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Long-Lasting Disinfection Evaluation Test Results August 10, 2020

Experimental Approach

A standard EPA-approved method does not currently 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”.

In general, 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. These contact time points do not indicate the longevity or durability of the coating. Testing on aged, weathered, and abraded coated coupons is necessary to assess the continued viricidal activity to support claims of activity over days, weeks, or months.

Several different enveloped and non-enveloped viruses will be used in this study; current viruses planned to be used are listed in 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. The impact of the material and the product on analysis methods was assessed for each method.

Table 1Virus Types used in this Study 


Virus Description 

Host Cell 

Analysis Method 

MS2 (ATCC 15597-B1) 

Non-enveloped virus; bacteriophage 

Escherichia coli 

Plaque assay, E. coli C-300 (ATCC 15597) 


Enveloped virus; bacteriophage 

Pseudomonas syringae 

Plaque assay, P. syringae LM2489 


Enveloped virus; beta coronavirus 

Mouse; Murine 17 clone 1 cell line 17Cl1 


SARS-CoV-2 Isolate USA-WA1/2020 (BEI or ATCC (BEI NR-52281)) 

Enveloped virus; human beta coronavirus 

Human; Vero cells (ATCC CCL-81) 


To date, the majority of the microbiostats that have been tested contain single active ingredient (3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride) in varying weight percentages by productOne product contains additional ammonium chloride-based active ingredients. Table 2 lists the tested productsreported active ingredient concentrations; 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 or trigger-pull spray onto pre-cleaned and sterilized stainless-steel couponsProduct 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 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 contained either 5% heat inactivated fetal bovine serum (FBS) in phosphate buffered saline (PBS), in accordance with soil load described in ASTM 1053, or 10% beef extract in PBS added to the inoculum (instead of 5% FBS). As presented in the August 4, 2020 update, beef extract resulted in increased stability of the phi6 virus on the control (uncoated) coupons over the 48-hour time period used in this study. This increased stability enabled differences in recovery of infectious phi6 to be discerned between test coupons (coated with product) and control coupons at the longer contact times (particularly at 8, 24, and 48 hours after inoculation). 


Table 2: Product information and neutralization methods 


Current EPA Registration 

Active ingredients 


Neutralization methods 

Product A 



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 



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 



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),​ 


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 



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 



3- (trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (1.3%) 

Used as received; applied by trigger-pull sprayer 

phi6: 10% Dey-Engley broth in PBS 

Product G 



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 that is part of the product packaging 

phi6: 50% Dey-Engley broth in PBS 


Current Results 

Figure 1 presents the log reductions of viable phi6 virus at the 2-hour contact time as a function of product, application method, and inoculum soil load (FBS or beef extract) 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. 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 conditionsNot all products have been tested with both application methods and both soil load additives at this time. These two parameters (application method, soil load) did not result in statistically significant differences in log reductions by product at the 2-hour timepoint.  

In the draftProtocol for the Evaluation of Bactericidal Activity of Hard, Non-porous Copper Containing Surface Product”, the current performance standard to support registration is a 3-log reduction (99.9%) within a one-hour contact time. Consistent with current registrations for copper containing surface products, EPA intends to revise the protocol to allow up to two hours for the contact time. Along with simulated touches and cleanings, this contact time and minimum log reduction are currently being considered as a potential performance standard for testing long-lasting efficacy against viruses. The testing reported here does not equate to the 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. 


Microbiostat Efficacy agains Phi6 2-Hour Contact Time Stainless Steel

Figure 1: Calculated log reduction values for products at the 2-hour time point  
(ESS = electrostatic sprayer application; Trigger = trigger-pull sprayer application;  
Airbrush = application via airbrushSubmerged = application via submersion; 
Trigger+wipe = excess product wiped off after 3 minutes post Trigger-spray application) 

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