United States Environmental Protection Agency Office of Chemical Safety and Pollution Prevention (7503P) EPA XXX

- X

- XX

- XXX April 2015 

# Product Performance Test Guidelines

OCSPP 810.2200 : Disinfectants for Use on Hard Surfaces

- E fficacy Data RecommendationsNOTICE

This guideline is one of a series of test guidelines established by the United States Environmental Protection Agency’s Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic Substances (OPP TS). To distin g uish these guidelines from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS as part of the guideline’s number. Any test guideline developed after April 22, 2010 will use the

new a cronym (OCSPP) in their title.

The OCSPP test guidelines serve as a compendium of accepted standardized methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA. This document provide s guidance for conducting the test, and is also used by EPA, the public, and companies that are subject to data submission requirements under TSCA, FIFRA and/or the FFDCA

. As a guidance document, these guidelines are not binding on either EPA or any outsi de parties, and the EPA may depart from the guidelines where circumstances warrant and without prior notice. At places in this guidance, the Agency uses the word “should.” In this guidance, use of “should” with regard to an action means that the action is recommended rather than mandatory. The procedures contained in this guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. You may pr opose alternatives to the recommendations described in these guidelines, and the Agency will assess them for appropriateness on a case

- by

- case basis.

For additional information about these test guidelines and to access the guidelines electronically, pl ease go to http://www.epa.gov/ocspp and select the topic entitled, “Test Methods and Guidelines

. ” You may also access the guidelines in http://www.regulations.gov grouped by Series under Docket ID #s: EPA

- HQ

- OPPT

2009

- 0150 through EPA

- HQ

- OPPT

- 2009

- 0159, and EPA

- HQ

- OPPT

- 2009

- 0576.

DRAFT DOCUMENT DISCLAIMER: This draft document is distributed solely for the purpose of external review. It has not been formally disseminated by the EPA and should not be construed to represent any Agency determination or poli cy. The information correction process under the Agency’s Information Quality Guidelines does not apply until this document is formally disseminated by the EPA in its final form. This draft document should only be cited or quoted in the context of providin g comments.

2OCSPP 810.2200 : Disinfectants for use on hard surfaces

- efficacy data recommendations

(a) Scope

.
(1) A pplicability . This guideline describes test methods that EPA believes will generally satisfy testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA )( 7U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a) . It addresses testing to demonstrate the effectiveness of antimicrobial pesticides bearing claims as disinfectants, fungicides, virucides, and tuberculocides. Refer to OCSPP 810.2000: General Considerations for Public Health Uses of Antimicrobial Agents for guidance.

(2) Background . The source materials used in dev eloping this OCSPP test guideline are OPP guidelines 91

- 2 : Products for use on hard surfaces and 91

- 30 : Acceptable methods (Pesticide Assessment Guidelines, Subdivision G, Product Performance. EPA report 540/9

- 82

- 026, October 1982).

(b) Purpose

. This guideline addresses efficacy testing for antimicrobial pesticides intended to be used on hard surfaces, namely disinfectants, fungicides, virucides, and tuberculocides in a variety of product types (water

- soluble powders, liquids, sprays, towelettes, etc.).

(c) General considerations

(1) Efficacy claims. This guideline provides guidance for developing data solely for the purpose of supporting the following claims for hard surface disinfectants ( limited spectrum disinfectants, general or broad spectrum disi nfectants, hospital or healthcare disinfectants ) and additional claims ( internal toilet and urinal bowl surface disinfectants, and disinfectants with fungicidal, virucidal, and/or tuberculocidal claims ) . Refer to OCSPP Test Guideline 810.2000 for general testing recommendations prior to testing

. See Tables 1 and 2 for a summary of the efficacy claims and recommendations for testing

.
(2) Test Methods. Refer to Table s 1 and 2 for information on efficacy test method s recommended to support disinfectant and additional label claim s , respectively

. The Agency recomm ends the use of the most recently published version of the standard method s (e.g., AOAC International [AOAC] , ASTM) . S ee standard methods for control ca rrier count s (mean number of via b le microbes per carrier ) and other method associated information

. Agency Standard Operating Procedures for many of the test procedures referenced in the table s are available online at: http://www.epa.gov/pesticides/methods/atmpa2z.htm. Information regarding specifics such as the number of batches to test, performance standards, and control carrier counts is provided in the description for each type of label clai m.

(3) Spray product s . Spray products refere nced in Table 1 include aerosol , trigger, and

3pump dispensers.

(4) T owelette products

. For products formulated as impregnated wipes or towelettes, refer to Table 1. For evaluation of efficacy, t he Agency recommends th e use of a modified AOAC Method 961.02 ( Germicidal Spray Products as Disinfectants test ) or ASTM E2362 (Standard Practice for Evaluation of Pre

- saturated or Impregnated Towelettes for Hard Surface Disinfection) . Instead of spraying the inoculated surface of the carriers, the product should be tested by wiping the surface of the carriers with the saturated towelette, and then sub

- culturing the carriers after the specified holding time. The towelette should be removed from its container and handled with ster ile gloves. One towelette should be used to wipe 10 inoculated carriers (e.g., use 1 towelette for 10 carriers or 6 towelettes for 60 carriers )

. For each carrier, an unused area of the towelette should be used for wiping. Also, see section (j) of this docu ment on bridging for cases where an applicant formulates a disinfectant towelette using an existing EPA

- registered bulk liquid formulation

. N ote: A detailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report.

(5) Product batches. Refer to the claim

- specific sections below to identify the type and number of batches to be evaluated for efficacy. Depending on the claim, testing will be co nducted on batches (two or three) at or below the lower certified limit(s) (LCL) listed on the confidential statement of formula (CSF) of the product , or at the nominal concentration.

(6) Soil load. If a product is intended to be effective in the presence of organic soil ( i.e., a one

- step disinfectant product ), an appropriate organic soil, such as 5 percent blood serum , should be added to the inoculum prior to carrier inoculation

.
(7) Neutralization confirmation. Testing to verify the effectiveness of the neutral izer should be conducted in advance or concurrently with the efficacy test . The procedure should include conditions relevant to the efficacy test (e.g., g r owth medi um, product dilution and contact time, neutralizer, and microbe)

.
(8) Confirmatory data. Refer to OCSPP 810.2000: General Considerations for Public Health Uses of Antimicrobial Agents, section (b)(7) for situations (i.e., duplicated product formulations and minor formulation changes in a registered product) requiring confirmatory data . Specific info rmation related to c onfirmatory data is provided in the description for each label claim. For pressurized spray products, certification should be furnished specifying that all parts and materials used in manufacturing the container for pressurized spray di sinfectants are identical to those specified by the basic manufacturer.

(9) Additional microorganisms. For efficacy testing requirements related to broad

spectrum or hospital disinfectants which bear label claims against bacteria other

4than Salmonella enteric a (ATCC10708), Staphylococcus aureus (ATCC 6538)

, or Pseudo m onas aeruginosa (ATCC 15442) , see section (d)( 6 ).

(10) Contact Time. All p roducts should meet the performance standard associated with the method and microbe at ≤ 10 min utes of contact

.

5Table 1. Summary of Testing for Disinfectant C laims

Claim Formulation/ Test Methods Test Organisms No. of Batches/Carriers

Limited spectrum disinfectant/hard

non

- porous surfaces Water soluble powders/liquids AOAC Use

- Dilution Method (ref. 1)
Staphylococcus

aureus (ATCC 6538)

or Salmonella enterica (ATCC 10708) Three batches at LCL ; 60 carriers against either organism ( 180 total carriers) Spray products AOAC Germicidal Spray Products as Disinfectants Test (ref. 2 )

Towelettes AOAC Germicidal Spray Products as Disinfectants Test modified for towelettes or ASTM E2362 (ref. 3 )

Broad

- spectrum disinfectant/hard

non

- porous surfaces Water soluble powders/liquids AOAC Use

- Dilution Method
Staphylococcus

aureus (ATCC 6538) and Salmonella enterica (ATCC 10708) Three batches at LCL ; 60 carriers against each organism (360 total carriers) Spray products AOAC Germicidal Spray Products as Disinfectants Test

Towelettes AOAC Germicidal Spray Products as Disinfectants Test modified for towelettes or ASTM E2362

Hospital or healthcare disinfectant/hard

non

- porous surfaces Water soluble powders/liquids AOAC Use

- Dilution Method
Staphylococcus

aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442) Three batches at LCL : 60 carriers against each organism (360 total carriers) Spray products AOAC Germicidal Spray Products as Disinfectants Test

Towelettes AOAC Germicidal Spray Products as Disinfectants Test modified for towelettes

or ASTM E2362

6Table 2. Summary of Testing for Additional Label Claims

Claim Formulation/Test Methods Test Organisms No. of Batches/Carriers

Fungicidal disinfectant/hard

non

- porous surfaces Water soluble powders/liquids AOAC Use

- Dilution Test modified for fungi or AOAC Fungicidal Test (ref. 4)
Trichophyton mentag rophytes (ATCC 9533) Two batches at the LCL ; 1 0 carriers per batch (20 total carriers) for carrier

- based methods. Two batches for the AOAC Fungicidal Test. Spray products AOAC Germicidal Spray Products as Disinfectants Test modified for fungi

Towelettes AOAC Germicidal Spray Products as Disinfectants Test modified for towelettes or ASTM E2362

Virucidal disinfectant/hard

non

- porous surfaces Water soluble powders/liquids ASTM E1053 (ref. 5)

Virus claimed on the label or approved surrogate Two batches at the nominal concentration ; 10 carriers per batch. For non

- surrogates, test one surface per batch; for surrogates, test two surfaces per batch. Towelettes AOAC Germicidal Spray Products as Disinfectants Test modified for towelettes or ASTM E2362

Tuberculocidal disinfectant/hard

non

- porous surfaces. Water soluble powders/liquids AOAC Tuberculocidal Activity of Disinfectants (ref. 8), Quantitative Tuberculocidal Activity Test (ref. 9)

Mycobacterium bovis BCG Two batches at the LCL ; 10 carriers per batch.

Spray products AOAC Germicidal Spray Products Test modified for tuberculocidal activity Two batches at the LCL ; 10 carriers per batch

Towelettes AOAC Germicidal Spray Products as Disinfectants Test modified for towelettes or ASTM E2362 Two batches at the LCL ; 10 carriers per batch

Additional bacteria /hard

non

- porous surfaces. Water soluble powders/liquids AOAC Use

- Dilution Test Additional bacteria claimed on the label Two batches at the nominal concentration ; 10 carriers for each batch.

I nternal toilet and urinal bowl surfaces above and below the water line W ater

- soluble powders and

non

- volatile liquid products AOAC Use

- Dilution Test modified to include a 5% organic soil challenge added to the bacterial inoculum. S. enterica (ATCC 10708) or S.

aureus (ATCC 6538) for limited disinfectant products ; S. enterica and S.

aureus for broad

spectrum disinfectant products; S. aureus and P. aeru ginosa (ATCC 15442) for Test sixty carriers against each of three batches of the product at the LCL

7hospital d isinfectant s

(d) Disinfectants

(1) Limited spectrum products. This section addresses efficacy testing for disinfectant products with limited efficacy (effective against Gram

- negative or Gram

- positive bacteria , but not both ). Refer to Table 1 for test method s

. Conduct the following:

(i) Test organism. To demonstrate efficacy, testing should be conducted against Salmonella enterica ( S. enterica )

, formerly de signated as Salmonella choleraesuis

, American Type Culture Collection (ATCC ) 10708 for effectiveness against Gram

- negative bacteria, or Staphylococcus

aureus ( S. aureus ) (ATCC 6538) for effectiveness against Gram

- positive bacteria.

(ii) Batches/number of carriers. Test three batches of the product at the LCL. For each batch, test using sixty carriers.

(iii) Control carrier counts for AOAC Use

- Dilution Method s (ref. 1 )

. F or the AOAC Use

- Dilution Metho d s , the mean log density for S. enterica is to be at least 5.0 (corresponding to a geometric mean density of 1.0 x 10 5 ) and not above 6.0 (corresponding to a geometric mean density of 1.0 x 10 6 ). A mean log density <5.0 or >6.0 invalidates the test. T he mean log density for S. aureus is to be at least 6.0 (correspond ing to a geometric mean density of 1.0 x 10 6 ) and not above 7.0 (corresponding to a geometric mean density of 1.0 x 10 7 ). A mean log density <6.0 or >7.0 invalidates the test.

(iv) Control carrier counts for s pray p roducts and single

- use towelettes. T he mean log density for S. enterica is to be at least 4.0 (corresponding to a geometric mean density of 1.0 x 10 4 ) and not above 5.5 (corresponding to a geometric mean density of 3.2 x 10 5 ). A mean log density <4.0 or >5.5 invalidates the test. The mean lo g density for S. aureus is to be at least 5.0 (corresponding to a geometric mean density of 1.0 x 10 5 ) and not above 6.5 (corresponding to a geometric mean density of 3.2 x 10 6 ). A mean log density <5.0 or >6.5 invalidates the test.

(v) Performance measure.

(A) For the AOAC Use

- Dilution Methods, conduct three independent tests (i.e., three batches tested once on three different test days) against the test microbe at the LCL . The performance standard for S. aureus is 0

- 3 positive carriers out of sixty. The perfo rmance standard for S. enterica is 0

- 1 positive carriers out of sixty. Contamination of one carrier (culture tube) is allowed per 60

- carrier
8set; occurrence of more than one contaminated carrier invalidates the test results

.
(B) For the AOAC Germicidal Spray Products as Disinfectants test and towelette methods , the product should kill the test microorganisms on 59 out of each set of 60 carriers/slides

. Contamination of only one carrier (culture tube) is allowed per 60

- carrier set; occ urrence of more than one contaminated carrier invalidates the test results

.
(vi) Confirmatory testing for limited spectrum products

. For confirmatory testing, follow the above testing guidance in sections (c) and (d)(1), with the exception that ten carriers f or each of two different batches of product should be tested against S. aureus (ATCC 6538) or S. enterica (ATCC 10708). For all methods, the product should kill all the test microorganisms on all carriers

. Control carrier counts specifications should be me t. For a valid test, n o contamination of any carrier is allowed.

(2) General or broad spectrum efficacy products

. For disinfectants with label claims against both Gram

- negative and Gram

- positive bacteria, the product is considered a general or broad spectrum disinfectant. Refer to Table 1 for test method s

. Conduct the following:

(i) Test organism . To demonstrate efficacy, testing should be conducted against both S. enterica ( ATCC 10708 ) and S. aureus ( ATCC 6538.

(ii) Batches/number of carriers. For each organism, tes t sixty carriers against each of three batches of the product at the LCL

.
(iii) Control carrier counts for AOAC Use

- Dilution Methods

. F or the AOAC Use

- Dilution Methods, the mean log density for S. enterica is to be at least 5.0 (corresponding to a geometric mean density of 1.0 x 10 5 ) and not above 6.0 (corresponding to a geometric mean density of 1.0 x 10 6 ). A mean log density <5.0 or >6.0 invalidates the test. The mean log density for S. aureus is to be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10 6 ) and not above 7.0 (corresponding to a geometric mean density of 1.0 x 10 7 ). A mean log density <6.0 or >7.0 invalidates the test.

(iv) Control carrier counts for spray products and towelettes

. T he mean log density for S. enterica is to be at least 4.0 (corresponding to a geometric mean density of 1.0 x 10 4 ) and not above 5.5 (corresponding to a geometric mean density of 3.2 x 10 5 ). A mean log density <4.0 or >5.5 invalidates the test. The mean log density for S. aureus is to be at least 5.0 (corresponding to a geometric mean density of 1.0 x 10 5 ) and not above 6.5 (corresponding to a geometric mean density of 3.2 x 10 6 ). A mean log density <5.0 or >6.5 invalidates the test.

9(v) Performance measure.

(A) For the AOAC Use

- Dilution Methods, conduct three independent
tests (i.e., three batches tested at the LCL on three different test days) agai nst the test microbe. The performance standard for S. aureus is 0

- 3 positive carriers out of sixty. The performance standard for S. enterica is 0

- 1 positive carriers out of sixty. Contamination of only one carrier (culture tube) is allowed per 60

- carrier set; occurrence of more than one contaminated carrier invalid ates the test results

.
(B) For the AOAC Germicidal Spray Products as Disinfectants test and towelette method s, the product should kill the test microorganisms on 59 out of each set of 60 carriers/slides

. Contamination of only one carrier (culture tube) is all owed per 60

- carrier set; occurrence of more than one contaminated carrier invalidates the test results

.
(vi) Confirmatory testing for general or broad spectrum products. For confirmatory testing, follow the above testing guidance in sections (c) and (d) (2), w ith the exception that ten carriers for each of two different batches of product (within the CSF) should be tested against S. aureus (ATCC 6538) and S. enterica (ATCC 10708). For all methods, the product should kill all the test microorganisms on all carri ers. Control carrier counts specifications should be met. For a valid test, n o contamination of any carrier is allowed.

(3) Hospital or healthcare disinfectants. This section addresses efficacy testing for products recommended for use in hospitals, clinics, dental offices, nursing homes, sickrooms, or any other healthcare

- related facility. Refer to Table 1 for test methods. Conduct the following:

(i) Test organism. To demonstrate efficacy, testing should be conducted against S

. aureus (ATCC 6538) and P seudomonas aeruginosa (ATCC 15442).

(ii) Batches/number of carriers. For each organism, t est sixty carriers against each of three batches of the product at the LCL

.
(iii) Control carrier counts for AOAC Use

- Dilution Methods

. F or the Use

Dilution Methods, the mean log density for S. aureus and P. aeruginosa is to be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10 6 ) and not above 7.0 (corresponding to a geometric mean density of 1.0 x 10 7 ). A mean log density <6.0 or >7.0 invalidates the test.

(iv) Control carrier counts for spray products and to welettes. T he mean log density for both S. aureus and P. aeruginosa is to be at least 5.0 (corresponding to a geometric mean density of 1.0 x 10 5 ) and not above 6.5

10(corre sponding to a geometric mean density of 3.2 x 10 6 ). A mean log density <5.0 or >6.5 invalidates the test.

(v) Performance measure.

(A) For the AOAC Use

- Dilution Methods, conduct three independent
tests (i.e., three batches at the LCL tested on three different test days) against the test microbe. The performance standard for S. aureus is 0

- 3 positive carriers out of sixty. The performance standard for P. aeruginosa is 0

- 6 positive carriers out of sixty. Contamination of only one carrier (culture tube) is allowed per 60

- carrier set; occurrence of more than one contaminated carrier invalidates the test results

.
(B) For the AOAC Germicidal Spray Products as Disinfectants test and towelette methods , the product should kill the test microorganis ms on 59 out of each set of 60 carriers/slides

. Contamination of only one carrier (culture tube) is allowed per 60

- carrier set; occurrence of more than one contaminated carrier invalidates the test results

.
(vi) Confirmatory testing for products with hospital or healthcare disinfectant claim

. For confirmatory testing, follow the above testing guidance in sections (c) and (d) ( 3 ), with the exception that ten carriers for each of two different batches of product (within the CSF) should be tested against S. aureus (ATCC 6538) and P. aeruginosa (ATCC 15442). For all methods, the product should kill all the test microorganisms on all carriers. C ontrol carrier counts specifications should be met. For a valid test, n o contamination of any carrier is allowed.

(4) Disinfect ants for i nternal t oilet and u rinal b owl s urface s a bove and b elow the

w ater l ine. To add claims to existing limited, broad

- spectrum, or hospital products for internal toilet and urina l bowl surfaces , conduct the following :

(i) Test method. Regarding water

- soluble powders and non

- volatile liquid products t est procedure

, the Agency recommends the use of the AOAC Use

Dilution Methods modified to include a 5% organic soil challenge added to the bacterial inoculum

.
(A) The contained bowl water (96 fl oz of 200

- 40 0 ppm hard water

, which represents traditional high volume toilets) should be used to calculate the appropriate use dilution for testing. The contained bowl water for low volume toilets should be measured and used to calculate the appropriate use dilution using 200

- 400 ppm hard water for testing.

11(ii) Test organisms. Test against S

. enterica (ATCC 10708) or S

. aureus (ATCC 6538), for limited disinfectant products; S. enterica and S. aureus

, for broad

- spectrum disinfectant products; and S. aureus and P

. aerugino sa (ATCC 15442), for hospital disinfectant products. Follow the testing recommendations (i.e., control carrier coun t s, and performance measure) specified for limited, broad

- spectrum, and hospital disinfectant claims.

(iii) Batches/number of carriers. Test sixt y carriers against each of three batches of the product at the LCL

.
(iv) Performance Measure. Meet the performance measures specified for each claim (e.g., hospital claim) and specified microbe(s)

.
(5) Additional microorganisms. Label claims for additional microbes other than the designated test microorganism(s) is permitted, provided that the target microbe is likely to be present in or on the recommended use areas and surfaces. This section addresses efficacy testing for limited

, broad

- spectrum or hospital disinfectants which bear label claims against bacteria other than S

. enterica (ATCC 10708), S

. aureus (ATCC 6538) or P

. aeruginosa (ATCC 15442). Following recommendations for test methods, per the product formulation as provide d in Table 1

, conduct additional testing as described below:

(i) Test organism . To demonstrate efficacy, testing should be conducted against each specific bacterium to be claimed on the label.

(ii) Batches/number of carriers

. Test ten carriers against each of two batches of the product at the nominal concentration.

(iii) Control carrier counts for all methods. The minimum carrier count for a valid test s hould be a mean of 1 x 10 4 CFU/carrier

.
(iv) Performance measure. The product sho uld kill all the test microorganisms on all ten carriers

. For a valid test, n o contamination of any carrier is allowed.

(e) F ungicidal claims. A fungicidal claim may be added to an existing broad

- spectrum or hospital disinfectant product. Refer to Table 2 for test methods. Conduct the following:

(1) Test organism. To demonstrate efficacy , testing should be conducted against Trichophyton mentagrophytes ( ATCC 9533).

(2) Batches/number of carriers.

(i) For AOAC Fungicidal Activity of Disinfectants T est

. Two batches of product at the LCL should be evaluated for efficacy at 5, 10, and 15 minute

12exp o sure times. The m ethod is a suspension test.

(ii) For AOAC Use

- Dilution Method , AOAC Germicidal Spray Produc t s as Disinfectants Test, and towelettes

. T e st s may be m odified to conform to appropriate elements (e.g., media, growth conditions) in the AOAC Fungicidal Activity of Disinfectants test. Test ten carriers against each of two batches of product at the LCL

.
(3) Titer of conidial suspension /control counts

.
(i) A OAC Fungicidal Activity of Disinfectants test

. The inoculum employed should provide a concentration of ≥ 5 x 10 6 conidia/mL.

(ii) AOAC Use

- Dilution Method , AOAC Germicidal Spray Products as Disinfectants Test, and single

- use towelettes

. The inoculum employed shoul d provide a concentration of 1 x 10 4

– 1 x 10 5 conidia per carrier.

(4) Performance measure

. (i) AOAC International Fungicidal Activity of Disinfectants test

. All fungal spores at 10 and 15 minutes should be killed (no positive carriers)

.
(ii) AOAC Use

- Dilution Method, AOAC Germicidal Spray Products as Disinfectants Test, and towelettes

. All fungal spores on all 10 carriers should be killed (no positive carriers)

.

(f) V irucidal claims

. Virucidal claims may be added to existing broad

- spectrum or hospital disinfectant products (liquids, sprays, towelettes)

. Virucidal products are intended for use on dry inanimate surfaces; therefore, data are developed using carrier

- based methods. To simulate in

- use conditions, the specific virus to be treated (or surrogate ) should be inoculated onto hard surfaces (e.g., Petri dishes, glass carriers

, or other appropriate test surface), allowed to dry, and then treated with the product according to the directions for use on the product label. Refer to Table 2 for test methods. Conduct the following:

(1) Test organism

. Each specific virus listed on the label should be tested, unless there is an acceptable surrogate for the virus. For example, label claims against Hepatitis B virus, Hepatitis C virus, and Norovirus, the Duck Hepatitis B virus, Bovine Viral Diarrhea virus, and Feline Calicivirus, respectively, are considered acceptable surrogates for testing. Additional guidance and protocols for surrogate virus testing can be found at http://www.epa.gov/oppad001/regpolicy.htm

.
(2) In limited situations such as towelette bridging and confirmatory testing, the Agency will allow testing of the m ost resistant virus on the label based on d isinfection hierarchy

. The categories and the presumed levels of resistance are provided below:

13(i) Small n on - enveloped viruses (most resistant viruses )

. For l abel claims to inactivate small non

- enveloped viruses such as members of the Picornaviridae family (e.g., poliovirus, enterovirus, hepatitis A virus, rhinovirus), and the Caliciviridae family (e.g., parvovirus) select the most resistant representative virus (or acceptable surrogate) such as Feline calicivirus for testing

.
(ii) Large non

- enveloped viruses (intermediate resistant viruses )

. For label claims to inactivate large non

- enveloped virus es such as members of the members of the Adenoviridae family (e.g., adenovirus), Reoviridae family (e .g., rotavirus), and Papillomaviridae family (e.g., papillomavirus), select the most resistant representative virus (or acceptable surrogate) such as Adenovirus for testing.

(iii) E nveloped viruses (least resistant viruses)

. For label claims to inactivate

env eloped viruses such as members of the Coronaviridae family (e.g., coronavirus), Flaviviridae family (e.g., hepatitis C virus), Herpesviridae family (e.g., herpes virus), Poxviridae family (e.g., vaccinia), Hepadnaviridae family (e.g., hepatitis B virus), O rthomyxoviridae family (e.g., Influenza), Paramyxoviridae family (e.g., parainfluenza) and Retroviridae family (e.g., human immunodeficiency virus), select the most resistant representative virus (or acceptable surrogate) for testing

.
(3) Soil load

. If the product is intended to be represented as virucidal in the presence of organic soil (one

- step), an appropriate organic soil, such as 5 percent blood serum, should be included with the viral inoculum. When viral suspensions are grown in the presence of at least 5% serum, addition of serum to the inoculum is not expected as part of a study to support a one

- step label claim.

(4) Number of b atches

. Test two batches of product at the nominal concentration.

(5) Surfaces. For non

- surrogates, test one surface per batch; for surrogates, test two surfaces per batch

.
(6) Viral concentration

. Test each batch against a recoverable virus end point titer of ≥10 4 viable viral particles from the test surface for a specified exposure period

.
(7) Performance measure

. Following tr eatment of the test virus with the disinfectant product, the presence of remaining viable virus should then be assayed using a n appropriate virological technique (e.g., cytopathogenic effect, fluorescent antibody, plaque count, or animal response) . The protocol for the viral assay should provide the information identified below.

(i) The virus recovery ( control carrier counts ) should include a minimum of

14four determinations (i.e., four replications) per each dilution in the assay system (e.g., tissue culture , embryonated egg, animal infection, etc.)

.
(ii) Cytotoxicity control. The effect of the disinfectant (with addition of the neutralizer) on the viral assay system should include a minimum of four determinations (i.e., four replications) per each dilution. Note : Level of cytotoxicity may be determined in advance or for one test and is not required to be performed with each test.

(iii) The activity of the disinfectant (treated carriers) against the test virus should include a minimum of four determinations (i.e., four replications) per dilution in the assay system.

(iv) Neutralization control. N eutralization controls should be performed ( Ref. 6 ) and should include a minimum of four determinations (i.e., four replications) per each dilution. The neutralizer control may be determined in advance or for one test and is not required to be performed with each test.

(v) Any special methods (e.g., use of columns) which are used to increase the virus titer and to detoxify the residual disinfectant should be described in the protocol

.
(vi) The ID 50 values (control carrier counts) calculated for each assay should be provided.

(vii) The test results should be reported as the log reduction of the virus (ID 50 of the virus control carriers less the ID 50 of the tes t carriers) a s calculated by

a n appropriate statistical method (e.g., Reed and Munch, Most Probable Number , Spearman

- Karber )

.
(viii) The product should demonstrate complete inactivation of the virus at all dilutions.

(ix) If cytotoxicity is present

, the virus contro l titer should be i ncreased to demonstrate a ≥ 3 log 10 reduction in viral titer beyond the cytotoxic level.

(x) A laboratory report of a single test with one virus involving a tissue culture assay system should include the details of the methods employed ; see examples of information in Tables 2

- 1, 2

- 2 and 2

- 3 :

(A) Performance measure. The product should demonstrate complete inactivation of the virus at all dilutions. If cytotoxicity is present, a ≥ 3

- log reduction in viral titer should be demonstrated beyond the cytotoxic level recovered from the carrier surface.

15Table 2

- 1: Example of Virucidal Test Results for One Test

Dilution of Virus Virus

- Disinfectant* (treated carriers) Virus

- Control* (control carriers) Cytotoxicity

– Control (disinfectant and neutralizer without virus)
10

- 1 T T T T + + + + T T T T
10

- 2 T T T T + + + + T T T T
10

- 3 T 0 0 0 + + + + T 0 0 0
10

- 4 0 0 0 0 + + + + 0 0 0 0
10

- 5 0 0 0 0 + + + + 0 0 0 0
10

- 6 0 0 0 0 + + + 0 0 0 0 0
10

- 7 0 0 0 0 + 0 0 0 0 0 0 0
10

- 8 0 0 0 0 0 0 0 0 0 0 0 0 Note: T = toxic to cell line; + = virus recovered; 0 = no virus recovered *Recovery of virus from surfaces.

Table 2

- 2: Example Calculation (Spearman

- Karber) of the Tissue Culture Infective Dose 50 (TCID 50 )

– for the Control Counts Values Accumulated Values

Virus Dilution Inoculated No. Infected/ No. Inoculated No. Infected No. not Infected No. Infected No. not Infected No. Infected/ No. Inoc ulated % Infected

10

- 1 4/4 4 0 24 0 24/24 100
10

- 2 4/4 4 0 20 0 20/20 100
10

- 3 4/4 4 0 16 0 16/16 100
10

- 4 4/4 4 0 12 0 12/12 100
10

- 5 4/4 4 0 8 0 8/8 100
10

- 6 3/4 3 1 4 1 4/5 80
10

- 7 1/4 1 3 1 4 1/5 20
10

- 8 0/4 0 4 0 8 0/8 0
TCID 50 = 10 6.5 (6.5 logs)

16Table 2

- 3: Example Calculation (Spearman

- Karber) of the Tissue Culture Lethal Dose 50 (TCLD 50 )

– for the Treated Carriers
Values Accumulated Values
Virus Dilution Inoculated No. Toxic / No. Inoculated No. Toxic No. not Toxic No. Toxic No. not Toxic No. Toxic / No. Inoculated % Toxic

10

- 1 4/4 4 0 9 0 9/9 100
10

- 2 4/4 4 0 5 0 5/5 100
10

- 3 1/4 1 3 1 3 1/4 25
10

- 4 0/4 0 4 0 7 0/7 0
10

- 5 0/4 0 4 0 11 0/11 0
10

- 6 0/4 0 4 0 15 0/15 0
10

- 7 0/4 0 4 0 19 0/19 0
10

- 8 0/4 0 4 0 23 0/23 0
TCLD 50 = 10 2.7 Therefore: Virus inactivation = TCID 50 - TCLD 50 = 10 3.8 log 10 (a log reduction of 3.8)

(g) Tuberculocidal claims . Tuberculocidal claims may be added to broad

- spectrum or hospital disinfectant products. In the Agency’s “Data Call

- In Notice f or Tuberculocidal Effectiveness for All Antimicrobial Pesticides with Tuberculocidal Claims,” dated June 13, 1986 (Ref. 7), applicants were given the option of choosing from one of three test methods (AOAC Tuberculocidal Activity of Disinfectants test, a m odified AOAC Tuberculocidal Activity of Disinfectants test, or the Quantitative Tuberculocidal Activity Test) for conducting tuberculocidal efficacy tests. Based on experience since 1986, the Agency does not recommend the use of the Quantitative Tuberculoc idal Activity Test (a suspension test) for testing disinfectant formulations for use on hard surfaces. An exception to this is for glutaraldehyde

- based products, which have not been validated in the AOAC Tuberculocidal Activity of Disinfectants test (a car rier based test). Therefore, the Quantitative Tuberculocidal Activity Test should only be used for glutaraldehyde

- based products. The Agency strongly recommends for all other liquid formulations, use of the carrier

- based AOAC Tuberculocidal Activity of Dis infectants test. Refer to Table 2 for recommendations per formulation type

. Conduct the following:

(1) Test organism . To demonstrate efficacy, testing should be conducted against Mycobacterium bovis (BCG) ( M. bovis )

. Test cultures of M. bovis (BCG) may be grown using agitation; consult with EPA regarding the methodology for producing these cultures.

(2) Batches/number of carriers

. Test ten carriers against each of two different batches of product at the LCL.

(3) Control carrier counts for AOAC Tuberculocidal Activity of Disinfectants test and AOAC Tuberculocidal Activity of Disinfectants test with modifications

. Per the 2012 revisions for the AOAC Tuberculocidal Activity of Disinfectants test, the mean log density is to be at least 4.0 (corresponding to a geom etric mean density of 1.0 x 10 4 ) and not above 6.0 (corresponding to a geometric mean density of 1.0
17x 10 6 ). A mean log density < 4.0 or > 6.0 invalidates the test.

(4) Control carrier counts for AOAC Germicidal Spray Products as Disinfectants test and towele ttes

. The log density of M. bovis should be ≥ 4.0 (corresponding to a geometric mean density of ≥1.0 x 10 4 CFU/carrier) a mean log density of <4.0 invalidates the test.

(i) Evaluate the product against M. bovis BCG for the label recommended contact time.

(5) Performance Measure

. Mycobacterium on all carriers (in primary subculture medium) should be killed (no positive carriers) , and there should be no growth in any of the associated subculture media. For a valid test, no c ontamination of any carrier or seconda ry subculture medium is allowed.

(6) The spray and towelette test s should be modified to conform to appropriate elements (e.g., media, growth conditions, etc.) as in the AOAC Tuberculocidal Activity of Disinfectants test.

(7) Media Performance . The performance of the subculture media should be confirmed in advance or concurrently with testing. The results should be submitted with the product performance data.

(h) Glutaraldehyde formulations

(1) Test Procedure. For glutaraldehyde formulations, the Agency recommends the Quantitative Tuberculocidal Activity Test. This test has been referenced in the Agency’s “Data Call

- In Notice for Tuberculocidal Effectiveness for All Antimicrobial Pesticides with Tuberculocidal Claims,” dated June 13, 1986 ( Ref. 7 ). Two samples, representing two different batches of the product should each be uti lized in at least four separate studies (a total of at least eight studies), against M. bovis, so that upper 95 percent confidence lim its can be determined for each point on the survival curve. If the product is intended to be represented as tuberculocidal in the presence of organic soil (one

- step), an appropriate organic soil, such as 5 percent blood serum, should be included with the b acterial inoculum.

(2) Performance measure. For the Quantitative Tuberculocidal Activity Method, survival curves should be constructed from the average of four separate replicates so that the upper 95% Confidence Limit can be determined for each point on the

curve. The minimum time claimed for efficacy is determined by finding the point where the average survival curve intersects the probability of one survivor. If the data show a four

- log reduction, but the survivor curve does not intersect the one

survivor l ine, the minimal time is found by extrapolating the upper 95% confidence limit curve such that the value where it intersects the one survivor line is not 50%

18greater than when the survivor curve intersects the one survivor line. The test substance should d emonstrate > 1.0 x 10 4 CFU kill of the test organism at the stated contact time (i.e., a > 4 log 10 reduction of the test organism).

(3) The organism titer should be > 1 x 10 7 CFU/ml.

(i) Validation testing for Quaternary Ammonium Compounds. Products formulated solely with quaternary ammonium compounds as the active ingredient(s) should be supported with validation testing to confirm their tuberculocidal label claim. One additional product sample should be tested in a different laboratory fro m the original one, or in the same laboratory using different study director, technical staff and quality assurance unit, using the same test procedure and conditions as used in the first laboratory test.

(j) Bridging for EPA

- registered liquids and disinfecta nt towelettes

. In cases where an applicant formulates a disinfectant towelette using an existing EPA

- registered bulk liquid formulation, the following chemistry and efficacy studies should be conducted and submitted to EPA for review.

(1) Bridging: Chemical Analysis

.
(i) All active ingredients in the expressed liquid should evaluated for concentration and must be within the certified limits of the Confidential Statement of Formula of the liquid formula being referenced (bridged).

(ii) The disinfectant towelettes pa ckage should be filled according to the manufacturing specifications. Excess liquid in the bulk towelettes containers is not to be poured off for use in the chemical testing for data bridging, rather the liquid used in the chemical testing should only be t ha t expressed from the towelettes.

(iii) Two batches (expressed liquid) of the towelette should be tested.

(iv) Analytical data for the active ingredients in the expressed liquid should be submitted for review.

(v) T he means used to express the liquid should be includ ed in the study protocol.

(2) Bridging: E fficacy Testing . Efficacy testing should be conducted under the same testing conditions (e.g. soil load, contact time, temperature) on two or three batches (depending on claim) as used for the bulk liquid testing. A detailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. All testing requirements for controls, control carrier counts and pe rformance

19standards should be followed for each test method/microbe combination. For each type of claim, see specific details below:

(i) L imited, broad

- spectrum and h ospital disinfectant s . The Agency recommends the use of the AOAC Germicidal Spray Products as Disinfectants test modified for towelettes, using the test organisms (and control count requirements for a valid test) s pecified for the primary claim. Test sixty carriers against each of three batches of the product at the nominal concentration. The prod uct should kill the test microorganisms on 59 out of each set of 60 carriers/slides. This testing is intended to support bridging of all remaining vegetative bacteria listed (with the exception of the tuberculocidal claim) on the EPA registered liquid disinfectant used to saturate the towelette to the EPA registered towelette product.

(ii) Fungicidal claims . The Agency recommends the use of the AOAC Germicidal Spray Products as Disinfectants test modified for fungicidal towelette testing. The test should be modified to conform to appropriate elements (e.g., media, growth conditions) in the AOAC International Fungicidal Activity of Disinfectants test. Evaluate the product against T. mentagrophytes (ATCC 9533) for the label recommended contact time. Test ten carriers against each of two batches of product at the nominal concentration. The product should kill (no positive carriers) the test organism on all 10 carriers. This testing is intended to support bridging of all remaining fungi listed on the EPA regist ered liquid disinfectant used to saturate the towelette to the EPA registered towelette product.

(iii) Tuberculocidal Claims

. Bridging of data for towelettes is not allowed for tuberculocidal claims.

(iv) Virus Claims

. Based on the bulk liquid label, claims for sma ll non

enveloped, large non

- enveloped and enveloped viruses can be supported by selecting the most resistant virus on the existing label

– al l other vir us es on the label will be supported by this testing

. Test ten carriers for each of two batches of the product at the nominal concentration on one surface type. T he product should demonstrate complete inactivation of the virus at all dilutions. If cytotoxicity is present, a ≥ 3

- log reduction in viral titer should be demonstrated beyond the c ytotoxic level recovered from the carrier surface.

(k) Data collection and reporting

.

Refer to OCSPP 810.2000: General Considerations for Public Health Uses of Antimicrobial Agents for guidance. The applicant is encouraged to use the EPA’s standard efficac y report format, which may be found at http://www.epa.gov/oppad001/efficacystudystandards.htm

.

20(l) References

. The references in this paragraph may be consulted for addi tional background information :

(1) Official Methods of Analysis of the AOAC International , Chapter 6, Disinfectants, Use

- Dilution Methods (955.14, 955.15, & 964.02)

. Current edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417. \

(2) Off icial Methods of Analysis of the AOAC International, Chapter 6, Disinfectants, Official Method 961.02 Germicidal Spray Products as Disinfectants

. Current edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417.

(3) Standard Practice for Evaluation of Pre

- saturated or Impregnated Towelettes for Hard Surface Disinfection

, ASTM Designation E2362 . Current edition

. ASTM International , 100 Barr Harbor Drive, West Conshohocken, PA 19428

.
(4) Official Methods of Analysis of the AOAC International , Chapter 6, Disinfectants, Official Method 955.17 Fungicidal Activity of Disinfectants

. Current edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417.

(5) Test Method for Efficacy of Virucidal Agen ts Intended for Inanimate Environmental Surfaces, ASTM Designation E1053. , Current edition. ASTM International , 100 Barr Harbor Drive, West Conshohocken, PA 19428.

(6) Standard Test Method for Neutralization of Virucidal Agents in Virucidal Efficacy Evaluati ons, ASTM Designation E1483. Current edition. ASTM International , 100 Barr Harbor Drive, West Conshohocken, PA 19428.

(7) Environmental Protection Agency, Data Call

- in Notice for Tuberculocidal Effectiveness Data for All Antimicrobial Pesticides with Tubercul ocidal Claims (Registration Division, Office of Pesticide Pro grams, June 13, 1986).

(8) Official Methods of Analysis of the AOAC International , Chapter 6, Disinfectants, Official Method 965.12 Tuberculocidal Activity of Dis infectants. Current edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417.

(9) Ascenzi, J.M., et al., A More Accurate Method for Measurement of Tuberculocidal Activity of Disinfectants. Applied Environmental Microbiology , Vol. 53, No. 9, 1987, pp. 218 9

- 2192. (Suspension

- based assay).

21