FINAL REPORT STUDY TITLE

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1 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 (Notebook# ) TESTING FACILITY Southwest Research Institute (SwRI ) Applied Physics Division Applied Power Division 6220 Culebra Road San Antonio, TX , USA Tel: , Fax: STUDY SPONSOR BIOSENTA Inc Finch Ave West, Suite 503 Toronto, Ontario, M3J 3H7, CANADA, Tel: TEST GUIDELINE OCSPP TEST ORGANISMS: Methicillin-Resistant Staphylococcus aureus (MRSA) (ATCC 33592) TEST PRODUCT IDENTITY ZeroMold Plus TM (48538, BI# , Lot #3, >60 days) ZeroMold Plus TM (48539, BI# , 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

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5 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 5 of 17 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 OBJECTIVE TEST SUBSTANCE, MATERIALS, EQUIPMENT & PERSONNEL... 6 Test Substance... 6 Materials and Equipment... 7 Personnel PROCEDURES PROTOCOL CHANGE AND STUDY DEVIATION Protocol Amendments Study Deviations STUDY DESIGN CONTROLS AND ACCEPTANCE CRITERIA STUDY ACCEPTANCE CRITERIA CALCULATION AND STATISTICAL ANALYSIS RESULTS Methicillin-Resistant Staphylococcus aureus (MRSA) STUDY CONCLUSION RECORD AND SAMPLE RETENTION REFERENCES APPENDICES... 17

6 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 6 of 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 ) and Health Canada Therapeutic Products Directorate (TPD). AOAC Official Method (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 = 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 BI# , Lot #3, >60 days 0.44% BI# , Lot #4, >60 days 0.44% Concentration/Dilution Tested Ready-to-use, Trigger Spray

7 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 7 of 17 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 Becton Dickinson N/A 9/30/ ml Serological Pipet N/A Corning N/A 11/30/ ml Serological Pipet N/A Becton Dickinson N/A 10/31/2018 Cefoxitin N/A Oxoid/Fisher Scientific 30 mcg/disc June-17 CHROMagar MRSA II N/A Becton Dickinson Individual plates 12/25/2014 Coagulase Cryo N/A Hardy Diagnostics N/A 4/9/2015 Cotton applicators N/A Fisher Scientific N/A 9/26/2015 Anhydrous, Ethanol Fisher Scientific Oct-16 Histology Glycerol B Fisher/Acros Gram Stain Kit Hydrochloric acid Multiple, (see CoA) , , , SHBD5057 V Spectrophot ometric October-18 Becton Dickinson N/A 4/30/2015 Sigma-Aldrich HyPure Water N/A AZA HyClone %, Bio Reagent Molecular Biology Grade Oct-15 Jan-16 Inoculation loops N/A , Fisher Scientific N/A N/A Letheen Broth N/A Becton Dickinson N/A 2/29/2016 McFarland Remel/Fisher Turbidity N/A Scientific Standard /18/2015 Mueller Hinton Agar N/A Hardy Diagnostics Individual plates 12/30/2014 Nutrient Agar N/A Becton Dickinson Individual plates 12/17/2014 Nutrient Broth N/A Becton Dickinson N/A 2/29/2016

8 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 8 of 17 ph Buffer Kit Multiple, (see CoA) Phosphate Buffer N/A Saline Sodium Hydroxide Sodium Thiosulfate Staphylococcus aureus Staphylococcus aureus (MRSA) Staphylococcus epidermidis Trypticase Soy Agar Trypticase Soy Broth Fisher Scientific N/A Feb SLBH8376 V BCBM3765 V N/A Remel/Fisher Scientific Remel/Fisher Scientific Sigma-Aldrich Butterfield 8/13/ % 8/18/2015 Reagent grade, 98% pellets Oct-15 Sigma-Aldrich Anhydrous 9/15/2024 Hardy Diagnostics/Micro biologics/atcc #25923 Aug-15 N/A ATCC #33592 N/A N/A Hardy Diagnostics/Micro biologics/atcc N/A Becton Dickinson #12228 Nov-15 Individual plates 12/25/2014 N/A 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 N/A B244, L1.115 Check weights Class N/S /13/2015 B244, L1.115 Data Logging System N/A N/A N/A B244, L1.115 Incubator Lab-Line, Imperial III, N/A B244, L1.115 Incubator Lab-Line, Imperial III, N/A B244, L1.115 ph Meter Oakton ph/con, 510 series N/A B244, L1.115 Pipette Eppendorf Research Plus, P A 2/26/2015 B244, L1.115

9 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 9 of 17 Pipette Pipette Pipette Refrigerator Eppendorf Research Plus, P200 Eppendorf Research Plus, P1000 Socorex, Acura 815, P1000 Sanyo Medicool, MPR- 411F Z 2/26/2015 B244, L Z 2/26/2015 B244, L /26/2015 B244, L N/A B244, L1.115 Refrigerator/Freezer Fisher Scientific, Isotemp, A N/A B244, L1.111 Scale Sartorius, ED2202S /2/2015 B244, L1.115 Scale Mettler Toledo, AB104-S /2/2015 B244, L1.115 Temp./RH Probe Vaisala J /5/2015 B244, L1.115 Thermocouple Omega, type K, -5 to +105 C /29/2015 B244, L1.115 Thermocouple Thermometer Data Logger Timer Ultra Low -80 C Freezer Duro-Sense Corp., type K, -80 to +180 C 336 2/24/2015 B244, L1.115 Omega, HH /29/2015 B244, L1.115 Fisher Scientific/S /24/2015 B244, L1.115 Sanyo, MDF-C8V 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

10 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 10 of 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 was attached in Appendix 1, all TAPs were archived by QA) TAP Name Number Autoclave Operation and Validation for use in the Microbiology Laboratory Use of the Oakton ph/con Meter in the Microbiology Laboratory Verification and Use of Automatic Pipettes in the Microbiology Lab Application Sterilization ph measurement Pipetting Calibration and Verification of Analytical Balances Weighing Calibration of Thermocouples and Relative Humidity Sensors Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure. Temperature 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 in appendix 1. Brief test parameters are shown below: Carrier Type: Glass slides (25 mm 25 mm) Contact Time: 10 Minutes Test Temperature: C (room temperature) Growth Medium: Nutrient Media Sub-culture Medium: (Modified) Letheen Broth Diluent Type: N/A

11 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 11 of 17 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 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 Figure 1. Schematic diagram showing the study design of this GLP study.

12 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 12 of 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 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.

13 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 13 of 17 Table 5. Zone Diameter Standards for Staphylococcus aureus extracted from CLSI M100- S24 Tables 2C and 4A. Zone Diameter Antimicrobial Agent (nearest whole mm) Oxacillin Susceptible Resistant Staphylococcus aureus Staphylococcus aureus ATCC 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 log 10 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 x 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:

14 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 14 of 17 ( average# ofcolonies / plate@ dilution) neutralizervolume CFU/Carrier = ( volumeplated) x( dilutionfactor) Log10 X1+ Log10X 2 + Log10XN Average Log10 Carrier Population Control = N 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 BI# , Lot #3 BI# , Lot #4 Purity Control Acceptable Acceptable Carrier Sterility control No growth No growth Viability Control Neutralizing Subculture Medium Sterility Control Neutralization Confirmation Carrier population control SwRI test # Growth No growth Growth 3.98 x Growth No growth Growth 3.98 x 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 >60 days old >60 days old BioSenta Batch/Lot number BI# , Lot #3 BI# , Lot #4 Test organism Staphylococcus aureus (MRSA) #33592 Contact time Soil load Number of carriers tested Carriers positive Carrier negative 10 min None 10 min 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 (e) (3). A summary of these test results are stated in Table 9.

15 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 15 of 17 Table 9. Results of test and quality control strains for identification and antibiotic resistance tests performed. Test Gram stain Colony morphology: Nutrient agar/tsa Staphylococcus aureus MRSA ATCC a +, non-spore coccus, singly, pairs and clusters Entire, glistening, circular, low convex, yellow-white, opaque, and smooth Staphylococcus aureus ATCC b N/A Entire, glistening, circular, low convex, yellow-white, opaque, and smooth Staphylococcus epidermidis ATCC b N/A Entire, glistening, circular, convex, opaque, and smooth Coagulase + + CHROMagar MRSA II Mauve colony color No growth N/A CLSI Disk diffusion test Resistant zone diameter 30 µg Cefoxitin c 0 mm Susceptible mm 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 (e)(3)(i) through (e)(3)(iv), the following information should be included when using an antibiotic resistant test organism. N/A Source and Identity: American Type Culture Collection, Methicillin-resistant Staphylococcus aureus; subsp. aureus # 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 methicillinresistant 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.

16 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 16 of 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 U.S. Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention, Product Performance Test Guidelines, OCSPP : General Considerations for Public Health Uses of Antimicrobial Agents, March 12, EPA Product Performance Test Guidelines; OCSPP : Disinfectants for Use on Hard Surfaces-Efficacy Data Recommendations, September 4, AOAC Official Method Germicidal Spray Products as Disinfectants (2012). US Environmental Protection Agency, Office of Pesticide Programs, Lower Certified Limit Testing Guidance, Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Eleventh Edition, M02-A11, January Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Disk Susceptibility Tests; Twenty-Fourth Informational Supplement, M100-S24, January

17 Microencapsulation and Nanomaterials Department Protocol #: GLP-SP-240 Final Report: Germicidal Spray Test of ZeroMold Plus For Additional Bacteria Page 17 of 17 APPENDICES Appendix 1. TAP (Rev. 0): Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure used in this study. Appendix 2. GLP Study Protocol (GLP-SP-240).

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19 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure 1.0 SCOPE TAP Xingguo Cheng Effective Date: Revision Page November of 18 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 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 Additional bacteria disinfectant/hard nonporous surfaces Test Organism Methicillin-Resistant Staphylococcus aureus (MRSA) (ATCC 33592) No. of Batches/Carriers Two batches at LCL, 10 carriers/batch Evaluation of Success 10/10 carriers are negative for growth in ten minutes Table 1. Summary of testing for efficacy claims per EPA OCSPP for Germicidal Spray Product Test. Southwest Research Institute Proprietary

20 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure 3.0 SAFETY TAP Xingguo Cheng Effective Date: Revision Page November of 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: Laboratory coat Nitrile gloves Safety glasses 3.3 All culture inoculations/manipulations (stock tubes, test culture, carriers, subculture 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 Southwest Research Institute Proprietary

21 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure 5.5 CFU- Colony Forming Unit 5.6 DI- Deionized (water) 5.7 EPA- Environmental Protection Agency 5.8 MHA- Mueller-Hinton agar TAP MRSA- Methicillin-Resistant Staphylococcus aureus 5.10 PBDW Phosphate Buffered Dilution Water Xingguo Cheng Effective Date: Revision Page November of 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 log 10 density 5.14 TSA or TSB- Tryptic Soy Agar or Broth 6.0 REFERENCES U.S. Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention, Product Performance Test Guidelines, OCSPP : General Considerations for Public Health Uses of Antimicrobial Agents, March 12, EPA Product Performance Test Guidelines; OCSPP : Disinfectants for Use on Hard Surfaces-Efficacy Data Recommendations AOAC Official Method Germicidal Spray Products as Disinfectants (2012) Southwest Research Institute Proprietary

22 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP Xingguo Cheng Effective Date: Revision Page November of US Environmental Protection Agency, Office of Pesticide Programs, Lower Certified Limit Testing Guidance, CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Eleventh Edition, M02-A11, January CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests; Twenty-Fourth Informational Supplement, M100-S24, January PROCEDURE Test Organism ATCC # Growth Medium Sub-Culture Medium Incubation Parameters MRSA Nutrient media Modified Letheen Broth 36 C, aerobic Table 2: Summary of efficacy test organism and associated growth conditions. 7.1 Reagent Preparation Nutrient Broth Weigh out 8 grams of Nutrient media (TAP ) 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 Mix thoroughly Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. Southwest Research Institute Proprietary

23 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP Xingguo Cheng Effective Date: Revision Page November of Check the final ph and adjust, if needed, to 6.8 ± 0.2 (TAP ) Autoclave at 121 C for 1 hour (TAP ) 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 For test culture preparations, dispense 10 ml of media into appropriate number of 25 x 150 mm Kim-Kap tubes Trypticase Soy Broth 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 Mix thoroughly Warm gently until solution is complete Check the final ph and adjust, if needed, to 7.3 ± Autoclave at 121 C for 1 hour 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 Subculture/Neutralization Media: Modified Letheen Broth 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 Na 2 S 2 O 3 in 1L of the above media) Mix thoroughly. Southwest Research Institute Proprietary

24 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP Xingguo Cheng Effective Date: Revision Page November of Heat with frequent agitation and boil for 1 minute to completely dissolve the powder Check the final ph and adjust, if needed, to 7.0 ± Autoclave at 121 C for 1 hour 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 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 Carrier Preparation Table 3: Number of sub-culture tubes required per organism test Visually screen all carriers and discard those that are visibly damaged (scratched, chipped, or nicked) Clean the carrier of oil, dirt, and all debris by rinsing with 95% ethanol followed by a rinse with DI-water Allow all carriers to dry before sterilization 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 Steam sterilize the carrier pouches in the autoclave for a minimum of 20 minutes at 121 C with a 30 minute drying cycle. Southwest Research Institute Proprietary

25 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP Xingguo Cheng Effective Date: Revision Page November of 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 Culture Initiation and Preservation Open ampoule of freeze-dried organism as indicated by ATCC 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 Aseptically transfer the entire rehydrated pellet back into the broth tube. Mix well Streak a loopful for isolation on nutrient agar for QC of culture purity. Include a QC media only plate Incubate tube and plates at 36 ± 2 C for 24 hours 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 Aseptically pipet 0.5 ml (TAP ) of culture per 1 ml sterile cryovial, containing 0.5 ml of glycerol. Vortex cryovial(s) well Store cryovials at 60 C. 7.4 Test Culture Preparation Defrost a single cryovial to room temperature and briefly vortex to mix. Each cryovial should be single use only. Southwest Research Institute Proprietary

26 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP Xingguo Cheng Effective Date: Revision Page November of 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 Incubate at 36 ± 2 C for 24 ± 2 hours 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 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 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 hours. These will be used for the antibiotic resistance confirmation and culture identification testing in section After test culture tubes from have incubated h, vortex each culture tube 3-4 seconds and let stand for 10 minutes at room temperature 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 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 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. Southwest Research Institute Proprietary

27 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP Xingguo Cheng Effective Date: Revision Page November of Immediately spread the inoculum uniformly over the majority of the carrier surface using a sterile loop. Do not touch the edges of the carrier Cover the Petri dish immediately and repeat the operation for all test carriers, viability controls, and quantification of microbe on carriers After all the carriers have been inoculated, place them in the incubator at 36 ± 2 C and let dry for minutes. 7.6 Test Procedure 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 Distance = 6-8" Number of pumps = Contact Time = 10 minutes Exposure Temperature = C (room temperature) Maintain the carriers in a horizontal position for the specified contact time After the exposure time is complete, transfer the carriers sequentially into the subculture tubes containing the appropriate neutralizer 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 After the carrier is deposited in the subculture tube, recap the subculture tube and shake culture for a few seconds to mix Incubate the tubes at 36 ± 2 C for 48 ± 2hours. Southwest Research Institute Proprietary

28 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP Xingguo Cheng Effective Date: Revision Page November of 18 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 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 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. 7.7 Study Controls 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 Culture Purity Control 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 Isolation plates will be incubated at 36 ± 2 C for hours and examined for characteristic, individual colony morphology per the organism being tested 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. Southwest Research Institute Proprietary

29 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure Sterility Controls TAP Xingguo Cheng Effective Date: Revision Page November of 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 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 Viability Controls (Positive Control) 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 Verification of Positive Carriers 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 Examine the plates for colonial morphology characteristic of the test organism and check by Gram stain Enumeration of viable bacteria from carriers 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 Vortex immediately for 120 ± 5 seconds. Southwest Research Institute Proprietary

30 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP Xingguo Cheng Effective Date: Revision Page November of 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 Briefly vortex each serial dilution tube prior to plating 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 Incubate the plates inverted concurrently with the efficacy subculture tubes at 36 ± 2 C for up to 48 ± 2 hours 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. Organism Average LD Corresponding Mean Density MRSA x 10 4 Table 4: Acceptable values of mean density, required per organism, when performing the Germicidal Spray Product Test 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 (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 Southwest Research Institute Proprietary

31 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure Test Gram stain Colony morphology: Nutrient agar/tsa CHROMagar MRSA II Staphylococcus aureus MRSA ATCC a +, non-spore coccus, singly, pairs, in short chains or irregular clusters Entire, glistening, circular, low convex, yellow-white, opaque, and smooth TAP Xingguo Cheng Effective Date: Revision Page November of 18 Staphylococcus aureus ATCC b N/A Entire, glistening, circular, low convex, yellow-white, opaque, and smooth Staphylococcus epidermidis ATCC b N/A Entire, glistening, circular, convex, opaque, and smooth Coagulase + + Mauve colony color No growth or non-mauve colonies Disk diffusion zone Resistant diameter: 30 µg cefoxitin c 21 mm QC range mm 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 Quality Control Strains N/A N/A Staphylococcus epidermidis (ATCC 12228) and Staphylococcus aureus (ATCC 25923) Defrost a single cryovial to room temperature and briefly vortex to mix. Each cryovial should be single use only 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 Incubate at 36 ± 2 C for 24 ± 2 hours 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. Southwest Research Institute Proprietary

32 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP Xingguo Cheng Effective Date: Revision Page November of Incubate at 36 ± 2 C for hours. These will be the quality control cultures utilized in the following tests Culture Identification: Colony morphology: Observe and record colony morphology of the test organism from the test purity control plate 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 Tube Coagulase Method: Remove three Coagulase Cryo tubes from the freezer and thaw to room temperature Add approximately 50 µl of an hour TSB culture per vial. Inoculate one Coagulase Cryo tube per organism listed in Table Incubate tubes at 36 ± 2 C for 1-4 hours 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 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. Southwest Research Institute Proprietary

33 UNCONTROLLED COPY IF PRINTED SOUTHWEST RESEARCH INSTITUTE Test/Analytical Procedure (TAP) Group/Section TAP ID No. Revised By: Microbiology Laboratory (B244, Labs and 1.115C) Title: Germicidal Spray Product Test for Additional Bacteria: Disinfectant Testing Procedure TAP CHROMagar MRSA II: Xingguo Cheng Effective Date: Revision Page November of Warm CHROMagar plates to room temperature in the dark before inoculation Generate serial ten-fold dilutions of the hour TSB cultures from the first two organisms listed in Table 5, by pipetting 1 ml of the inoculum into 9 ml of PBDW Using the undiluted, 10-1, and 10-2 dilutions, streak each tube for isolation on a CHROMagar plate. Include a QC media only plate Incubate culture plates for hours and QC media only plate for 72 hours at 36 ± 2 C. NOTE: Avoid exposure to light during incubation Read plates against a white background. Colonies of MRSA will appear mauve Antibiotic Resistance Confirmation: CLSI Disk Diffusion Test Prepare separate, direct colony suspension in 0.85% saline of isolated colonies of the first two organisms listed in Table 5 from hour TSA plates 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 Dip a sterile cotton swab into the adjusted inoculum. Rotate the swab several times and press firmly on the Southwest Research Institute Proprietary