culture preparations, dispense 10 mL of media into appropriate number of 25 x 150 mm Kim-Kap tubes.

7.1.2 Trypticase Soy Broth

7.1.2.1 Weigh out 30 grams of Trypticase Soy media and suspend in 1L of

reagent grade water. For smaller volumes of broth, calculate required grams of media based on the above ratio per liter.


7.1.2.3 Warm gently until solution is complete.

7.1.2.4 Check the final pH and adjust, if needed, to 7.3 ± 0.2.

7.1.2.5 Autoclave at 121°C for 1 hour.

7.1.2.6 After the sterilization cycle, let the media cool under the BSC until

it can be capped tightly and store at room temperature for 1 month.

7.1.1 Subculture/Neutralization Media: Modified Letheen Broth

7.1.1.1 Weigh out 25.7 grams of the Letheen Broth media and suspend in 1L of reagent grade water. For smaller volumes of broth, calculate required grams of media based on the above ratio per liter. For NaClO-based disinfectants, Sodium Thiosulfate is added to the Letheen broth at 0.1% (w/v) (e.g., 1 g or equivalent volume of Na2S2O3 in 1L of the above media).















7.1.1.3 Heat with frequent agitation and boil for 1 minute to completely

dissolve the powder.

7.1.1.4 Check the final pH and adjust, if needed, to 7.0 ± 0.2.

7.1.1.5 Autoclave at 121°C for 1 hour.

7.1.1.6 After the sterilization cycle, let the media cool under the BSC until it can be capped tightly and store at 4°C for 1 month.

7.1.1.7 For testing preparation, dispense 20 mL of the subculture media

into the appropriate number of 38 x 200 mm Kim-Kap tubes.

Test Organism TEST Media Sterility

Carrier Sterility

Carrier Viability

Carrier Counts

Test Substance (Negative Control)

Total

MRSA 10 1 1 2 3 1 18

Table 3: Number of sub-culture tubes required per organism test.

7.2 Carrier Preparation 7.2.1 Visually screen all carriers and discard those that are visibly damaged

(scratched, chipped, or nicked).

7.2.2 Clean the carrier of oil, dirt, and all debris by rinsing with 95% ethanol followed by a rinse with DI-water.

7.2.3 Allow all carriers to dry before sterilization.

7.2.4 Place carriers, flat (not stacked or overlapping), into a sterilization pouch

or load carriers into an autoclavable glass cover slip rack which is wrapped in aluminum foil.

7.2.5 Steam sterilize the carrier pouches in the autoclave for a minimum of 20

minutes at 121°C with a 30 minute drying cycle.











7.2.6 Place individual carriers into a sterile Petri dish matted with 2 pieces of sterile 9.0 cm filter paper (Whatman No.2) and close with the lid.

7.3 Culture Initiation and Maintenance

7.3.1 Culture Initiation and Preservation

7.3.1.1 Open ampoule of freeze-dried organism as indicated by ATCC.

7.3.1.2 Using a tube containing 5 to 6 mL of nutrient broth, aseptically

withdraw approximately 0.5 to 1.0 mL with a transfer pipet and rehydrate pellet.

7.3.1.3 Aseptically transfer the entire rehydrated pellet back into the broth

tube. Mix well.

7.3.1.4 Streak a loopful for isolation on nutrient agar for QC of culture purity. Include a QC media only plate.

7.3.1.5 Incubate tube and plates at 36 ± 2°C for 24 hours.

7.3.1.6 After incubation, vortex tube and streak a loopful for isolation on

nutrient agar to verify the culture purity. Include a QC media only plate. Incubate plates at 36 ± 2°C for 24 hours.

7.3.1.7 Aseptically pipet 0.5 mL (TAP-01-0207-033) of culture per 1 mL

sterile cryovial, containing 0.5 mL of glycerol. Vortex cryovial(s) well.

7.3.1.8 Store cryovials at ≤ ̶ 60°C.

7.4 Test Culture Preparation

7.4.1 Defrost a single cryovial to room temperature and briefly vortex to mix.

Each cryovial should be single use only.












7.4.2 Add a 10 µL loopful of the thawed frozen stock to a tube containing 10 mL nutrient broth and vortex to mix. Include a QC purity plate struck for isolation on nutrient agar or TSA along with a QC media only plate.

7.4.3 Incubate at 36 ± 2°C for 24 ± 2 hours.

7.4.4 Briefly vortex the 24 hour culture prior to transfer. For the final subculture

step, inoculate a sufficient number of 25 x 150 mm tubes (e.g. six to eight) containing 10 mL nutrient broth with 10 µL per tube of the 24 hour nutrient broth culture; incubate at 36 ± 2°C for 48 - 54 hours. Include a QC plate to check for culture purity and a media only plate as above. NOTE: For each bacterium, one daily transfer is required prior to the inoculation of a final test culture. Daily cultures may be subcultured up to five days.

7.4.5 After at least 24 hours of incubation, subculture the purity plate from 7.4.4, using isolated colonies, to one 10 mL tube of TSB and 2 - 3 nutrient agar or TSA plates. Incubate at 36 ± 2°C for 18 - 24 hours. These will be used for the antibiotic resistance confirmation and culture identification testing in section 7.7.7.

7.4.6 After test culture tubes from 7.4.4 have incubated 48 - 54 h, vortex each

culture tube 3 - 4 seconds and let stand for 10 minutes at room temperature.

7.4.7 Remove the upper portion of each culture tube, leaving behind any debris or clumps and transfer into a sterile test tube. Swirl pooled culture to mix.

7.4.8 Streak a loopful of the pooled culture for isolation on nutrient agar or TSA

for purity QC along with a QC media only plate. Incubate plates at 36 ± 2°C for 24 ± 2 hours.

7.5 Carrier Inoculation

7.5.1 Using a calibrated 4.0 mm/10 µL loop, transfer a loopful of the test culture

onto approximately 1 square inch of the sterile test carrier in the Petri dish. NOTE: Vortex to mix the inoculum periodically during inoculation of the carriers.











7.5.2 Immediately spread the inoculum uniformly over the majority of the carrier surface using a sterile loop. Do not touch the edges of the carrier.

7.5.3 Cover the Petri dish immediately and repeat the operation for all test carriers, viability controls, and quantification of microbe on carriers.

7.5.4 After all the carriers have been inoculated, place them in the incubator at

36 ± 2°C and let dry for 30 - 40 minutes.

7.6 Test Procedure 7.6.1 After the required drying time, the slides are sequentially sprayed with the

test disinfectant in a horizontal position for a specified time, distance, and number of pumps. 7.6.1.1 Distance = 6 - 8"

7.6.1.2 Number of pumps = 4

7.6.1.3 Contact Time = 10 minutes

7.6.1.4 Exposure Temperature = 18 - 24°C (room temperature)

7.6.2 Maintain the carriers in a horizontal position for the specified contact time.

7.6.3 After the exposure time is complete, transfer the carriers sequentially into

the subculture tubes containing the appropriate neutralizer.

7.6.4 Use sterile forceps to pick up the carriers, allow the excess disinfectant to drain and transfer to the subculture tube. Flame forceps between each carrier. NOTE: The carrier can touch the interior sides of the subculture tube during transfer, but avoid this contact as much as possible.

7.6.5 After the carrier is deposited in the subculture tube, recap the subculture tube and shake culture for a few seconds to mix.

7.6.6 Incubate the tubes at 36 ± 2°C for 48 ± 2hours.











NOTE: If a secondary subculture tube is deemed necessary to achieve neutralization and support growth, then transfer the carrier from the primary subculture tube to a secondary subculture tube within 25 - 60 minutes of the initial transfer and in sequential order. Thoroughly shake the subculture tubes after all carriers have been transferred. Incubate both the primary and secondary subculture tubes and record the results from both tubes.

7.6.7 After the period of incubation is complete, report the results as + (growth) or – (no growth) as determined by presence or absence of turbidity. For tubes with growth, representative positive tubes should be checked by streak plate and Gram stain to check for characteristic morphology of the test organism.

NOTE: Once the results are recorded, it is important that the carriers be reprocessed, including visual screening, washing and sterilization, before use in another study.

7.7 Study Controls

7.7.1 Culture Purity Control

7.7.1.1 On the day of testing, the bacterial culture will be struck for

isolation on nutrient agar or TSA to verify purity. A QC media only plate with be incubated along with purity plate.

7.7.1.2 Isolation plates will be incubated at 36 ± 2°C for 24 - 48 hours and examined for characteristic, individual colony morphology per the organism being tested.

7.7.2 Test Substance Control (Negative Control)

7.7.2.1 To verify compatibility and any visual abnormalities that may

occur with exposure of the test substance to the neutralization media, a carrier exposed to the test substance only, for the specified 10 minute contact time will be placed within a subculture tube of neutralization media.

7.7.2.2 The tube will be incubated with test cultures and monitored for development of color change, precipitation and/or any other visual effect that would interfere with the reading of the final + or – turbidity results.











7.7.3 Sterility Controls 7.7.3.1 On the day of testing, a blank subculture tube with 20 mL

neutralization media should be incubated along with the test cultures to confirm sterility of media.

7.7.3.2 On the day of testing, place a sterile, uninoculated carrier into a tube of the neutralizing media and incubate with the test cultures to confirm sterility of the carriers prior to inoculation.

7.7.4 Viability Controls (Positive Control)

7.7.4.1 On the day of testing, place two dried inoculated carriers into

separate tubes of neutralizing media and incubate with the test cultures to confirm viability of the bacterial organism after drying/attachment period to the carrier surface.

7.7.5 Verification of Positive Carriers 7.7.5.1 Following incubation, a random selection of positive tubes (if

applies per test results) will be examined for the test organism by inoculating onto nutrient agar or TSA for isolation and confirmation of the organism. The inoculated plates are incubated as in the test.

7.7.5.2 Examine the plates for colonial morphology characteristic of the test organism and check by Gram stain.

7.7.6 Enumeration of viable bacteria from carriers

7.7.6.1 After the carriers have dried from the inoculation step above, select

three for confirming the biological load per carrier by placing each individually into a sterile 50 mL tube containing 20 mL of the neutralizing media.

7.7.6.2 Vortex immediately for 120 ± 5 seconds.











7.7.6.3 After vortexing, make serial 10-fold dilutions in 9 mL of phosphate-buffered dilution water.

NOTE: If the serial dilutions are not made and plated immediately, keep the vortexed tubes at 2 - 5°C until this step can be done; however, dilution and plating should be performed within 2 hours of vortexing.

7.7.6.4 Briefly vortex each serial dilution tube prior to plating.

7.7.6.5 Plate 0.1 mL aliquots of appropriate dilutions in duplicate on TSA; dilutions of 10-1 through 10-5 should result in plates with a countable range of colonies.

7.7.6.6 Incubate the plates inverted concurrently with the efficacy

subculture tubes at 36 ± 2°C for up to 48 ± 2 hours.

7.7.6.7 After incubation, count the colonies by hand. Use dilutions yielding counts up to 300 colonies for enumeration. Ensure that the mean LD is acceptable via the criteria stated in Table 4.


MRSA ≥4.0 ≥ 1 x 104 Table 4: Acceptable values of mean density, required per organism, when performing the Germicidal Spray Product

Test.

7.7.7 Antibiotic Resistance Confirmation and Culture Identification On the day of testing, the antibiotic resistance (CLSI disk diffusion test) and identity (e.g. biochemical test, Gram stain, morphology) of the test organism utilized should be confirmed per EPA OCSPP 810.2000 (e) (3). Expected test results are stated in Table 5. NOTE: The confirmation may also be conducted within the usual transfer cycle or other appropriate transfer depending upon organism’s growth requirements











Test Staphylococcus aureus

MRSA ATCC 33592a

Staphylococcus aureus ATCC 25923b

Staphylococcus epidermidis

ATCC 12228b

Gram stain +, non-spore coccus, singly, pairs, in short

chains or irregular clusters N/A N/A

Colony morphology: Nutrient agar/TSA

Entire, glistening, circular, low convex, yellow-white,

opaque, and smooth

Entire, glistening, circular, low convex, yellow-white,

opaque, and smooth

Entire, glistening, circular, convex, opaque, and

smooth Coagulase + + ̶

CHROMagar MRSA II Mauve colony color No growth or non-mauve colonies N/A

Disk diffusion zone diameter:

30 µg cefoxitinc

Resistant ≤ 21 mm

QC range 23-29 mm N/A

Table 5: Expected results of test and quality control strains for identification and antibiotic resistance tests. a Test organism, b Quality control strain, c Cefoxitin is tested as a surrogate for oxacillin. Oxacillin is not reliable for testing and

is the replacement antimicrobial agent for methicillin, which is no longer commercially available in the United States.

7.7.7.1 Quality Control Strains Staphylococcus epidermidis (ATCC 12228) and Staphylococcus aureus (ATCC 25923)

7.7.7.1.1 Defrost a single cryovial to room temperature and

briefly vortex to mix. Each cryovial should be single use only.

7.7.7.1.2 Add a 10 µL loopful of the thawed frozen stock to a

tube containing 10 mL TSB and vortex to mix. Streak for isolation on TSA and include a QC media only plate.

7.7.7.1.3 Incubate at 36 ± 2°C for 24 ± 2 hours.

7.7.7.1.4 Briefly vortex 24 hour culture and add a 10 µL loopful to a tube containing 10 mL TSB and vortex to mix. Select isolated colonies from the 24 hour TSA plate and streak for isolation on 2 - 3 TSA plates and include a QC media only plate.











7.7.7.1.5 Incubate at 36 ± 2°C for 18 - 24 hours. These will be the quality control cultures utilized in the following tests.

7.7.7.2 Culture Identification:

7.7.7.2.1 Colony morphology: Observe and record colony

morphology of the test organism from the test purity control plate.

7.7.7.2.2 Gram stain: Perform a Gram stain per kit instructions

on an isolated colony of the test organism from the test purity control plate. View slide under 100 x oil immersion lens and record observations.

7.7.7.2.3 Tube Coagulase Method:

7.7.7.2.3.1 Remove three Coagulase Cryo tubes from the freezer and thaw to room temperature.

7.7.7.2.3.2 Add approximately 50 µL of an 18 - 24 hour TSB culture per vial. Inoculate one Coagulase Cryo tube per organism listed in Table 5.

7.7.7.2.3.3 Incubate tubes at 36 ± 2°C for 1 - 4 hours.

7.7.7.2.3.4 Tubes should be observed hourly during the first four hours. Avoid shaking the tubes while reading the test. Any degree of clotting is a positive test.

7.7.7.2.3.5 Negative tests at 4 hours should be held at room

temperature for 24 hours before reporting results. NOTE: A flocculent or string like precipitation should not be considered a true clot, and should be reported as negative.











7.7.7.2.4 CHROMagar MRSA II:

7.7.7.2.4.1 Warm CHROMagar plates to room temperature in the dark before inoculation.

7.7.7.2.4.2 Generate serial ten-fold dilutions of the 18 - 24 hour TSB cultures from the first two organisms listed in Table 5, by pipetting 1 mL of the inoculum into 9 mL of PBDW.

7.7.7.2.4.3 Using the undiluted, 10-1, and 10-2 dilutions,

streak each tube for isolation on a CHROMagar plate. Include a QC media only plate.

7.7.7.2.4.4 Incubate culture plates for 20 - 26 hours and QC

media only plate for 72 hours at 36 ± 2°C.

NOTE: Avoid exposure to light during incubation.

7.7.7.2.4.5 Read plates against a white background. Colonies of MRSA will appear mauve.

7.7.7.3 Antibiotic Resistance Confirmation: CLSI Disk Diffusion Test 7.7.7.3.1 Prepare separate, direct colony suspension in 0.85%

saline of isolated colonies of the first two organisms listed in Table 5 from 18 - 24 hour TSA plates.

7.7.7.3.2 Adjust the suspensions to achieve a turbidity equivalent to a 0.5 McFarland standard. Invert the turbidity standard gently to fully suspend. Visually compare the turbidity using adequate light and read the tubes against a white card with contrasting black lines.

NOTE: Bacterial suspension tubes should be of similar diameter as the turbidity standard.

7.7.7.3.3 Dip a sterile cotton swab into the adjusted inoculum. Rotate the swab several times and press firmly on the











inside wall of the tube above the fluid level to remove excess fluid from the swab. NOTE: Optimally, perform this step within 15 minutes after adjusting the turbidity of the inoculum suspension.

7.7.7.3.4 Inoculate the surface of three Mueller-Hinton agar

(MHA) plates for each adjusted suspension by streaking the swab over the entire agar surface. Repeat this procedure two more times, rotating the plate approximately 60° each time. Finally, swab the rim of the agar.

7.7.7.3.5 Leave the lid ajar for 3 - 5 minutes but, no more than 15

minutes.

7.7.7.3.6 Dispense one 30 µg cefoxitin disk/plate onto the center surface of the inoculated agar plate. Press each disk down firmly.

NOTE: A disk should not be relocated once it has come in contact with the agar surface.

7.7.7.3.7 Within 15 minutes after disks are applied, invert the

plates and incubate at 35 ± 2°C for 24 hours. NOTE: 24 hours incubation, instead of the normal 16 - 18 hours, is specific for testing cefoxitin against Staphylococcus spp.

7.7.7.3.8 Measure the diameters of the zones of complete inhibition, including the diameter of the disk. Measure zones to nearest whole millimeter using a ruler, which is held on the back of the inverted plate. Hold plate a few inches above a black, nonreflecting background illuminated with reflected light. NOTE: Zone margin should be considered the area showing no obvious growth visible with the unaided eye. NOTE: If discrete colonies grow within the clear zone of inhibition, the test should be repeated. If discrete colonies











continue to grow within the zone of inhibition, measure the colony-free inner zone.

7.7.7.3.9 Report the zones of inhibition for 30 µg cefoxitin disks

(surrogate test of oxacillin) as susceptible or resistant based on CLSI M100 Table 2C for Staphylococcus aureus for antimicrobial agent oxacillin (Table 6).

Antimicrobial Agent Oxacillin

Zone Diameter (nearest whole mm)

Susceptible Resistant Staphylococcus aureus ≥ 22 ≤ 21

Table 6: Zone Diameter Standards for Staphylococcus aureus extracted from CLSI M100-S24 Table 2C.

NOTE: Cefoxitin is tested as a surrogate for oxacillin. Oxacillin is not reliable for testing and is the replacement antimicrobial agent for methicillin, which is no longer commercially available in the United States.

7.7.8 Neutralization Confirmation 7.7.8.1 Perform one neutralization confirmation test per organism,

inoculating with 10 - 100 CFU/tube.

7.7.8.2 Grow a bacterial test culture as in Section 7.4.

7.7.8.3 Generate serial ten-fold dilutions of the final pooled culture by pipetting 1 mL of the inoculum into 9 mL of PBDW.

7.7.8.4 Use four dilutions, (1.0 x 10-5, 1.0 x 10-6, 1.0 x 10-7, and 1.0 x 10-8)

to inoculate the neutralizer/subculture media tubes below.

7.7.8.5 To estimate the CFU/mL, plate 0.1 mL of each of the four dilutions in duplicate on TSA. Briefly vortex each tube prior to plating.

7.7.8.6 Incubate the plates at 36 ± 2°C for 24 - 48 hours and count and

record the colonies (CFU) per dilution.











7.7.8.7 Expose four sterile carriers to the test substance/disinfectant for the specified contact time in the same manner as the product efficacy testing.

7.7.8.8 Transfer the carriers individually into subculture tubes of the

neutralizing media. Drain excess liquid from the carrier prior to transfer. This set of tubes represents the Neutralizer Primary Subculture Treatment.

7.7.8.9 Then transfer each carrier to subculture tubes containing the secondary media. This set of tubes represents the Secondary Subculture Treatment.

7.7.8.10 Each of the Primary and Secondary subculture tubes is

inoculated with 0.1 mL of the corresponding four inoculum dilutions.

7.7.8.11 Incubate the tubes for 48 ± 2 hours at 36 ± 2°C.

8.0 RECORDS

8.1 All documentation pertaining to this procedure and associated GLP-protocol will be documented in the assigned GLP-project notebook.

8.2 Applicable records generated through this procedure shall be maintained in

accordance with Division 01 SOP-01-4.2.4, Storage and Maintenance of Quality Records

Revision Number

Effective Date

Description of Changes


http://nt01a.chem.swri.edu/sops/SOPs/SOP4.2.4.pdf�

Southwest Research Institute Project 01.20792 Chemistry and Chemical Engineering Division GLP-SP-240 GLP Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 1 of 11

GLP-SP-240

STUDY PROTOCOL

GLP GERMICIDAL SPRAY TEST of ZEROMOLD PLUS for ADDITIONAL BACTERIA

SOUTHWEST RESEARCH INSTITUTE STUDY NUMBER

01.20792.01.001

DATA REQUIREMENTS AOAC Germicidal Spray Method

TEST ORGANISM

Methicillin-Resistant Staphylococcus aureus (MRSA)

(ATCC 33592)

TESTING FACILITY

Southwest Research Institute (SwRI) 6220 Culebra Road

San Antonio, TX 78238-5166 USA

Tel: 210-522-6354 Fax: 210-522-4565

SPONSOR

BIOSENTA Inc. 1120 Finch Ave, West Suite 503

Toronto, Ontario, M3J 3H7 CANADA

PH: 416-410-2019


1. OBJECTIVE The purpose of this study is to determine the effectiveness of the Sponsor’s product: ZeroMold Plus disinfectant, for a hard, nonporous surface following the Association of Analytical Communities (AOAC) Germicidal Spray Method in accordance with the Environmental Protection Agency (EPA) 40 CFR 160 Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Good Laboratory Practice (GLP) Standards. This method is in compliance with the requirements of EPA and Health Canada Therapeutic Products Directorate (TPD). AOAC Official Method 961.02 (2012) is applicable for testing spray and pressurized spray disinfectants to determine effectiveness as disinfectant for contaminated hard, nonporous, inanimate environmental surfaces. 2. TEST SUBSTANCE CHARACTERIZATION Two lots of ZeroMold Plus at Lower Certified Limit (LCL) will be tested. BIOSENTA previously provided the ZeroMold Plus to SwRI. SwRI performed the titration and dilution of the products to LCL according to GLP protocol (GLP-SP-210) to ensure the LCL compliance. Product information is as follows: Name: ZeroMold Plus Active ingredient: NaClO Lower certified limit (LCL): 0.43% Acceptable active ingredient concentration for efficacy testing: 0.43% - 0.44% (2% above LCL) Storage Conditions: Ambient Temperature, cap closed (all lots tested)

Efficacy Claim Test Organism No. of

Batches/Carriers Evaluation of Success

Additional bacteria disinfectant/hard non-

porous surfaces MRSA (ATCC 33592)

Two batches at LCL, 10 carriers/batch

10/10 carriers are negative for growth in

≤ ten minutes 3. TEST PRINCIPLE A film of bacterial cells dried on a surface of glass slide carriers is exposed to the test substance for a specified exposure time. After exposure, the carriers are transferred to vessels containing neutralizing subculture media and assayed for survivors. Appropriate viability, culture purity, sterility, carrier bio-load, as well as, neutralization and antibiotic resistance confirmation controls are performed.


4. ABBREVIATIONS AOAC- Association of Analytical Communities ATCC- American Type Culture Collection CLSI- Clinical Laboratory Standards Institute CFU- Colony Forming Unit MRSA- Methicillin-Resistant Staphylococcus aureus LD- log10 density LCL- lower certified limit 5. TEST SYSTEM/ANALYTICAL METHOD The detailed test procedure for each test organism is described in SwRI TAP 01-0207-037, Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure. This protocol only describes the general test procedure.

5.1 Efficacy Tested Organisms

Test Organism ATCC # Growth Medium Sub-Culture

Medium Incubation Parameters

MRSA 33592 Nutrient media Modified Letheen

Broth 36°C, aerobic

Table 2. Summary of organisms to be tested and growth conditions. 5.2 Carriers Glass slides, of dimensions 25 x 25 mm (or comparable size) will be utilized as the carrier (surface for biological attachment) for this assay. The carriers will be inspected, cleaned, and sterilized by means of steam sterilization at 121°C for a minimum of 20 minutes. Individual sterile plastic Petri dishes will be matted with 2 pieces of sterile filter paper. One sterile glass carrier will be placed into each sterile dish to serve as the testing platform/surface. 5.3 Preparation of Test Organism(s) A test culture will be generated by adding a 10 µL volume from a thawed cryogenically stored stock culture to a fresh media tube and incubated at 36 ± 2°C for 24 ± 2 hours. For the final sub-culture step, a sufficient number of tubes will be inoculated and incubated for 48-54 hours at 36 ± 2°C. After incubation, all tubes will be mixed well and allowed to settle out. The upper portion of each culture tube will be removed, eliminating debris, and pooled together to generate the official bacterial inoculum.


5.4 Inoculation of Carriers Each glass carrier (contained within a Petri dish) will be inoculated with 10 µL of the prepared bacterial suspension using a 4.0 mm loop or calibrated pipette and uniformly spread over the test surface. Each dish will then be covered and all at once placed at 36 ± 2°C and allowed to dry for 30 to 40 minutes. 5.5 Preparation of Test Substance The test substance(s) to be assayed will be used as directed by the Sponsor. ZeroMold Plus is a ready-to-use product. Products at the lower certified limit will be used for the testing. 5.6 Exposure/Test Substance Application Conditions Dried bacterial films, attached to the glass carriers will be exposed at room temperature, in a horizontal position via use of a spray dispenser (bottle). The spray time (interval), spray distance, number of pumps, and contact time (undisturbed) will be specified by the Sponsor. 5.7 Test System Recovery Following the spray treatment, each treated carrier will be held at room temperature for the specified contact exposure time. After the exposure time is complete, the carriers are drained of excess liquid. Sequentially, each carrier is aseptically placed into a tube containing 20 mL of neutralizing medium. If necessary, carriers are transferred into individual secondary sub-culture tubes within 30 minutes of the end of the initial transfer. 5.8 Incubation and Observation All neutralized subculture tubes are incubated for 48 ± 2 hours at 36 ± 2°C. After incubation, the subculture tubes are visually examined for growth by presence or absence of turbidity. For tubes with growth, representative positive tubes should be plated and Gram stained to check for characteristic morphology of the test organism. 5.9 Results Results will be reported as number of tubes positive per total number of tubes tested. Enumeration of bacteria per carrier will be calculated as log10 density (LD) per carrier.


5.10 Study Controls 5.10.1 Purity Control A ‘streak plate for isolation’ will be performed on each 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 of the test organism. 5.10.2 Carrier Sterility Control On the day of testing, a sterile, uninoculated carrier will be placed into a tube of neutralizing subculture media. This subculture tube will be incubated as said ‘test’ carrier tubes and examined for growth. The acceptance criterion for this study control is lack of growth to confirm the sterility of the carriers prior to test inoculation. 5.10.3 Viability Control On the day of testing, two dried inoculated carriers will be placed into individual tubes of neutralizing subculture media. These subculture tubes will be incubated as said ‘test’ carrier tubes and examined for growth. The acceptance criterion for this study control is growth which confirms exposure to the test organism. 5.10.4 Neutralizing Subculture Medium Sterility Control A representative sample of uninoculated neutralizing subculture medium will be incubated with said ‘test’ carrier tubes and other controls and examined. The acceptance criterion for this study control is lack of growth which confirms sterility of prepared neutralizing subculture media. 5.10.5 Test Substance Control (Negative Control) To verify compatibility and any visual abnormalities that may occur with exposure of the test substance to the neutralization media, a carrier exposed to the test substance only, for the specified 10 minute contact time will be placed within a subculture tube of neutralization media. The tube will be incubated with test cultures and monitored for development of color change, precipitation and/or any other visual effect that would interfere with the reading of the final + or – turbidity results.


5.10.6 Neutralization Confirmation

Effective neutralization of the test substance will be confirmed by exposing sterile carriers to the test substance and transferring them to subculture tubes containing the neutralizing media. The subculture tubes are inoculated with 10-100 CFU of the test organism, incubated under test conditions and visually examined for the presence of growth. This control will be performed with multiple replicates using different dilutions of the test organism and performed for each test organism the test substance is tested against. A standardized spread plate will be run concurrently in order to enumerate the number of CFU actually added. The acceptance criterion for this study control is growth in the subculture tube, minimally, following inoculation with ≤ 100 CFU. 5.10.7 Antibiotic Resistance Confirmation Confirmation of the antibiotic resistance of the test organism will be conducted to include numerical values for all antibiotics tested and the method used to obtain the results. The acceptance criterion for this study control is organism resistance to the antibiotic(s) being tested per Clinical and Laboratory Standard Institute (CLSI) antimicrobial susceptibility breakpoints. Additionally, the test organism will be characterized according to the following: the source and identity, the transfer history, the method used to confirm the identity, and the method of preservation/storage. The acceptance criterion for this study control is a pure culture demonstrating the colony morphology, Gram stain, and biochemical/identification test(s) results typical of the test organism. 5.10.8 Enumeration of Viable Bacteria on Carrier

Three inoculated carriers will be individually placed into a tube containing the subculture media. Each carrier tube will be vortexed to remove the bacteria from the carrier surface and then a series of 10-fold dilutions will be generated using phosphate-buffered dilution water. A sub-set of the dilution series will be plated, incubated, and the resulting colonies enumerated to determine the CFU per carrier. The log10 density (LD) for each carrier will be determined and used to calculate the average LD for the test. The mean LD acceptance criteria for this study control are organism dependent:


MRSA ≥4.0 ≥1.0 x 104

Table 3. Acceptable values of mean density, required per organism, when performing the Germicidal Spray Product test.


6. STUDY ACCEPTANCE CRITERIA 6.1 Test Substance Performance Criteria The U.S. EPA efficacy performance requirements for ‘Additional Bacteria’ claimed on the label in addition to the base broad-spectrum claim, requires 10 out of 10 carriers are negative for growth of the test organism for each lot of product. 6.2 Control Acceptance Criteria The study controls must perform according to the criteria detailed in the study controls description section. If any of the control acceptance criteria are not met, the test may be repeated under the current protocol number. 7. JUSTIFICATION OF TEST SYSTEM Regulatory agencies require that a specific bacterial claim for a test substance intended for use on hard surfaces be supported by appropriate scientific data demonstrating the efficacy of the test substance against the claimed bacteria. This is accomplished by treating the target organism with the test substance under conditions which simulate as closely as possible, in the laboratory, the actual conditions under which the test substance is designed to be used. For disinfectant spray products intended for use on hard surfaces (dry, inanimate environmental surfaces), a germicidal spray method is used in the generation of the supporting data. The experimental design in this protocol meets these requirements. 8. STUDY PERSONNEL

Xingguo Cheng, Sr. Research Scientist, Study Director, Materials and Bioengineering Section (Div. 01)

Amy De Los Santos, Research Scientist, Co-PI, Applied Power Division

Spring Cabiness, Research Scientist, Co-PI, Applied Physics Division

9. MAJOR EQUIPMENT

Imperial III Lab-Line Incubators, SN 04033589 /09045401, Laboratory 1.115/C, Bldg. 244

Tuttnauer 3870M Autoclave, SN 2304553, Laboratory 1.115C, Bldg. 244

Analytical Balances, Laboratory 1.115, Bldg. 244


Omegaette ® HH306 Data Logger Thermometer, SN 050501588, Laboratory 1.115 & 1.115C, Bldg. 244

Thermocouple, SN 18096, Laboratory 1.115 & 1.115C, Bldg. 244

Oakton pH/CON 510 Series meter, SN 181287, Laboratory 1.115, Bldg. 244

LabConco Purifier Class II Biological Safety Cabinet, SN 050942438AK, Laboratory 1.115C, Bldg. 244

Fisher Scientific Vortex Mixer, SN 120919031, Laboratory 1.115C, Bldg. 244

10. ASSOCIATED TECHNICAL ANALYTICAL PROCEDURES (TAP’s)

01-0207-030 Autoclave Operation and Validation for use in the Microbiology Laboratory

01-0207-028 Use of the Oakton® ph/CON Meter in the Microbiology Laboratory

01-0207-033 Verification and Use of Automatic Pipettes in the Microbiology Lab

01-0207-29 Calibration and Verification of Analytical Balances

01-0207-031Calibration of Thermocouples and Relative Humidity Sensors

01-0207-037 Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure

11. CALCULATIONS Carrier Population Calculation:

CFU/Carrier = rvolumeneutralizectordilutionfaxedvolumeplatdilutionplateofcoloniesaverage

×)()(

)@/#(

Average Log10 Carrier Population Control = N

XNLogXLogXLog 10210110 ++

X= CFU/carrier N= number of control carriers 12. QUALITY ASSURANCE/GOOD LABORATORY PRACTICES


SwRI maintains a Quality Assurance Unit/Division Quality Assurance (QAU/DQA) in compliance with EPA's current FIFRA Good Laboratory Practices Standard (40 CFR 160). The QAU/DQA will conduct periodic inspections of this study to assure conformance to the study protocol, standard operating procedures and GLP. The inspection results will be reported to SwRI management and the SwRI study director through the Quality Reporting System. The final report will be reviewed by the QAU/DQA, and a signed Quality Assurance Statement will be included which specifies the date inspections/audits were made and reported to SwRI management and the SwRI study director.

13. RECORDS TO BE MAINTAINED In accordance with SwRI TAP 01-0103-020, Conduct of a GLP Study, copies of the protocol, appendices, amendments, and the analytical method shall be available to the analysts, technicians and chemists involved in the study at SwRI. The analysts at SwRI shall maintain laboratory notebooks or equivalent documents in which they will record all procedures, weighing, observations, etc., relevant to the experimental work. Chromatograms, computer printouts, etc., will be clearly labeled and notebooks will remain in the analyst's possession throughout the study. Records shall be archived as written in SwRI TAP 01-0103-023, Project Setup and Record Archival for GLP. Training records shall be maintained for appropriate procedures and Protocols. At the completion of the analytical portion of the study, a report shall be prepared as specified in SwRI TAP 01-0103-022, Final Report for GLP Studies, and delivered to the sponsor for final approval. Following completion of the final report for the study, all methodology, raw data sheets, and original chromatograms will be inspected by the QAU/DQA at SwRI. At the sponsor's discretion, all original records will be sent to the sponsor for archiving in accordance with 40 CFR Part 160.

14. SAMPLE AND RECORD RETENTION The study for a research or marketing permit approved by EPA shall be maintained for five years from the period during which the sponsor holds that research or marketing permit to which the study is pertinent unless all originals are provided to the client. For samples remaining at the conclusion of the study SwRI will either return samples to the sponsor or dispose the sample after which the sponsor holds that research or marketing permit to which the study is pertinent unless all originals are provided to the client (EPA). Materials that degrade will not be maintained.


15. REFERENCES http://www.epa.gov/pesticides/methods/atmpmethods/MB-06-05.pdf U.S. Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention, Product Performance Test Guidelines, OCSPP 810.2000: General Considerations for Public Health Uses of Antimicrobial Agents, March 12, 2012. EPA Product Performance Test Guidelines; OCSPP 810.2200: Disinfectants for Use on Hard Surfaces-Efficacy Data Recommendations http://www.epa.gov/oppad001/dis_tss_docs/dis-01.htm http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series810.htm AOAC Official Method 961.02 Germicidal Spray Products as Disinfectants (2012) US Environmental Protection Agency, Office of Pesticide Programs, Lower Certified Limit Testing Guidance, http://www.epa.gov/oppad001/lower-cert-limit-test-guidance.pdf CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Eleventh Edition, M02-A11, January 2012. CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests; Twenty-Fourth Informational Supplement, M100-S24, January 2014. http://www.cdc.gov/MRSA/lab/index.html http://www.atcc.org/~/media/PDFs/mrsa09.ashx

http://www.epa.gov/pesticides/methods/atmpmethods/MB-06-05.pdf�

http://www.epa.gov/oppad001/dis_tss_docs/dis-01.htm�

http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series810.htm�


http://www.cdc.gov/MRSA/lab/index.html�

http://www.atcc.org/~/media/PDFs/mrsa09.ashx�

Documents

Final report GLP-SP-240 - Biosenta Inc.Southwest Research Institute Project 01.20792 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal