Evaluation of Residual Efficacy against Viruses on Surfaces Test Results March 8, 2021

Experimental Approach

In October 2020, EPA released for public comment (comment period closed on December 21, 2020) the “Interim Method for Evaluating the Efficacy of Antimicrobial Surface Coatings” applicable for registration-based testing of antimicrobial coating products. EPA is evaluating the comments and intends to finalize the method. At the initiation of this research effort, a standard EPA-approved method did not exist for testing long-lasting efficacy (ability to kill or eliminate the target pathogen) against viruses on surfaces. A standardized approach was developed to achieve the objective of this project by combining elements of ASTM 2197-17, ASTM 1053, and EPA’s “Protocol for the Evaluation of Bactericidal Activity of Hard, Non-porous Copper Containing Surface Product”. The interim guidance is highly consistent with the essential elements of the above referenced methods.

For the screening efficacy testing conducted in this research effort, uniform pieces of surface material (coupons) were prepared, cleaned, rinsed, sterilized, and coated with the product being tested in accordance with product manufacturer’s directions, and allowed to dry overnight. On the following day, testing “Day 0”, virus was inoculated onto product-coated (coated) and uncoated coupons. Sets of coupons (three coated and three uncoated) were then extracted to recover infective virus at defined contact times (0, 2, 4, 8, 24, and 48 hours) after inoculation. These contact time points represent the amount of time the virus is in contact with the coating. In the current testing, the coatings have not undergone any extended aging or durability testing; the durability testing specified in the interim guidance is an essential component for demonstrating the residual efficacy claims (for days to weeks to months) of these type of antimicrobial coating products. The initial assessment without durability provides for a screening assessment of the products. The contact time points used for this initial screening do not indicate the longevity or durability of the coating; they are the contact time between the applied virus and the coating.

Several different enveloped and non-enveloped viruses are being used in this study (Table 1). Non-enveloped viruses (e.g., bacteriophage MS2) are expected to be more resistant to chemical inactivation than enveloped viruses (e.g., SARS-CoV-2). Viable virus was quantitatively determined using methods appropriate for each virus (see Table 1) and described in a pre-approved quality assurance project plan.

Table 1: Virus Types used in this Study

Virus	Virus Description	Host Cell	Analysis Method
MS2 (ATCC 15597-B1)	Non-enveloped virus; bacteriophage	Escherichia coli	Plaque assay, E. coli C-3000 (ATCC 15597)
Phi6	Enveloped virus; bacteriophage	Pseudomonas syringae	Plaque assay, P. syringae LM2489
MHV-A59	Enveloped virus; beta coronavirus	Mouse; Murine 17 clone 1 cell line 17Cl1	TCID₅₀; RT-qPCR
SARS-CoV-2 Isolate USA-WA1/2020 (BEI or ATCC (BEI NR-52281))	Enveloped virus; human beta coronavirus	Human; Vero cells (ATCC CCL-81)	TCID₅₀; RT-qPCR

The products that have been included in this research effort to date are shown in Table 2. The primary focus of this work is on antiviral coating products that are applied to surfaces via spraying and claim to provide residual antimicrobial activity for extended periods of time (days, weeks, months). The research also incorporated copper alloy materials and peel and stick antimicrobial film-based products, primarily for comparison with respect to the test method. Table 2 lists the products, reported active ingredient concentrations, and how the product was applied; product names are currently being withheld until additional testing is complete.

Effective neutralization of the product during the viral analysis is an essential component of the efficacy assessment protocol. A neutralization panel was conducted based upon the volume of product applied to each coupon via electrostatic sprayer (approximately 100 µL) or trigger-pull sprayer (approximately 1 mL). Table 2 also lists the neutralization methods developed for each product and virus combination.

Current testing assessed the recovery of phi6 virus from stainless steel (304 stainless steel with milled finish) test coupons coated with products listed in Table 2. This testing provided an initial assessment of the product’s ability to inactivate virus at various contact times – it did not assess the long-term claims of any products. Log reduction of viable phi6 virus was calculated as the difference in recovery of viable phi6 from product-coated coupons and the recovery from uncoated stainless-steel control coupons.

Further, this update provides results for products primarily applied via electrostatic sprayer (ESS) or a trigger-pull spray onto pre-cleaned and sterilized stainless-steel coupons, and in accordance with manufacturer’s label directions. Product A was also tested via application by submerging the coupons in the product for 3 minutes and by application via trigger-sprayer followed by wiping excess product off after 3 minutes. These two additional application methods were part of the product’s label (direction for use). Product D was applied via an airbrush by the product manufacturer.

Each product was applied to a batch of test coupons. Electrostatic sprayer application used a 10 second application operated at a four-foot distance from the coupon surface and having a nominal (reported) 60-micron droplet size. Trigger-spray application was done 4-6 inches from the coupon surfaces and applied until coupons had an even coating. Coated coupons were dried overnight. All products except Product D via the airbrush application and Product H were applied by the EPA in accordance with the manufacturer’s directions. Product D (airbrush) and Product H were applied by the product manufacturers to stainless-steel coupons supplied by the EPA.

The phi6 inoculum for the results reported in this update contained 5% heat inactivated fetal bovine serum (FBS) in phosphate buffered saline (PBS).

Table 2: Product information and neutralization methods

Products	Current EPA Registration	Active ingredients	Application	Neutralization methods
Product A	Microbiostat	3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (0.75%)	Used as received; applied by electrostatic sprayer, trigger sprayer, trigger plus wiping post-application, and submersion	MS2/phi6: 10% Dey-Engley broth in PBS
Product B	Microbiostat	3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (<1%)	Used as received; applied by electrostatic sprayer and trigger sprayer	MS2/phi6: 10% Dey-Engley broth in PBS
Product C	Microbiostat	3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (<3.6%)	Used as received; applied by electrostatic sprayer and trigger sprayer	phi6: 10% Dey-Engley broth in PBS
Product D	Disinfectant (non-residual), Microbiostat	3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (36.60%); n-alkyl (50% C14, 40% C12, 10% C16) dimethyl benzyl ammonium chloride (6.40%); Octyl decyl dimethyl ammonium chloride (4.80%); Didecyl dimethyl ammonium chloride (2.88%); Dioctyl dimethyl ammonium chloride (1.92%)	Diluted 1:1 as requested by manufacturer; applied by airbrush by the manufacturer and electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS
Product E	Microbiostat	3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (0.84%)	Used as received; applied by electrostatic sprayer and trigger sprayer	phi6: 10% Dey-Engley broth in PBS
Product F	Microbiostat	3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (1.3%)	Used as received; applied by trigger-pull sprayer	phi6: 10% Dey-Engley broth in PBS
Product G	Microbiostat	3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (1%)	Used as received; applied by trigger-pull sprayer	phi6: 10% Dey-Engley broth in PBS
Product H	Not currently registered	3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (1.3%)	Product applied to stainless steel carriers by company via electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS
Product I	Disinfectant (bactericide, fungicide, virucide), 24-hr sanitizer (bacteria), mildewstat	Alkyl* dimethyl benzyl ammonium chloride (*50%C14, 40%C12, 10%C16) (0.200%); Octyl decyl dimethyl ammonium chloride (0.150%); Didecyl dimethyl ammonium chloride (0.075%); Dioctyl dimethyl ammonium chloride (0.075%)	Used as received; applied by trigger-pull sprayer supplied within the product packaging	phi6: 50% Dey-Engley broth in PBS
Product J	Microbiostat	3 (Trihydroxysilyl) Propyldimethyl Octadecyl Ammonium Chloride (5.0%)	Diluted 1:5 as requested by manufacturer; applied by electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS
Product K	Not currently registered	Octadecylaminodimethyltrimethoxysilylpropylammonium chloride (<1%); Quaternaryammonium componds benzyl-C12-C16-Alkyldimethyl, chloridees (<5%); Alkyldimethylbezylammonium chloride (<2%); Alkyl (C12-C14) dimethyl(ethylbenzyl) ammonium chloride (<2%)	Used as received per manufacturer; applied by electrostatic sprayer	phi6: 50% Dey-Engley broth in PBS
Product L	Not currently registered	3 (Trihydroxysilyl) Propyldimethyl Octadecyl Ammonium Chloride (1.0%); other ingredients (99%)	Used as received per manufacturer; applied by electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS
Product M	Microbiostat	Octadecylaminodimethyltrihydroxylsilyl propyl ammonium chloride (0.75%)	Used as received per manufacturer; applied by electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS
Product N	Hospital disinfectant, Microbiostat	Octadecylaminodimethyltrihydroxylsilyl propyl ammonium chloride (0.75%)	Diluted (0.5 ounce in 1 quart) as per manufacturer’s directions; applied by electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS
Product O		Copper alloy, 99.9%	coupons cleaned per manufacturer’s instructions prior to use in testing	Phi6: 10 mM EDTA in 10% DE Broth
Product P	Not currently registered	n-Propanol [n-Proyl alcohol] (35-45%); Toluene [Methyl-benzene] (35-45%)	Peel and stick onto stainless steel per manufacturer’s instructions	phi6: 10% Dey-Engley broth in PBS
Product Q	Not currently registered	Cylohexane (40-60%); Naphtha (Petroleum) Hydrotreated Light (20-40%)	Peel and stick onto stainless steel per manufacturer’s instructions	phi6: 10% Dey-Engley broth in PBS
Product R	Disinfectant, Microbiostat	Octyl decyl dimethyl ammonium chloride (0.0375%); Dioctyl dimethyl ammonium chloride (0.0150%); Didecyl dimethyl ammonium chloride (0.0225%); Alkyl (C14, 50%; C12, 40%; C16, 10%) dimethyl benzyl ammonium chloride (0.0500%); 3- (trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (0.3280%); Other Inert Ingredients (99.5470%) Active Ingredient is hydrolyzed to produce methanol. The final concentration of the active ingredient and methanol are: 3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (0.299%); Methanol approx. (0.066%)	Used as received; applied by electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS
Product S	Not currently registered	Octadecyl dimethyl (3-trihydroxysilyl propyl) ammonium chloride (0.05%); Octyl decyl dimethyl ammonium chloride (0.005%)	Used as received; applied by electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS
Product T	Hospital disinfectant, 24-hour residual disinfectant (bacteria), Microbiostat	Alkyl dimethyl benzyl ammonium chloride (50%C14, 40%C12, 10%C16) (0.276%), Didecyl dimethyl ammonium chloride (0.104%), Octyl decyl dimethyl ammonium chloride (0.207%), Dioctyl dimethyl ammonium chloride (0.104%), Ethanol (68.610%)	Used as received; applied by trigger-pull sprayer	phi6: 50% Dey-Engley broth in PBS
Product U	Not currently registered	Octadecylaminodimethyltrihydroxylsilyl propyl ammonium chloride(~5%)	Diluted 1:3 as requested by manufacturer; applied by electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS
Product V	Not currently registered	3-(Trimethoxysilyl) propyldimethyloctadecyl chloride(<3%); Methanol (<1%); Poly(ethylene oxide) (<0.1%)	Diluted 1:4 as requested by manufacturer; applied by trigger pull sprayer supplied by manufacturer	phi6: 10% Dey-Engley broth in PBS
Product W		Copper alloy, 90%	coupons cleaned per manufacturer’s instructions prior to use in testing	Phi6: 10 mM EDTA in 10% DE Broth
Product X		Copper alloy, 70%	coupons cleaned per manufacturer’s instructions prior to use in testing	Phi6: 10 mM EDTA in 10% DE Broth
Product Y	Not currently registered	Alkyl dimethyl benzyl ammonium Chloride (C12-16) (<0.5%)	Used as received; applied by electrostatic sprayer	phi6: 10% Dey-Engley broth in PBS

Current Results

Table 3 presents the log reductions of viable phi6 virus at the 2-hour contact time as a function of product and application method. Note that this 2-hr contact time reflects the amount of time the virus is in contact with the product coating on the stainless-steel coupons. The viral efficacy component of the EPA’s interim guidance for antimicrobial coatings requires a 3-log reduction within a contact time of 2 hours. These results do not reflect the longevity/durability of the product over time: coatings were typically one day old and were not subjected to any wear and tear by chemicals or physical abrasion or exposure to alternative environmental conditions. Results from the durability testing will be presented in a subsequent update.

In EPA's Interim Method for Evaluating the Efficacy of Antimicrobial Surface Coatings”, the current performance standard to support registration is a 3-log reduction (99.9%) against Pseudomonas aeruginosa and Staphylococcus aureus. The method can subsequently be adapted for registration against other bacteria and/or viruses, with supporting test data demonstrating efficacy (3-log reduction) against the additional microorganisms. Demonstrating efficacy includes testing on unabraded coated surfaces and surface coatings that have been subjected to dry and wet abrasion cycles.

The testing reported here does not equate to these entire testing requirements for registration of a product; the actual requirements may vary based on product claims and end use. These results provide an initial screening of products and their potential residual efficacy against viruses. Claims for longevity of the product (e.g., for days, weeks, or months) would require similar testing on aged, weathered, and abraded coupons. Additional testing is essential to indicate the effectiveness of the product over time, its residual efficacy durability, and as a function of material types.

Table 3: Product efficacy results against phi6 for unabraded surface coating

Product	Application Method	Efficacy (Log₁₀ Reduction)	Standard Error
Product A	ESS	0.4	0.18
Product A	ESS	0.9	0.15
Product A	ESS	1.0	0.07
Product A	Submerged	1.2	0.22
Product A	Trigger spray	1.4	0.31
Product A	Trigger spray + wipe off	0.8	0.50
Product B	ESS	1.5	0.15
Product B	ESS	0.7	0.03
Product B	Trigger spray	0.0	0.11
Product B	Trigger spray	0.5	0.18
Product C	ESS	1.1	0.46
Product C	Trigger spray	1.2	0.20
Product C	Trigger spray	1.5	0.25
Product D	Airbrush	>3.0	0.19
Product D	ESS	>3.0	0.19
Product E	ESS	>3.0	0.16
Product E	Trigger spray	>3.0	0.01
Product F	ESS	>3.0	0.29
Product F	Trigger spray	>3.0	0.01
Product G	Trigger spray	3.0	0.09
Product G	Wipe on	-0.5	0.22
Product H	ESS	>3.0	0.25
Product H	ESS	>3.0	0.26
Product I	Trigger spray	>3.0	0.18
Product J	ESS	>3.0	0.09
Product K	ESS	>3.0	0.09
Product L	ESS	>3.0	0.01
Product M	ESS	1.6	0.55
Product N	Trigger spray	1.2	0.73
Product O	Copper alloy coupon	>3.0	0.18
Product P	Peel and stick film	>3.0	0.11
Product Q	Peel and stick film	>3.0	0.11
Product R	ESS	>3.0	0.11
Product S	ESS	>3.0	0.11
Product T	Trigger spray	>3.0	0.10
Product U	ESS	>1.9	0.20
Product V	Trigger spray	>3.0	0.30
Product W	Copper alloy coupon	>3.0	0.08
Product X	Copper alloy coupon	>3.0	0.08
Product Y	ESS	>3.0	0.15

Residual efficacy testing specifies that a product must demonstrate a 3-log reduction in recovered viable virus from test coupons compared to control coupons within a 2-hour time period post-inoculation. Demonstrating a 3-log reduction against a specific virus is part of the required testing for registration; however, it does not represent the entirety of the testing requirements for product registration (e.g., for registration of a product with residual claims against SARS-CoV-2). Long-lasting claims (e.g., days, weeks, months) would require additional testing on aged, weathered, and abraded coupons with a 2-hour contact time limit on those coupons. Further, results shown here are against phi6 (not a coronavirus), therefore serve only as an indicator of residual efficacy potential. They are not intended to support label claims.

It should also be noted that the products were tested as received. Active ingredient percentages and other product constituents were not verified by chemical analysis. Active ingredients (in Table 2) are listed as they appear on the product labels.

Additional testing is being conducted to continue to assess the potential of products to provide residual effectiveness on surfaces against viruses. Upcoming testing is intended to include:

Testing against coronavirus (MHV-A59 and SARS-CoV-2)
Additional inoculum types (e.g., simulated saliva)
Additional product testing against phi6
Product chemical analysis (verification of active ingredients)
Durability testing to determine factors impacting long-term (or long-lasting) residual effectiveness
Additional testing on different materials
Assessment of the impact of application methods

Disclaimer

EPA is conducting several studies to inform the response to the COVID-19 public health emergency. The summary here is intended to provide a simple representation of the results of on-going testing; therefore, only a brief description of the purpose of the study, methods, and interim results are provided. This study is being conducted in accordance with an approved Quality Assurance Project Plan. The interim results have been reviewed by internal EPA technical experts, quality assurance staff, and management. No interpretation of the interim results is provided. Once complete, the study and its results will be described in detail in a publication subjected to external, expert peer review. EPA does not endorse the purchase or sale of any commercial products or services.

Please visit the EPA webpage for more information on the research EPA is conducting in support of COVID-19 response capabilities.

Evaluation of Residual Efficacy against Viruses on Surfaces Test Results March 8, 2021

Experimental Approach

Emergency Response Research