Test Method Modified Association of Analytical Communities Test Method Modified Germicidal Spray Products as Disinfectants

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1 Study Title Antibacterial Activity and Efficacy of E-Mist Innovations' Electrostatic Sprayer Product with Multiple Disinfectants Method Modified Association of Analytical Communities Method Modified Germicidal Spray Products as Disinfectants Study Identification Number NG7772 Study Sponsor Brandi Whiteley E-Mist Innovations, Inc. Facility Microchem Laboratory 1304 W. Industrial Blvd Round Rock, TX (512)

2 AOAC Germicidal Spray Products : General Information Formerly known as the Association of Official Analytical Chemists, AOAC International is a globally recognized, third party, not-for-profit association that provides education and facilitates the development of test methods and standards for a wide range of industries. The AOAC Germicidal Spray method is a semi-quantitative test method designed to assess the performance of liquid spray disinfectants. The method typically has a maximum contact time of ten minutes, during which the spray disinfectant must eliminate all test microorganisms present on at least 59 out of 60 contaminated test surfaces (carriers). The method is extremely technique sensitive. False-positive results may occur if critical steps are not carried out properly. The Germicidal Spray method is a benchmark method for disinfectants applied via spray and is recognized by the United States Environmental Protection Agency (EPA) for claim substantiation. Laboratory Qualifications Specific to AOAC Germicidal Spray Products Method Microchem Laboratory began conducting the Germicidal Spray Products as Disinfectants method in Since then, the laboratory has performed hundreds of spray tests on a broad array of test substances, against a myriad of bacterial and viral species. The laboratory is also experienced with regard to method modifications, such as quantifying the number of surviving microorganisms on test surfaces. Every Spray test at Microchem Laboratory is performed in a manner appropriate to the test substances submitted by the Study Sponsor, while maintaining the integrity of the method. Study Timeline Culture Initiated Carriers Inoculated Carriers Sprayed Carriers Incubated Tubes Evaluated Report Delivered S. aureus, P. aeruginosa, S. aureus (MRSA), E. facealis (VRE) 02OCT OCT OCT OCT OCT OCT2016 C. difficile Spore Prep 03OCT OCT OCT OCT OCT2016 Page 2 of 14

3 Substance Information The test substances were received on 30SEP2016 and the following picture was taken. (note: photos depict the test substances that were analyzed in this study) Substances Received were Virex 256, Oxivir TB, Clorox Bleach. Oxycide was brought by E-Mist on the day of testing. Substances arrived as ready to use ready to use. substances were not diluted prior to use in the study. Microorganism Information The test microorganism(s) selected for this test: Staphylococcus aureus 6538 This bacterium is a Gram-positive, spherical-shaped, facultative anaerobe. Staphylococcus species are known to demonstrate resistance to antibiotics such as methicillin. S. aureus pathogenicity can range from commensal skin colonization to more severe diseases such as pneumonia and toxic shock syndrome (TSS). S. aureus is commonly used in several test methods as a model for gram positive bacteria. It can be difficult to disinfect but does demonstrate susceptibility to low level disinfectants. Enterococcus faecalis (VRE) This bacteria is a Gram-positive, spherical-shaped strain of Enterococcus faecalis that has developed resistance to the antibiotic vancomycin. E. faecalis (VRE) can cause a variety of local and systemic infections including endocarditis, bacteremia, and urinary tract infections, which are exceptionally difficult to treat because of this strain's acquired drugresistance. Due to this bacterium's robust survival factors and resistance to commonly used antimicrobial agents, this bacterium is very challenging to disinfect. Page 3 of 14

4 Staphylococcus aureus (MRSA) This bacteria is a Gram-positive, cocci shaped, aerobe which is resistant to the penicillin-derivative antibiotic methicillin. MRSA can cause troublesome infections, and their rapid reproduction and resistance to antibiotics makes them more difficult to treat. MRSA bacteria are resistant to drying and can therefore survive on surfaces and fabrics for an extended period of time and therefore makes this bacteria an excellent representative for antimicrobial efficacy testing on surfaces. Clostridium difficile (endospores) This bacteria is a Gram-positive, rod shaped, endospore generating obligate anaerobe. Clostridium species are part of the normal human gut flora that produce spores which are highly resistant to chemical and environmental conditions. C. diff is commonly associated with hospital acquired infections and is know to cause antibiotic assisted colitis. Because of it's high resistance to antimicrobials, C. difficile is a benchmark bacteria for sporicidal and sterilant activity of chemicals. Pseudomonas aeruginosa ATCC This bacteria is a Gram-negative, rod-shaped microorganism with a single flagellum. It grows optimally under aerobic conditions, however, it can use a host of electron receptors to respire anaerobically. P. aeruginosa can be found almost anywhere in nature and it is an opportunistic pathogen. Like many other bacterial-related diseases, the ability to form resilient biofilms within human tissues under anaerobic conditions is thought to be the primary cause for pathogenicity. Page 4 of 14

5 Diagram of the Procedure Substance Received by Laboratory Microorganism Grown in Culture Culture Diluted per Method/Sponsor Instructions and Control Carriers Inoculated, Dried Substance Sprayed on Surface of Carriers and Control Carriers Harvested After Contact Time Carriers Assessed for Growth After Incubation Presumptive Positive/Negative Results Confirmed Page 5 of 14

6 Summary of the Procedure The test microorganism is grown in a liquid broth culture. culture is prepared by conducting one daily transfer into fresh liquid culture medium. The test culture can be supplemented with an artificial soil load for one-step cleaner disinfectant claims. Sterilized glass slides (carriers) are inoculated with a volume of the prepared test culture. Inoculated slides are dried in an incubator. Only completely dried carriers are used in the test. substance is prepared prior to test, as needed. Dried inoculated glass carriers are then treated with the test substance noting the distance, angle, and number of sprays applied. Treated carriers are allowed to incubate for a predetermined contact time. At the conclusion of the contact time, treated carriers are aseptically harvested and carefully placed into individual tubes containing neutralization/growth buffer. Neutralization/growth buffer tubes are enumerated and incubated. After the incubation period, plates assessed for growth of the test microorganism. Results are reported based on the number of colony conforming units on the plate. Page 6 of 14

7 Criteria for Scientific Defensibility of a Germicidal Spray Study For Microchem Laboratory to consider a Germicidal Spray Products study to be scientifically defensible, the following criteria must be met: 1. The average number of viable bacteria recovered from the dried untreated carriers before and after testing must be greater than approximately 1 x 10 5 cells/carrier or greater for S. aureus and P. aeruginosa, and greater than 1 x 104 cells/carrier for S. enterica. 2. Neutralization must be confirmed using a low level inoculum (<100 CFU) on a treated carrier. 3. Positive/Growth controls must demonstrate growth of appropriate test microorganism. 4. Negative/Purity controls must demonstrate no growth of test microorganism. Passing Criteria Passing criteria for this study is determined by the study sponsor. ing Parameters used in this Study Substance Diluent: See Notes Carriers per : See Notes Spray Distance: & Culture Growth Media: Culture Supplement: Inoculation Concentration: Carrier Dry Time: Contact Time: Neutralization Media: Tube Incubation Time: Carrier Type: Spray Application Spray Angle: Synthetic Broth N/A ~1 x minutes See Notes See Notes ± 2 hours Page 7 of x 36 mm glass slides 5 second trigger hold <45 Culture Growth Time: 48 hours Carrier Inoculum Volume: ml Carrier Inoculation Area: 1 inch2 Carrier Dry Temperature: 36 C ± 1 C Contact Temperature: Ambient Tube Incubation Temp: 36 C ± 1 C

8 Study Modifications Study was modified for an electrostatic sprayer. Electrostatic sprayer was operated on-site by the study sponsor. Study Notes The neutralizer used for the study was 20 ml Dey/Engley Broth supplemented with 0.1% sodium thiosulfate and 0.1% Catalase. One carrier was evaluated per spray distance per test microorganism. 2 carriers were evaluated for pre and post test controls per test microorganism. substances were diluted, as appropriate, prior to use in the study. The following dilution schemes were used for each respective test substance: Virex 256 was diluted 0.5 oz/gallon or ml/l with sterile tap water. Oxycide was diluted 3 oz/gallon or ml/l with sterile tap water. Bleach was diluted 4 oz/gallon or ml/l with sterile tap water for S. aureus ATCC 6538, P. aeruginosa ATCC 15442, E. faecalis (VRE) ATCC 51575, and for S. aureus (MRSA) ATCC Bleach was diluted 1 part bleach to 8 part sterile tap water for C. difficile ATCC For Substance Virex 256, the contact times were 1 minute for P. aeurginosa ATCC and 10 minutes for S. aureus ATCC 6538, E. faecalis (VRE) ATCC 51575, and S. aureus (MRSA) ATCC For Substance Oxycide, the contact time was 3 minutes for P. aeruginosa ATCC 15442, S. aureus ATCC 6538, E. faecalis (VRE) ATCC 51575, C. difficile ATCC 43598, and for S. aureus (MRSA) ATCC For Substance Oxivir TB, the contact time was 1 minute for P. aeruginosa ATCC 15442, S. aureus ATCC 6538, E. faecalis (VRE) ATCC 51575, and for S. aureus (MRSA) ATCC For Substance Bleach, the contact time was 5 minutes for S. aureus ATCC 6538, E. faecalis (VRE) ATCC 51575, C. difficile ATCC 43598, and for S. aureus (MRSA) ATCC For P. aeruginosa ATCC 15442, the contact time was 10 minutes. Page 8 of 14

9 Control Results Neutralization Method: Verified Growth Confirmation: Confirmed Media Sterility: Sterile Calculations To calculate CFU/ml of broth containing control carriers, the following equation is used: Where 10-x, 10-y, and 10-z are dilutions plated. Page 9 of 14

10 Results for S. aureus ATCC 6538 Microorganism Substance Time Point Distance Pre Control 1.82E+05 Post Control 1.77E+05 Control Virex 256 S. aureus ATCC 6538 Oxycide Oxivir TB Bleach (4oz/gallon) Percent Reduction Average Compared to Log/Carrier Control at Time Zero 1.80E+05 N/A 10 minutes 3 minutes 1 minute 5 minutes The limit of detection for this assay was 10 CFU/carrier. Values observed below the limit of detection are noted in the table as. Page 10 of 14

11 Results for P. aeruginosa ATCC Microorganism Substance Time Point Distance Pre Control 1.90E+04 Post Control Control Virex 256 P. aeruginosa ATCC Oxycide Oxivir TB Bleach (4oz/gallon) Percent Reduction Average Compared to Log/Carrier Control at Time Zero 9.50E+03 N/A 1 minute 3 minutes 1 minute 10 minutes The limit of detection for this assay was 10 CFU/carrier. Values observed below the limit of detection are noted in the table as. Page 11 of 14

12 Results for S. aureus (MRSA) ATCC Microorganism Substance Time Point Distance Pre Control 2.20E+04 Post Control 2.80E+04 Control Virex 256 S. aureus (MRSA) ATCC Oxycide Oxivir TB Bleach (4oz/gallon) Percent Reduction Average Compared to Log/Carrier Control at Time Zero 2.50E+04 N/A 10 minutes 3 minutes 1 minute 5 minutes The limit of detection for this assay was 10 CFU/carrier. Values observed below the limit of detection are noted in the table as. Page 12 of 14

13 Results for E. faecalis (VRE) ATCC Microorganism Substance Time Point Distance Pre Control 1.32E+06 Post Control 2.06E+06 Control Virex 256 E. faecalis (VRE) ATCC Oxycide Oxivir TB Bleach (4oz/gallon) Percent Reduction Average Compared to Log/Carrier Control at Time Zero 1.69E+06 N/A > % > % > % >99.995% > % > % > % > % 10 minutes 3 minutes 1 minute 5 minutes The limit of detection for this assay was 10 CFU/carrier. Values observed below the limit of detection are noted in the table as. Page 13 of 14

14 Results for C. difficile (endospores) ATCC Microorganism Substance Time Point Distance Pre Control 3.40E+05 Post Control 3.20E+05 Control C. difficile (endospores) ATCC Oxycide Bleach (1:8) Percent Reduction Average Compared to Log/Carrier Control at Time Zero 3.30E+05 N/A 2.10E E % E E % 2.66 >99.997% >4.52 >99.997% > minutes 5 minutes The limit of detection for this assay was 10 CFU/carrier. Values observed below the limit of detection are noted in the table as. The results of this study apply to the tested substances(s) only. Extrapolation of findings to related materials is the responsibility of the Sponsor. Copyright Microchem Laboratory, Reproduction and ordinary use of this study report by the entity listed as Sponsor is permitted. Other copying and reproduction of all or part of this document by other entities is expressly prohibited, unless prior permission is granted in writing by Microchem Laboratory. Page 14 of 14

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