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 - Efficacy 

Data Recommendations
NOTICE

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 (OPPTS). To 

distinguish 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 acronym (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 provides 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 

outside 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 propose 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, please 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-OPPT2009-0150 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 policy. 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 providing 

comments. 

2
OCSPP 810.2200: Disinfectants for use on hard surfaces - efficacy data recommendations

(a) Scope.

(1) Applicability. 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 developing 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 disinfectants, 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 Tables 1 and 2 for information on efficacy test methods

recommended to support disinfectant and additional label claims, respectively. The 

Agency recommends the use of the most recently published version of the standard 

methods (e.g., AOAC International [AOAC], ASTM). See standard methods for 

control carrier counts (mean number of viable microbes per carrier) and other 

method associated information. Agency Standard Operating Procedures for many 

of the test procedures referenced in the tables 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 claim.

(3) Spray products. Spray products referenced in Table 1 include aerosol, trigger, and

3
pump dispensers. 

(4) Towelette products. For products formulated as impregnated wipes or towelettes, 

refer to Table 1. For evaluation of efficacy, the Agency recommends the 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 sterile 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 document on bridging for cases where an applicant formulates a 

disinfectant towelette using an existing EPA-registered bulk liquid formulation. 

Note: 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 conducted 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 neutralizer 

should be conducted in advance or concurrently with the efficacy test. The 

procedure should include conditions relevant to the efficacy test (e.g., growth 

medium, 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 information related to confirmatory 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 disinfectants are identical to 

those specified by the basic manufacturer.

(9) Additional microorganisms. For efficacy testing requirements related to broadspectrum or hospital disinfectants which bear label claims against bacteria other 

4
than Salmonella enterica (ATCC10708), Staphylococcus aureus (ATCC 6538), or 

Pseudomonas aeruginosa (ATCC 15442), see section (d)(6).

(10) Contact Time. All products should meet the performance standard associated with 

the method and microbe at ≤10 minutes of contact. 

5
Table 1. Summary of Testing for Disinfectant Claims

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

6
Table 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 

mentagrophytes 

(ATCC 9533)

Two batches at the LCL; 10 

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.

Internal toilet 

and urinal bowl 

surfaces above 

and below the 

water line

Water-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 broadspectrum 

disinfectant 

products; S. aureus

and P. aeruginosa

(ATCC 15442) for 

Test sixty carriers against 

each of three batches of the 

product at the LCL

7
hospital 

disinfectants

(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 methods. Conduct 

the following:

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

against Salmonella enterica (S. enterica), formerly designated 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 Methods (ref. 1). For 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 105

) and 

not above 6.0 (corresponding to a geometric mean density of 1.0 x 106

). 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 106) and not above 7.0 (corresponding to a geometric mean density of 

1.0 x 107). A mean log density <6.0 or >7.0 invalidates the test. 

(iv) Control carrier counts for spray products and single-use towelettes.

The mean log density for S. enterica is to be at least 4.0 (corresponding to 

a geometric mean density of 1.0 x 104) and not above 5.5 (corresponding to 

a geometric mean density of 3.2 x 105). 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 105) and not above 6.5 

(corresponding to a geometric mean density of 3.2 x 106). 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 performance 

standard for S. enterica is 0-1 positive carriers out of sixty.

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

8
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 microorganisms 

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 limited spectrum products. For confirmatory 

testing, follow the above testing guidance in sections (c) and (d)(1), with 

the exception that ten carriers for 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 met. For a 

valid test, no 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 methods. 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, test sixty carriers against 

each of three batches of the product at the LCL.

(iii) Control carrier counts for AOAC Use-Dilution Methods. For 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 105

) and not above 

6.0 (corresponding to a geometric mean density of 1.0 x 106

). 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 106

) 

and not above 7.0 (corresponding to a geometric mean density of 1.0 x 107). 

A mean log density <6.0 or >7.0 invalidates the test. 

(iv) Control carrier counts for spray products and towelettes. The mean log 

density for S. enterica is to be at least 4.0 (corresponding to a geometric 

mean density of 1.0 x 104) and not above 5.5 (corresponding to a geometric 

mean density of 3.2 x 105). 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 105) and not above 6.5 (corresponding 

to a geometric mean density of 3.2 x 106). 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) 

against 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 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; 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), 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 S. enterica (ATCC 10708). For all methods, the product should kill all 

the test microorganisms on all carriers. Control carrier counts specifications 

should be met. For a valid test, no 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 Pseudomonas aeruginosa (ATCC

15442).

(ii) Batches/number of carriers. For each organism, test sixty carriers against 

each of three batches of the product at the LCL.

(iii) Control carrier counts for AOAC Use-Dilution Methods. For the UseDilution 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 106

) 

and not above 7.0 (corresponding to a geometric mean density of 1.0 x 107). 

A mean log density <6.0 or >7.0 invalidates the test. 

(iv) Control carrier counts for spray products and towelettes. The 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 105

) and not above 6.5 

10
(corresponding to a geometric mean density of 3.2 x 106

). 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 microorganisms 

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. 

Control carrier counts specifications should be met. For a valid test, no 

contamination of any carrier is allowed.

(4) Disinfectants for internal toilet and urinal bowl surfaces above and below the 

water line. To add claims to existing limited, broad-spectrum, or hospital products

for internal toilet and urinal bowl surfaces, conduct the following:

 

(i) Test method. Regarding water-soluble powders and non-volatile liquid 

products test procedure, the Agency recommends the use of the AOAC UseDilution Methods modified to include a 5% organic soil challenge added to 

the bacterial inoculum.

(A) The contained bowl water (96 fl oz of 200-400 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. aeruginosa

(ATCC 15442), for hospital disinfectant products. Follow the testing 

recommendations (i.e., control carrier counts, and performance measure) 

specified for limited, broad-spectrum, and hospital disinfectant claims.

(iii) Batches/number of carriers. Test sixty 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 provided 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 should be a mean of 1 x 104 CFU/carrier. 

(iv) Performance measure. The product should kill all the test microorganisms 

on all ten carriers. For a valid test, no contamination of any carrier is 

allowed. 

(e) Fungicidal 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 Test. Two batches of 

product at the LCL should be evaluated for efficacy at 5, 10, and 15 minute 

12
exposure times. The method is a suspension test.

(ii) For AOAC Use-Dilution Method, AOAC Germicidal Spray Products 

as Disinfectants Test, and towelettes. Tests may be modified 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) AOAC Fungicidal Activity of Disinfectants test. The inoculum employed 

should provide a concentration of ≥ 5 x 106 conidia/mL.

(ii) AOAC Use-Dilution Method, AOAC Germicidal Spray Products as 

Disinfectants Test, and single-use towelettes. The inoculum employed 

should provide a concentration of 1 x 104 – 1 x 105 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) Virucidal 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 most resistant virus on the label based on 

disinfection hierarchy. The categories and the presumed levels of resistance are 

provided below:

13
(i) Small non-enveloped viruses (most resistant viruses). For label 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 viruses 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) Enveloped viruses (least resistant viruses). For label claims to inactivate 

enveloped 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), Orthomyxoviridae 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 batches. 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 

≥104 viable viral particles from the test surface for a specified exposure period.

(7) Performance measure. Following treatment of the test virus with the disinfectant 

product, the presence of remaining viable virus should then be assayed using an 

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 

14
four 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. Neutralization 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 ID50 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 (ID50 of 

the virus control carriers less the ID50 of the test carriers) as calculated by 

an 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 control titer should be increased to 

demonstrate a ≥3 log10 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.

15
Table 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 (TCID50) – 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. 

Inoculated

% 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

TCID50 = 106.5 (6.5 logs)

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Table 2-3: Example Calculation (Spearman-Karber) of the Tissue Culture Lethal Dose 50 

(TCLD50) – 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

TCLD50 = 102.7 Therefore: Virus inactivation = TCID50 - TCLD50 = 103.8 log10 (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 for 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 modified 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 Tuberculocidal 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 carrier 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 Disinfectants 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 geometric mean density 

of 1.0 x 104

) and not above 6.0 (corresponding to a geometric mean density of 1.0 

17
x 106

). 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 towelettes. The log density of M. bovis should be ≥ 4.0 (corresponding to 

a geometric mean density of ≥1.0 x 104 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 contamination of any 

carrier or secondary subculture medium is allowed.

(6) The spray and towelette tests 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 

utilized in at least four separate studies (a total of at least eight studies), against M.

bovis, so that upper 95 percent confidence limits 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 bacterial 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 onesurvivor line, 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% 

18
greater than when the survivor curve intersects the one survivor line. The test 

substance should demonstrate >1.0 x 104 CFU kill of the test organism at the stated 

contact time (i.e., a >4 log10 reduction of the test organism). 

(3) The organism titer should be > 1 x 107 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 from 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 disinfectant 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 package 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 that 

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) The means used to express the liquid should be included in the study 

protocol.

(2) Bridging: Efficacy 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 performance 

19
standards should be followed for each test method/microbe combination. For each 

type of claim, see specific details below:

(i) Limited, broad-spectrum and hospital disinfectants. 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) specified for the primary claim. 

Test sixty carriers against each of three batches of the product at the 

nominal concentration. The product 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 registered 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 small nonenveloped, large non-enveloped and enveloped viruses can be supported by 

selecting the most resistant virus on the existing label– all other viruses 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. 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.

(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 

efficacy 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 additional 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) Official 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 Agents 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 

Evaluations, 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 Tuberculocidal Claims 

(Registration Division, Office of Pesticide Programs, June 13, 1986).

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

Official Method 965.12 Tuberculocidal Activity of Disinfectants. 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. 2189-2192. (Suspension-based assay).

21