United States

Environmental Protection

Agency

Office of Chemical Safety and 

Pollution Prevention

(7510P)

EPA 712-C-07-074

September 4, 2012

Product Performance 

Test Guidelines

OCSPP 810.2200:

Disinfectants for Use on 

Hard Surfaces—Efficacy 

Data Recommendations
2

NOTICE

This guideline is one of a series of test guidelines established by the Office of 

Chemical Safety and Pollution Prevention (OCSPP) (formerly the Office of 

Prevention, Pesticides and Toxic Substances (OPPTS) prior to April 22, 2010), 

United States Environmental Protection Agency 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), referred to 

hereinafter as the harmonized test guidelines.

The harmonized test guidelines serve as a compendium of accepted scientific 

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 appropriate tests, and is also used by EPA, the public, and the 

companies that are subject to data submission requirements under TSCA, FIFRA 

and/or the FFDCA. 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. 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. The methods 

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 methods recommended in 

these guidelines, with your supporting rationale. The Agency will assess such 

proposals on a case-by-case basis. 

For additional information about OCSPP harmonized test guidelines and to 

access the guidelines electronically, please go to http://www.epa.gov/ocspp and select 

“Test Methods & Guidelines” on the left side navigation menu. 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-HQOPPT-2009-0576.
1

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. 

(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) This guideline recommends methods for use in tests to be conducted to address the

data requirements for pesticide registration. Good Laboratory Practice Standards (GLP) as 

defined in 40 CFR Part 160 apply to studies to support disinfection on hard, non-porous surfaces. 

According to 40 CFR §160.17: “EPA may refuse to consider reliable for purposes of supporting 

an application for a research or marketing permit any data from a study which was not conducted 

in accordance with this part.” 40 CFR §160.12 (b) requires with any submitted research data “[a] 

A statement that the study was conducted in accordance with this part; [b] A statement 

describing in detail all differences between the practices used in the study and those required by

this part; or [c] A statement that the person was not a sponsor of the study, did not conduct the 

study, and does not know whether the study was conducted in accordance with this part.” Note: 

The Association of Official Analytical Chemicals (AOAC) recommended tests are designed to 

be conducted as written. For deviations (e.g., cultures grown with shaking instead of static, 

dilution of culture prior to drying on carriers) proposed to be used in the conduct of these tests, 

obtain written approval from the Agency and document such deviations in the study reports 

submitted to the Agency. The Agency may consult with the AOAC prior to accepting 

modifications to their standardized methods. Refer to OCSPP Test Guideline 810.2000 for 

general testing recommendations prior to initiating tests.

(2) Confirmatory testing. In certain situations an applicant may rely on previously 

submitted efficacy data to support an application or amendment for registration of a product and 

submit only confirmatory efficacy data on his own product to demonstrate his ability to produce 
2

an effective formulation. These situations are as listed in paragraphs (C)(3)(i) through (C)(3)(iii) 

of this guideline:

(i) Duplicated Product Formulations. In this situation, the applicant manufactures a 

formulation which duplicates a product that is already registered with complete supporting 

efficacy data. The chemical composition, manufacturing procedure, label claims, and directions 

for use are identical in substance to those of the original registration, and specific references 

(Master Record ID Numbers [MRID]) to the supporting data developed for the original product 

are cited by the applicant.

(ii) Minor Formulation Change in a Registered Product. In this situation, the change in 

the formulation is relatively minor, e.g., a change of an inert ingredient. The label claims and 

directions for use are unchanged from those accepted for the registered formulation, and specific 

references (MRID) to the supporting data developed for the original formulation are cited by the 

applicant. If the only change in the formulation is the addition of a fragrance or dye, 

confirmatory data do not need to be submitted. However, when the product is an aerosol 

formulation, confirmatory data should be submitted for all formulation changes, including the 

addition of fragrances and dyes.

(iii) The confirmatory data are to be developed from testing the applicant's own finished 

product. When the test methodology utilized in deriving the original supporting efficacy data 

were modified to include additional elements not specified in the recommended method, such as 

organic soil, hard water, longer or shorter contact time, etc., the confirmatory data should be 

produced under similarly modified conditions.

(4) Efficacy claims. Table 1 provides a quick reference guide to testing for basic claims 

described in this guideline. Consult the text for detailed testing descriptions.

Table 1. Testing for basic efficacy claims

Level of 

Efficacy

Test Methods Test 

Organisms

No. of 

Batches/Carriers

Evaluation 

of Success

Limited spectrum 

disinfectant/hard 

non-porous 

surfaces.

Water soluble 

powders/liquids

AOAC UseDilution 

Method or

AOAC Hard 

Surface 

Carrier Test 

(distilled 

water only)

Staphylococcus 

aureus (ATCC 

6538) or Salmonella 

enterica (ATCC 

10708)

Three batches, one at least 

60 days old. 60 carriers 

against either organism 

claimed (180 carriers).

59/60 carriers are 

negative for each 

batch tested for all 

methods except 

AOAC Hard 

Surface Carrier 

Test, which is 

58/60 carriers are 

negative for each 

batch.

Spray products AOAC 

Germicidal 

Spray 

Products Test

Towelettes Modified

AOAC

Germicidal 

Spray 

Products Test

Broad-spectrum 

disinfectant/hard 

Water soluble 

powders/liquids

AOAC UseDilution Staphylococcus 

aureus (ATCC 

Three batches, one at least 

60 days old. 60 carriers 

59/60 carriers are 

negative for each 
3

Level of 

Efficacy

Test Methods Test 

Organisms

No. of 

Batches/Carriers

Evaluation 

of Success

non-porous 

surfaces.

Method or

AOAC Hard 

Surface 

Carrier Test 

(distilled water 

only)

6538)and 

Salmonella enterica

(ATCC 10708)

against each organism (360 

carriers).

batch tested for all 

methods except 

AOAC Hard 

Surface Carrier 

Test, which is 

58/60 carriers are 

negative for each 

batch.

Spray products AOAC 

Germicidal 

Spray 

Products Test

Towelettes Modified 

Germicidal 

Spray Test

Hospital or 

healthcare 

disinfectant/hard 

non-porous 

surfaces.

Water soluble 

powders/liquids

AOAC UseDilution 

Method or

AOAC Hard 

Surface 

Carrier Test 

(distilled water 

only)

Staphylococcus 

aureus (ATCC 

6538)and 

Pseudomonas 

aeruginosa (ATCC 

15442)

Three batches, one at least 

60 days old. 60 carriers 

against each organism (360 

carriers).

59/60 carriers are 

negative for each 

batch tested for all 

methods except 

AOAC Hard 

Surface Carrier 

Test, which is 

58/60 carriers are 

negative against 

Staphylococcus 

aureus for each 

batch, and 57/60 

carriers are 

negative against 

Pseudomonas 

aeruginosa.

Spray products AOAC 

Germicidal 

Spray 

Products Test

Towelettes Modified 

Germicidal 

Spray Test

Fungicidal 

disinfectant/hard 

non-porous 

surfaces.

Water soluble 

powders/liquids

AOAC UseDilution Test 

modified for 

fungi or

AOAC 

Fungicidal 

Test

Trichophyton 

mentagrophytes 

(ATCC 9533)

Two batches, ten carriers 

per batch for the modified 

AOAC Use Dilution Test, 

the modified AOAC 

Germicidal Spray Products 

Test, and the EPA 

Towelette Test. Two 

batches for the AOAC 

Fungicidal Test.

All fungal spores 

on all carriers 

should be killed. 

For the AOAC 

Fungicidal Test, 

all fungal spores 

should be killed at 

10 and 15 minutes 

to support a 10 

minute label 

claim.

Spray products AOAC 

Germicidal 

Spray 

Products Test 

modified for 

fungi

Towelettes Modified 

Germicidal 

Spray Test

Virucidal 

disinfectant/hard 

non-porous 

surfaces.

Water soluble 

powders/liquids

AOAC UseDilution Test 

modified for 

viruses or

ASTM E1053-

Virus claimed on 

the label or 

approved surrogate.

Two batches. One surface 

per batch.

Complete 

inactivation of the 

virus. Where 

cytotoxicity is 

present, 

demonstrate a 3

log10 reduction.

Spray products AOAC 

Germicidal 

Spray 

Products Test 

modified for 

viruses or 

ASTM E1053-

Towelettes Modified 

Germicidal 

Spray Test

Tuberculocidal 

disinfectant/hard 

non-porous 

Water soluble 

powders/liquids

AOAC 

Tuberculocidal 

Activity of 

Two batches, ten carriers 

per batch. 

10/10 carriers are 

negative for 

growth and there 
4

Level of 

Efficacy

Test Methods Test 

Organisms

No. of 

Batches/Carriers

Evaluation 

of Success

surfaces. Disinfectants, 

Quantitative 

Tuberculocidal 

Activity Test Mycobacterium 

bovis BCG

Two batches, 4 replicates 

per batch.

Two batches, ten carriers 

per batch.

Two batches, ten carriers 

per batch.

is no growth in the 

additional test 

media. 

Survival Curve 

constructed from 

4 separate 

replicates at the 

95% confidence 

level to show 

probability of one 

survivor.

Spray products AOAC 

Germicidal 

Spray 

Products Test 

modified for 

tuberculocidal 

activity

10/10 carriers are 

negative for 

growth and there 

is no growth in the 

additional test 

media. 

Towelettes Modified 

Germicidal 

Spray Test

10/10 carriers are 

negative for 

growth and there 

is no growth in the 

additional test 

media. 

Additional 

bacteria/hard nonporous surfaces.

Water soluble 

powders/liquids

AOAC UseDilution Test 

or

AOAC Hard 

Surface 

Carrier Test 

(distilled water 

only)

Bacteria claimed on 

the label in addition 

to the base broadspectrum claim.

Two batches, ten carriers 

for each batch.

10/10 carriers are 

negative for 

growth of the test 

organism.

Spray products AOAC 

Germicidal 

Spray 

Products Test

(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). 

(i) Water-soluble powders and non-volatile liquid products test procedure. The Agency 

recommends the use of the AOAC International Use-Dilution Methods (Ref. 1) or the AOAC 

International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2). Sixty carriers for 

each of three samples, representing three different batches, one of which should be ≥60 days old, 

should be tested against Salmonella enterica (S. enterica)(formerly designated as Salmonella 

choleraesuis)(American Type Culture Collection)(ATCC 10708) for effectiveness against Gramnegative bacteria, or Staphylococcus aureus (S. aureus)(ATCC 6538) for effectiveness against 

Gram-positive bacteria. If the product is intended to be represented as bactericidal 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.
5

(ii) Germicidal spray products (aerosol or pump) and volatile liquid products test 

procedure. The Agency recommends use of the AOAC International Germicidal Spray Products 

as Disinfectants test (Ref. 3). Sixty carriers for each of three samples, representing three different 

batches, one of which should be ≥60 days old, should be tested against S. enterica (ATCC 

10708) for effectiveness against Gram-negative bacteria, or S. aureus (ATCC 6538) for 

effectiveness against Gram-positive bacteria. If the product is intended to be represented as 

bactericidal 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.

(iii) Single-use towelettes test procedure. The Agency recommends the use of a modified 

AOAC Germicidal Spray Products as Disinfectants test (Ref. 3) or ASTM E2362 (Ref 4). Sixty

carriers for each of three samples, representing three different batches, one of which should be 

≥60 days old, should be tested against S. enterica (ATCC 10708) for effectiveness against Gramnegative bacteria, or S. aureus (ATCC 6538) for effectiveness against Gram-positive bacteria. If 

the product is intended to be represented as bactericidal in the presence of organic soil (onestep), an appropriate organic soil, such as 5 percent blood serum, should be included with the 

bacterial inoculum. 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 subculturing 

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 a minimum of 10 

inoculated carriers for a total of 6 towelettes for all 60 carriers. Alternatively, one carrier with a 

surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set 

per batch. The area of the towelette used for wiping should be rotated so as to expose a 

maximum amount of its surface in the course of wiping a set of carriers. 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. 

(iv) Evaluation of limited disinfectant success. For the AOAC International Use-Dilution 

Methods, the Germicidal Spray Products as Disinfectants test, and single-use towelettes, the 

product should kill the test microorganisms on 59 out of each set of 60 carriers/slides in≤ ten 

minutes. In addition, per the 2009 AOAC revisions for the Use-Dilution Method, 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); a mean log density <6.0 invalidates the test. For the AOAC International Hard Surface 

Carrier Test Methods, the product should kill the test microorganisms on 58 out of each set of 60 

carriers for S. enterica or S. aureus in ≤ten minutes. For the Hard Surface Carrier Test, the dried 

carrier counts should be 0.5 – 2.0 x 106for Salmonella enterica and 1 – 5 x 106for 

Staphylococcus aureus.

(2) Confirmatory testing for limited spectrum products

(i) Water-soluble powders and non-volatile liquid products test procedure. The Agency 

recommends the use of the AOAC International Use-Dilution Methods (Ref.1) or the AOAC 

International Hard Surface Carrier Test Methods (distilled water only) (Ref. 2). Ten carriers for 

each of two product samples, representing two different batches of the product, should be tested 
6

against either S. aureus (ATCC 6538) or S. enterica (ATCC 10708)(depending on whether the 

product is claimed to be effective against Gram-positive or Gram-negative bacteria). If the 

product is intended to be represented as bactericidal 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.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid products test

procedure. The Agency recommends the AOAC International Germicidal Spray Products as 

Disinfectants test (Ref. 3). Ten carriers for each of two product samples, representing two 

different batches of the product, should be tested against either S. aureus (ATCC 6538) or S. 

enterica (ATCC 10708)(depending on whether the product is claimed to be effective against 

Gram-positive or Gram-negative bacteria). If the product is intended to be represented as 

bactericidal 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. 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.

(iii) Single-use towelettes test procedure. The Agency recommends the use of a modified 

AOAC Germicidal Spray Products as Disinfectants test (Ref. 3) or ASTM E2362 (Ref 4). Ten

carriers for each of two samples, representing two different batches, should be tested against S.

enterica (ATCC 10708) for effectiveness against Gram-negative bacteria, or S. aureus (ATCC 

6538) for effectiveness against Gram-positive bacteria. If the product is intended to be 

represented as bactericidal 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. 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 subculturing 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 a minimum of 10 inoculated carriers for a total of 1 

towelette for all 10 carriers. Alternatively, one carrier with a surface area equivalent to ten 1 x 1

inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette 

used for wiping should be rotated so as to expose a maximum amount of its surface in the course 

of wiping a set of carriers. 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. 

(iv) Evaluation of confirmatory limited disinfectant success. The product should kill all 

the test microorganisms on all carriers in ≤ten minutes. In addition, per the 2009 AOAC 

revisions for the Use-Dilution Method, 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); a mean log density <6.0 invalidates the 

test. For the Hard Surface Carrier Test, the dried carrier counts should be 0.5 – 2.0 x 106for 

Salmonella enterica and 1 – 5 x 106for Staphylococcus aureus. 

(3) General or broad spectrum efficacy products. When a disinfectant is represented in 
7

labeling as having efficacy against both Gram-negative and Gram-positive bacteria, the product 

is considered a general or broad spectrum disinfectant.

(i) Water-soluble powders and non-volatile liquid products test procedure. The Agency 

recommends the use of the AOAC International Use-Dilution Methods (Ref. 1) or the AOAC 

International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2). Sixty carriers for 

each of three samples, representing three different batches, one of which should be ≥60 days old, 

should be tested against both S. enterica (ATCC 10708) and S. aureus (ATCC 6538). If the 

product is intended to be represented as bactericidal 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.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid products test 

procedure. The Agency recommends the AOAC International Germicidal Spray Products as 

Disinfectants test (Ref. 3). Sixty carriers for each of three samples, representing three different 

batches, one of which should be ≥60 days old, should be tested against both S. enterica (ATCC 

10708) and S. aureus (ATCC 6538). If the product is intended to be represented as bactericidal 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. 

(iii) Single-use towelettes test procedure. The Agency recommends the use of a modified 

AOAC Germicidal Spray Products as Disinfectants test (Ref. 3) or ASTM E2362 (Ref 4). Sixty

carriers for each of three samples, representing three different batches, one of which should be 

≥60 days old, should be tested against S. enterica (ATCC 10708) for effectiveness against Gramnegative bacteria, and S. aureus (ATCC 6538) for effectiveness against Gram-positive bacteria. 

If the product is intended to be represented as bactericidal in the presence of organic soil (onestep), an appropriate organic soil, such as 5 percent blood serum, should be included with the 

bacterial inoculum. 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 subculturing 

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 a minimum of 10 

inoculated carriers for a total of 6 towelettes for all 60 carriers. Alternatively, one carrier with a 

surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set 

per batch. The area of the towelette used for wiping should be rotated so as to expose a 

maximum amount of its surface in the course of wiping a set of carriers. 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. 

 

(iv) Evaluation of general or broad spectrum disinfectant success. For the AOAC 

International Use-Dilution Methods, the Germicidal Spray Products as Disinfectants test, and 

single-use towelettes, the product should kill the test microorganisms on 59 out of each set of 60 

carriers/slides in ≤ten minutes. In addition, per the 2009 AOAC revisions for the Use-Dilution 

Method, 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); a mean log density <6.0 invalidates the test. For the AOAC 
8

International Hard Surface Carrier Test Methods, the product should kill the test microorganisms 

on 58 out of each set of 60 carriers in ≤ten minutes. For the Hard Surface Carrier Test, the dried 

carrier counts should be 0.5 – 2.0 x 106for Salmonella enterica and 1 – 5 x 106for 

Staphylococcus aureus.

(4) Confirmatory testing for general or broad spectrum products

(i) Water-soluble powders and non-volatile liquid products test procedure. The Agency 

recommends the use of the AOAC International Use-Dilution Methods (Ref. 1) or the AOAC 

International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2). Ten carriers for 

each of two product samples, representing two different batches of the product, should be tested 

against both S. aureus (ATCC 6538) and S. enterica (ATCC 10708). If the product is intended to 

be represented as bactericidal 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.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid products test 

procedure. The Agency recommends the AOAC International Germicidal Spray Products as 

Disinfectants test (Ref. 3). Ten carriers for each of two product samples, representing two 

different batches of the product, should be tested against both S. aureus (ATCC 6538) and S. 

enterica (ATCC 10708). If the product is intended to be represented as bactericidal 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. 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.

(iii) Single-use towelettes test procedure. The Agency recommends the use of a modified 

AOAC Germicidal Spray Products as Disinfectants test (Ref. 3) or ASTM E2362 (Ref 4). Ten

carriers for each of two samples, representing two different batches, should be tested against both 

S. enterica (ATCC 10708) and S. aureus (ATCC 6538). If the product is intended to be 

represented as bactericidal 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. 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 subculturing 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 a minimum of 10 inoculated carriers for a total of 1 

towelette for all 10 carriers. Alternatively, one carrier with a surface area equivalent to ten 1 x 1

inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette 

used for wiping should be rotated so as to expose a maximum amount of its surface in the course 

of wiping a set of carriers. 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. 

(iv) Evaluation of confirmatory general or broad spectrum disinfectant success. The 

product should kill all the test microorganisms on all carriers in ≤ten minutes. In addition, per the 
9

2009 AOAC revisions for the Use-Dilution Method, 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); a mean log density <6.0 

invalidates the test. For the Hard Surface Carrier Test, the dried carrier counts should be 0.5 –

2.0 x 106for Salmonella enterica and 1 – 5 x 106for Staphylococcus aureus.

(5) 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.

(i) Water-soluble powders and non-volatile liquid product test procedure. The Agency 

recommends the use of the AOAC International Use-Dilution Methods (Ref. 1) or the AOAC 

International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2). Sixty carriers for 

each of three samples, representing three different batches, one of which should be ≥60 days old, 

should be tested against S. aureus (ATCC 6538), and Pseudomonas aeruginosa (P. 

aeruginosa)(ATCC 15442). If the product is intended to be represented as bactericidal 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.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid products test 

procedure. The Agency recommends the use of the AOAC International Germicidal Spray 

Products as Disinfectants test (Ref. 3). Sixty carriers for each of three samples, representing three 

different batches, one of which should be ≥60 days old, should be tested against: S. aureus 

(ATCC 6538), and P. aeruginosa (ATCC 15442). If the product is intended to be represented as 

bactericidal 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.

(iii) Single-use towelettes test procedure. The Agency recommends the use of a modified 

AOAC Germicidal Spray Products as Disinfectants test (Ref. 3) or ASTM E2362 (Ref. 4). Sixty

carriers for each of three samples, representing three different batches, one of which should be 

≥60 days old, should be tested against S. aureus (ATCC 6538), and P. aeruginosa (ATCC 

15442). If the product is intended to be represented as bactericidal 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. 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 

subculturing 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 a minimum 

of 10 inoculated carriers for a total of 6 towelettes for all 60 carriers. Alternatively, one carrier 

with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per 

carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a 

maximum amount of its surface in the course of wiping a set of carriers. 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. 

 

(iv) Evaluation of hospital or healthcare disinfectant success. For the AOAC International 
10

Use-Dilution Methods, the Germicidal Spray Products as Disinfectants test, and single-use 

towelettes, the product should kill the test microorganisms on 59 out of each set of 60 carriers in

≤ten minutes. In addition, per the 2009 AOAC revisions for the Use-Dilution Method, 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); a mean log density <6.0 invalidates the test. For the AOAC 

International Hard Surface Carrier Test Methods, the product should kill the test microorganisms 

on 58 out of each set of 60 carriers for S. aureus, and 57 out of each set of 60 carriers for P. 

aeruginosa within ten minutes or less. For the Hard Surface Carrier Test, the dried carrier counts 

should be 1 – 5 x 106for both Staphylococcus aureus and Pseudomonas aeruginosa.

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

(i) Water-soluble powders and non-volatile liquid products test procedure.The Agency 

recommends the use of the AOAC International Use-Dilution Methods (Ref. 1) or the AOAC 

International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2). Ten carriers for 

each of two product samples, representing two different batches of the product, should be tested 

against S. aureus (ATCC 6538) P. aeruginosa (ATCC 15442). If the product is intended to be 

represented as bactericidal 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.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid products test 

procedure.The Agency recommends the AOAC International Germicidal Spray Products as 

Disinfectants test (Ref. 3). Ten carriers for each of two product samples, representing two 

different batches of the product, should be tested against S. aureus (ATCC 6538) and P. 

aeruginosa (ATCC 15442). If the product is intended to be represented as bactericidal 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. 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.

(iii) Single-use towelettes test procedure. The Agency recommends the use of a modified 

AOAC Germicidal Spray Products as Disinfectants test (Ref. 3) or ASTM E2362 (Ref. 4). Ten 

carriers for each of two product samples, representing two different batches of the product, 

should be tested against S. aureus (ATCC 6538) and P. aeruginosa (ATCC 15442). If the 

product is intended to be represented as bactericidal 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. 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 subculturing 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 a minimum of 10 inoculated 

carriers for a total of 1 towelette for all 10 carriers. Alternatively, one carrier with a surface area 

equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. 

The area of the towelette used for wiping should be rotated so as to expose a maximum amount 

of its surface in the course of wiping a set of carriers. Note: A detailed description of the wiping 
11

procedure, including the towelette folding and rotation process should be included in the test 

protocol and documented in the raw data and final report. 

 

(iv) Evaluation of confirmatory hospital or healthcare disinfectant success. The product 

should kill all the test microorganisms on all carriers in ≤ten minutes. In addition, per the 2009 

AOAC revisions for the Use-Dilution Method, 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); a mean 

log density <6.0 invalidates the test. For the Hard Surface Carrier Test, the dried carrier counts 

should be 1 – 5 x 106for both Staphylococcus aureus and Pseudomonas aeruginosa.

(7) Bridging for disinfectant towelettes. In some cases, disinfectant towelette 

formulations are identical to registered liquid formulations. In order to bridge efficacy data from 

the EPA registered bulk liquid disinfectant used to saturate a towelette or other related product 

form, the studies in paragraphs (d)(7)(i) and (d)(7)(ii) of this guideline should be conducted and 

submitted to EPA for review.

(i) Chemical Testing - Comparison of Expressed Liquid from the Towelette(s) to the EPA 

Registered Liquid Disinfectant Formulation to which it is being bridged: All active ingredients in 

the expressed liquid should be within the certified limits of the Confidential Statement of 

Formula of the liquid formula being referenced/bridged. The disinfectant towelettes package 

should be filled according to the manufacturing specifications. Excess liquid in the bulk 

towelette containers cannot be poured off for use in the chemical testing for bridging of the 

efficacy data. The liquid used in the chemical testing should only be that expressed from the 

towelettes. Three batches (one of which is 60 days old) should be tested. Analytical data for the 

active ingredients in the expressed liquid should be submitted for review.

(ii) Efficacy Testing - Efficacy testing should be conducted under the same testing 

conditions (e.g. soil load, contact time, temperature) as used for the bulk liquid testing. 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. For

limited disinfectants, broad-spectrum disinfectants, and hospital disinfectants, to bridge bacterial 

disinfection claims:

(A) Test Procedure. The Agency recommends the use of the AOAC Germicidal Spray 

Products as Disinfectants test modified for towelettes, using the test organisms specified for 

limited, broad-spectrum or hospital disinfectant testing. Sixty carriers for each organism should 

be tested against three different batches of the product (one of which should be ≥60 days old). 

Instead of spraying the inoculated surface of the carrier, the product should be tested by wiping 

the surface of the carrier with the saturated towelette, and then subculturing the carriers after the 

specified holding time. One towelette should be used to treat 10 carriers. Alternatively, one 

carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette 

per carrier set per batch.

(B) Evaluation of bactericidal towelette success. The product should kill the test 
12

organism on 59 out of 60 carriers. This testing is intended to support bridging of all vegetative 

bacteria listed on the EPA registered liquid disinfectant used to saturate the towelette to the EPA 

registered towelette product.

(8) Disinfectants for Internal Toilet and Urinal Bowl Surfaces Above and Below the 

Water Line. This section addresses efficacy testing for products bearing label claims as 

disinfectants (limited, broad-spectrum, or hospital) for internal toilet and urinal bowl surfaces. 

Regarding water-soluble powders and non-volatile liquid products test procedure, the Agency

recommends the use of the AOAC International Use-Dilution Methods (Ref. 1) modified to 

include a 5% organic soil challenge added to the bacterial inoculum. Sixty carriers for each of 

three samples, representing three different batches, one of which is ≥60 days old, should be 

tested against Salmonella enterica (ATCC 10708) or Staphylococcus aureus (ATCC 6538), for 

limited disinfectant products; S. enterica and S. aureus, for broad-spectrum disinfectant products; 

and S. aureus and Pseudomonas aeruginosa (ATCC 15442), for hospital disinfectant products. 

The contained bowl water (-96 fl oz, 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 for testing.

(i) Evaluation of disinfectant success for internal toilet bowl and urinal bowl surfaces.

For the AOAC International Use-Dilution Methods and the Germicidal Spray Products as 

Disinfectants test, the product should kill the test microorganisms on 59 out of each set of 60 

carriers/slides within ten minutes or less. In addition, per the 2009 AOAC revisions for the UseDilution Method, 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); a mean log density <6.0 invalidates the 

test.

(9) Additional microorganisms. This section addresses efficacy testing for limited, 

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

Salmonella enterica (ATCC 10708), Staphylococcus aureus (ATCC 6538) or Pseudomonas 

aeruginosa (ATCC 15442).

(i) Water-soluble powders and non-volatile liquid products test procedure. The Agency 

recommends the use of the AOAC International Use-Dilution Methods (Ref. 1) or the AOAC 

International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2). Ten carriers 

should be tested against each specific bacterium for each of two samples representing two 

different batches. If the product is intended to be represented as bactericidal 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.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid products test 

procedure. The Agency recommends the use of the AOAC International Germicidal Spray 

Products as Disinfectants test (Ref. 3). Ten carriers should be tested against each specific 

bacterium for each of two samples representing two different batches. If the product is intended 

to be represented as bactericidal in the presence of organic soil (one-step), an appropriate organic 
13

soil, such as 5 percent blood serum, should be included with the bacterial inoculum.

(iii) Single-use towelettes test procedure. The Agency recommends the use of a modified 

AOAC modified Germicidal Spray Products as Disinfectants test (Ref.3) or ASTM E2362 (Ref. 

4). Ten carriers should be tested against each specific bacterium for each of two samples 

representing two different batches. If the product is intended to be represented as bactericidal 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. Instead of spraying the inoculated surface 

of the carrier, the product should be tested by wiping the surface of the carrier with the saturated 

towelette, and then subculturing 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 a minimum of 10 inoculated carriers. Alternatively, one carrier with a surface area 

equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. 

The area of the towelette used for wiping should be rotated so as to expose a maximum amount 

of its surface in the course of wiping a set of slides. 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. 

(iv) Evaluation of disinfectant success for additional microorganisms. The product should 

kill all the test microorganisms on all carriers in ≤ten minutes. The minimum carrier count to 

make the test valid should be 1 x 104. 

(e) Disinfectants with fungicidal claims. This section addresses efficacy testing for 

broad-spectrum or hospital disinfectant products which bear label claims of efficacy against 

pathogenic fungi.

(1) Water soluble powders and non-volatile liquid products

(i) Test procedures. The Agency recommends the use of the AOAC International 

Fungicidal Activity of Disinfectants test (Ref. 5). The test should be conducted at 5, 10, and 15 

minute exposure times. Two samples representing two different batches of the product should be 

evaluated for efficacy against Trichophyton mentagrophytes (T. mentagrophytes)(ATCC 9533).

The inoculum employed should provide a concentration of ≥5 x 106conidia/mL. If the product is 

intended to be represented as fungicidal in the presence of organic soil (one-step), an appropriate 

organic soil, such as 5% blood serum, should be included with the fungal inoculum. The Agency 

also recommends the use of the AOAC International Use-Dilution Methods (Ref. 1). This test 

may be modified to conform to appropriate elements (e.g., media, growth conditions, etc.) in the 

AOAC International Fungicidal Activity of Disinfectants test. Ten carriers for each of two 

samples representing two different batches of the product should be evaluated against T. 

mentagrophytes (ATCC 9533). The inoculum employed should provide a concentration of 1 x 

104– 1 x 105conidia per carrier. If the product is intended to be represented as fungicidal in the 

presence of organic soil (one-step), an appropriate organic soil, such as 5 percent blood serum, 

should be included with the fungal inoculum.
14

(ii) Evaluation of fungicidal success. For the AOAC International Fungicidal Activity of 

Disinfectants test, all fungal spores at 10 and 15 minutes should be killed to support a 10 minute

exposure time. For the AOAC International Use-Dilution Methods, all fungal spores on all 10 

carriers should be killed in ≤ten minutes.

(2) Germicidal spray products (aerosol or pump) and volatile liquid products—(i) 

Test procedures. The Agency recommends the use of the AOAC International Germicidal Spray 

Products as Disinfectants test (Ref. 3).This test may be modified to conform to appropriate 

elements (e.g., media, growth conditions, etc.) in the AOAC International Fungicidal Activity of 

Disinfectants test. Ten carriers for each of two samples representing two different batches of the 

product should be evaluated against T. mentagrophytes (ATCC 9533). The inoculum employed 

should be modified to provide a concentration of 1 x 104– 1 x 105conidia per carrier. If the 

product is intended to be represented as fungicidal in the presence of organic soil (one-step), an 

appropriate organic soil, such as 5 percent blood serum, should be included with the fungal 

inoculum.

(ii) Evaluation of fungicidal success. All fungal spores on all 10 carriers should be killed 

in ≤ten minutes.

(3) Single-Use Towelettes

(i) Test Procedure. The Agency recommends the use of a modified AOAC Germicidal 

Spray Products as Disinfectants test (Ref. 3) or ASTM 2362 (Ref. 4). Ten carriers for each of 

two samples representing two different batches of the product should be evaluated against T. 

mentagrophytes (ATCC 9533). The inoculum employed should be modified to provide a 

concentration of 1 x 104– 1 x 105conidia per carrier. If the product is intended to be represented 

as fungicidal in the presence of organic soil (one-step), an appropriate organic soil, such as 5 

percent blood serum, should be included with the fungal inoculum. Instead of spraying the 

inoculated surface of the carrier, the product should be tested by wiping the surface of the carrier

with the saturated towelette, and then subculturing 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 a minimum of 10 inoculated carriers. Alternatively, one carrier 

with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per 

carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a 

maximum amount of its surface in the course of wiping a set of slides. 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. 

 

(ii) Evaluation of fungicidal towelette success. All fungal spores on all 10 carriers should 

be killed in ≤ten minutes. 

(4) Bridging for disinfectant towelettes. In some cases, disinfectant towelette 

formulations are identical to registered liquid formulations. In order to bridge efficacy data from 

the EPA registered bulk liquid disinfectant used to saturate a towelette or other related product 
15

form, the studies in paragraphs (e)(4)(i) and (e)(4)(ii) of this guideline should be conducted and 

submitted to EPA for review:

(i) Chemical Testing - Comparison of Expressed Liquid from the Towelette(s) to the EPA 

Registered Liquid Disinfectant Formulation to which it is being bridged: All active ingredients in 

the expressed liquid should be within the certified limits of the Confidential Statement of 

Formula of the liquid formula being referenced/bridged. The disinfectant towelettes package 

should be filled according to the manufacturing specifications. Excess liquid in the bulk 

towelette containers cannot be poured off for use in the chemical testing for bridging of the 

efficacy data. The liquid used in the chemical testing should only be that expressed from the 

towelettes. Two batches should be tested. Analytical data for the active ingredients in the 

expressed liquid should be submitted for review.

(ii) Efficacy Testing - Efficacy testing should be conducted under the same testing 

conditions (e.g., soil load, contact time, temperature) as used for the bulk liquid testing. This 

testing allows bridging of data from the registered bulk liquid used to saturate the towel for each 

type of organism in this paragraph. 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. For fungicidal test procedure, the Agency

recommends the use of the AOAC International Germicidal Spray Products as Disinfectants 

(Ref. 3) 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. Ten carriers for each of two samples, representing two batches of 

the product should be evaluated against T. mentagrophytes (ATCC 9533) for the label 

recommended contact time. The inoculum employed should be at a count to achieve 1 x 104– 1

x 105conidia per carrier. Instead of spraying the inoculated surface of the carrier, the product 

should be tested by wiping the surface of the carrier with the saturated towelette, and then 

subculturing the carriers after the specified holding time. One towelette should be used to wipe a 

minimum of 10 inoculated carriers. Alternatively, one carrier with a surface area equivalent to 

ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch.

(A) Evaluation of Fungicidal towelette success. The product should kill the test organism 

on all 10 carriers in ≤ten minutes. 

(B) Bridging. This testing is intended to support bridging of all fungal test organisms 

from the EPA registered bulk liquid disinfectant used to saturate the towelette to the EPA 

registered towelette product.

(f) Disinfectants with virucidal claims. This section addresses efficacy testing for 

broad-spectrum or hospital disinfectant products that bear label claims of effectiveness against 

viruses. Virucidal products are intended for use on dry inanimate surfaces; therefore, virological 

data are usually developed by carrier methods. Each specific virus listed on the label should be 

tested, unless there is an acceptable surrogate for the virus. For label claims against Hepatitis B

virus, Hepatitis C virus, and Norovirus, the Duck Hepatitis B virus, Bovine Viral Diarrhea virus, 
16

and Feline Calicivirus, respectively, are currently considered acceptable surrogates for testing. 

Additional guidance and protocols for surrogate virus testing can be found at 

http://www.epa.gov/oppad001/regpolicy.htm. To simulate in-use conditions, the specific virus to 

be treated (or surrogate as noted in this paragraph) 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. 

(1) Water soluble powders and non-volatile liquid products test procedures. The 

Agency recommends the use of either the AOAC International Use-Dilution Methods (Ref 1) 

modified for virucidal testing or the ASTM E1053 Test Method for Efficacy of Virucidal Agents 

Intended for Inanimate Environmental Surfaces (Ref. 6). One surface for each of two samples, 

representing two different batches of disinfectant, should be tested against a recoverable virus 

end point titer of ≥104viable viral particles from the test surface for a specified exposure period 

(≤10 minutes) at room temperature. 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.

(2) Germicidal spray products (aerosol or pump) and volatile liquid products test 

procedure. The Agency recommends the use of a AOAC International Germicidal Spray 

Products as Disinfectants test (Ref. 3) modified for virucidal testing or the ASTM E1053 

Virucidal Test Method (Ref. 6). One surface for each of two samples, representing two different 

batches of disinfectant, should be tested against a recoverable virus endpoint titer of at least 104

viable viral particles from the test surface for the exposure period specified on the label. 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.

(3) Single-use towelettes test procedure. The Agency recommends the use of the 

modified AOAC Germicidal Spray Products as Disinfectants test (Ref. 3) or ASTM E1053 (Ref. 

6). One surface for each of two samples, representing two different batches of disinfectant, 

should be tested against a recoverable virus end point titer of ≥104viable viral particles from the 

test surface for a specified exposure period (≤10 minutes) at room temperature. 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. Instead of spraying 

the inoculated surface of the carrier, the product should be tested by wiping the surface of the 

carrier with the saturated towelette, and then subculturing 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 a minimum of 10 inoculated carriers. Alternatively, one carrier 

with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per 
17

carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a 

maximum amount of its surface in the course of wiping a set of slides. 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. 

 

(4) Evaluation of virucidal success. 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 in paragraphs (f)(4)(i)through (f)(4)(ix) of this guideline.

(i) The virus recovery (titer) should include a minimum of four determinations per each 

dilution in the assay system (e.g., tissue culture, embryonated egg, animal infection, etc.).

(ii) Cytotoxicity controls. The effect of the disinfectant on the viral assay system should 

include a minimum of four determinations per each dilution. For EPA approved protocols for 

surrogate virus testing, two determinations per each dilution should be included.

(iii) The activity of the disinfectant against the test virus should include a minimum of 

four determinations per dilution in the assay system.

(iv) Neutralization controls. Neutralization controls should be performed (Ref. 7) and 

should include a minimum of four determinations per each dilution. For EPA approved protocols 

for surrogate virus testing, two determinations per each dilution should be included.

(v) Any special methods which are used to increase the virus titer and to detoxify the 

residual disinfectant should be described.

(vi) The ID50 values calculated for each assay should be provided.

(vii) The test results should be reported as the reduction of the virus titer by the activity 

of the disinfectant (ID50 of the virus control less the ID50 of the test system) expressed as the 

logarithm to the base 10 and calculated by a 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. 

If cytotoxicity is present, the virus control titer should be increased to demonstrate a ≥3 log10

reduction in viral titer beyond the cytotoxic level. Table 1 provides an example of a typical 

laboratory report of a single test with one virus, assayed in a tissue culture system.

(ix) A laboratory report of a single test with one virus (recovered from a treated surface) 

involving a tissue culture assay system should include the details of the methods employed and 

the information included in Tables 2-1, 2-2 and 2-3: 
18

Table 2-1: Test Results

Dilution of Virus Virus - Disinfectant* Virus - Control* Cytotoxic - Control

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

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

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

10-40 0 0 0 + + + + 0 0 0 0

10-50 0 0 0 + + + + 0 0 0 0

10-60 0 0 0 + + + 0 0 0 0 0

10-70 0 0 0 + 0 0 0 0 0 0 0

10-80 0 0 0 0 0 0 0 0 0 0 0

Note: T = toxic; + = virus recovered; 0 = no virus recovered

Table 2-2: Calculation of the Tissue Culture Infective Dose 50 (TCID50)

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

10-14/4 4 0 24 0 24/24 100

10-24/4 4 0 20 0 20/20 100

10-34/4 4 0 16 0 16/16 100

10-44/4 4 0 12 0 12/12 100

10-54/4 4 0 8 0 8/8 100

10-63/4 3 1 4 1 4/5 80

10-71/4 1 3 1 4 1/5 20

10-80/4 0 4 0 8 0/8 0

TCID50 = 106.5

Table 2-3: Calculations of the Tissue Culture Lethal Dose 50 (TCLD50)

Values Accumulated Values
19

Virus 

Dilution 

Inoculated

No. Toxic / 

No. 

Inoculated

No. Toxic No. not 

Toxic

No. Toxic No. not 

Toxic

No. Toxic / 

No. 

Inoculated

% Toxic

10-14/4 4 0 9 0 9/9 100

10-24/4 4 0 5 0 5/5 100

10-31/4 1 3 1 3 1/4 25

10-40/4 0 4 0 7 0/7 0

10-50/4 0 4 0 11 0/11 0

10-60/4 0 4 0 15 0/15 0

10-70/4 0 4 0 19 0/19 0

10-80/4 0 4 0 23 0/23 0

TCLD50 = 102.7 Therefore: Virus inactivation = TCID50 - TCLD50 = 103.8 log 10

(5) Bridging for disinfectant towelettes. In some cases, disinfectant towelette 

formulations are identical to registered liquid formulations. In order to bridge efficacy data from 

the EPA registered bulk liquid disinfectant used to saturate a towelette or other related product 

form, the studies in paragraphs (f)(5)(i) and (f)(5)(ii) of this guideline should be conducted and 

submitted to EPA for review.

(i) Chemical Testing—Comparison of Expressed Liquid from the Towelette(s) to the 

EPA Registered Liquid Disinfectant Formulation to which it is being bridged: All active 

ingredients in the expressed liquid should be within the certified limits of the Confidential 

Statement of Formula of the liquid formula being referenced/bridged. The disinfectant towelettes 

package should be filled according to the manufacturing specifications. Excess liquid in the bulk 

towelette containers cannot be poured off for use in the chemical testing for bridging of the 

efficacy data. The liquid used in the chemical testing should only be that expressed from the 

towelettes. Two batches should be tested. Analytical data for the active ingredients in the 

expressed liquid should be submitted for review.

(ii) Efficacy Testing: Efficacy testing should be conducted under the same testing 

conditions (e.g. soil load, contact time, temperature) as used for the bulk liquid testing. This 

testing allows bridging of data from the registered bulk liquid used to saturate the towel for each 

type of virus in paragraphs (f)(5)(ii)(A)(1) through (f)(5)(ii)(A)(3) of this guideline. 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. 

(A) Virucidal Test Procedure. The Agency recommends the use of either the AOAC 

International Germicidal Spray Products as Disinfectants (Ref. 3) modified for virucidal 

towelette testing or the ASTM E1053 (Ref. 6) modified for virucidal towelette testing. 
20

(1) To support bridging of all viral claims, the most difficult to inactivate small-sized 

non-enveloped virus, from the viral strains registered for the bulk liquid, should be selected for 

testing. Examples of small-sized non-enveloped viral families include members of the 

Picornaviridae family (e.g., poliovirus, enterovirus, hepatitis A virus, rhinovirus), and 

Parvoviridae family (e.g., parvovirus). 

(2) To support bridging of viral claims for large-sized non-enveloped and enveloped viral 

strains, the most difficult to inactivate large-sized non-enveloped virus, from the viral strains 

registered for the bulk liquid, should be selected for testing. Examples of large-sized nonenveloped viral families include members of the Adenoviridae family (e.g., adenovirus), 

Reoviridae family (e.g., rotavirus), and Papillomaviridae family (e.g., papillomavirus).

(3) To support bridging of viral claims for enveloped viral strains, the most difficult to 

inactivate enveloped virus, from the viral strains registered for the bulk liquid, should be selected 

for testing. Examples of enveloped viral families include 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).

(B) Ten carriers for each of two samples, representing two batches of disinfectant, should 

be tested against a recoverable dried virus end point titer of ≥104viral particles from the test 

surface for a specified exposure period at room temperature. Instead of spraying the inoculated 

surface of the carrier, the product should be tested by wiping the surface of the carrier with the 

saturated towelette, and then subculturing the carriers after the specified holding time. One 

towelette should be used to wipe a minimum of 10 inoculated carriers. Alternatively, one carrier 

with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per 

carrier set per batch. 

The protocol for the viral assay should provide the information identified in paragraphs 

(f)(5)(ii)(B)(1) through (f)(5)(ii)(B)(7) of this guideline:

(1) The virus recovery (titer) should include a minimum of four determinations for each 

dilution in the assay system (e.g., cell culture, embryonated egg, animal infection).

(2) Cytotoxicity controls. The effect of the test substance on the viral assay system 

should include a minimum of four determinations for each dilution. For EPA approved protocols 

for surrogate virus testing, two determinations per each dilution should be included.

(3) The activity of the test substance against the test virus should include a minimum of 

four determinations for each dilution in the assay system.

(4) Neutralization controls. Neutralization controls should be performed (Ref. 7) and 

should include a minimum of four determinations per each dilution. For EPA approved protocols 
21

for surrogate virus testing, two determinations per each dilution should be included.

(5) Any special methods which are used to increase the virus titer and to detoxify the 

residual test substance should be described.

(6) The LD50 values calculated for each assay should be provided.

(7) The test results should be reported as the reduction of the virus titer by the activity of 

the test substance (LD50 of the virus control less the LD50 of the test system) expressed as the 

logarithm to the base 10 and calculated by a statistical method (e.g., Reed and Munch, Most 

Probable Number, Spearman-Karber).

(C) Evaluation of virucidal success. 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.

(g) Disinfectants with tuberculocidal claims. This section addresses efficacy testing for 

broad-spectrum or hospital disinfectant products which bear label claims of effectiveness as 

tuberculocides. In the Agency’s “Data Call-In Notice for Tuberculocidal Effectiveness for All 

Antimicrobial Pesticides with Tuberculocidal Claims,” dated June 13, 1986 (Ref. 8), 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. In 

general, the Agency does not believe that the Quantitative Tuberculocidal Activity Test (a 

suspension test) is appropriate for disinfectant formulations used on hard surfaces. An exception 

to this is for glutaraldehyde-based products, which have never 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 all other formulations to use the carrier-based AOAC 

Tuberculocidal Activity of Disinfectants test.

(1) Water-soluble powders and non-volatile liquid products test procedures. The 

Agency recommends the test procedures in paragraphs (g)(1)(i) through (g)(1)(iv) of this 

guideline.

(i) AOAC International Tuberculocidal Activity of Disinfectants test. The AOAC 

International Tuberculocidal Activity of Disinfectants test (Ref. 9) employing a 10 minute

contact time and 20 ̊C temperature. Ten carriers for each of two samples representing two 

different batches of the product should be tested against Mycobacterium bovis (BCG)(M.bovis).

If the product is intended to be represented as tuberculocidal in the presence of organic soil (onestep), an appropriate organic soil, such as 5 percent blood serum, should be included with the 

bacterial inoculum. 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.
22

(ii) AOAC International Tuberculocidal Activity of Disinfectants test with modifications. 

The AOAC International Tuberculocidal Activity of Disinfectants test with modifications to the 

10 minute contact time and/or 20 ̊C temperature (Ref. 9). Ten carriers for each of two samples 

representing two different batches of the product should be tested against M. bovis (BCG). 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. 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.

(iii) Evaluation of tuberculocide success. For the AOAC International Tuberculocidal 

Activity of Disinfectants test, all organisms on all carriers should be killed, and there should be 

no growth in any of the inoculated subculture media.

(iv) 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.

(2) Glutaraldehyde formulations

(i) Test Procedure. For glutaraldehyde formulations, the Agency recommends the 

Quantitative Tuberculocidal Activity Test. This test has been published in the Agency’s “Data 

Call-In Notice for Tuberculocidal Effectiveness for All Antimicrobial Pesticides with 

Tuberculocidal Claims,” dated June 13, 1986 (Ref. 8). 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.

(ii) Evaluation of tuberculocide success. 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 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% greater than when the survivor curve intersects the one survivor 

line.

(3) Germicidal spray products and volatile liquid products—(i) Test procedure. The 
23

Agency recommends the AOAC International Germicidal Spray Products as Disinfectants test 

(Ref. 3), using the media, test culture, and other elements described in the AOAC International 

Tuberculocidal Activity of Disinfectants test. Ten carriers for each of two samples representing 

two different batches of the product should be tested against M. bovis (BCG). 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. 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.

(ii) Evaluation of tuberculocide success. When using the AOAC International Germicidal 

Spray Products as Disinfectants test, all organisms on all carriers/slides should be killed, and 

there should be no growth in any of the inoculated subculture media.

(4) Single-Use Towelettes

(i) Test Procedure. The Agency recommends the use of a modified AOAC Germicidal 

Spray Products as Disinfectants test (Ref.3) or ASTM 2362 (Ref. 4). Ten carriers for each of two 

samples representing two different batches of the product should be evaluated against M. bovis

(BCG). 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. Instead of spraying the inoculated surface of the carrier, the product 

should be tested by wiping the surface of the carrier with the saturated towelette, and then 

subculturing 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 a minimum 

of 10 inoculated carriers. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 

inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette 

used for wiping should be rotated so as to expose a maximum amount of its surface in the course 

of wiping a set of slides. 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. 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. 

 

(ii) Evaluation of tuberculocidal towelette success. All organisms on all carriers should 

be killed, and there should be no growth in any of the inoculated subculture media.

(5) Bridging for disinfectant towelettes. In some cases, disinfectant towelette 

formulations are identical to registered liquid formulations. In order to bridge efficacy data from 

the EPA registered bulk liquid disinfectant used to saturate a towelette or other related product 

form, the studies in paragraphs (g)(5)(i) and (g)(5)(ii) of this guideline should be conducted and 

submitted to EPA for review.

(i) Chemical Testing - Comparison of Expressed Liquid from the Towelette(s) to the EPA 

Registered Liquid Disinfectant Formulation to which it is being bridged: All active ingredients in 
24

the expressed liquid should be within the certified limits of the Confidential Statement of 

Formula of the liquid formula being referenced/bridged. The disinfectant towelettes package 

should be filled according to the manufacturing specifications. Excess liquid in the bulk 

towelette containers cannot be poured off for use in the chemical testing for bridging of the 

efficacy data. The liquid used in the chemical testing should only be that expressed from the 

towelettes. Two batches should be tested. Analytical data for the active ingredients in the 

expressed liquid should be submitted for review.

(ii) Efficacy Testing: Efficacy testing should be conducted under the same testing 

conditions (e.g. soil load, contact time, temperature) as used for the bulk liquid testing. This

testing allows bridging of data from the registered bulk liquid used to saturate the towel for each 

type of organism in paragraph (g)(5)(ii)(A) of this guideline. 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. 

(A) Test Procedure. The Agency recommends the use of the modified AOAC Germicidal 

Spray Products as Disinfectants test (Ref. 3) or ASTM 2362 (Ref. 4). The test should be 

modified to conform to appropriate elements (e.g., media, growth conditions, etc) in the AOAC 

International Tuberculocidal Activity of Disinfectants test. Ten carriers for each of two samples, 

representing two batches of the product, should be tested against M. bovis BCG. Instead of 

spraying the inoculated surface of the carrier, the product should be tested by wiping the surface 

of the carrier with the saturated towelette, and then subculturing the carriers after the specified 

holding time. One towelette should be used to wipe a minimum of 10 inoculated carriers.

Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped 

using one towelette per carrier set per batch. 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.

(B) Evaluation of tuberculocidal towelette success. All organisms on all carriers should 

be killed, and there should be no growth in any of the inoculated subculture media.

(C) This testing is intended to support bridging of all mycobacteria listed on the EPA 

registered liquid disinfectant used to saturate the towelette to the EPA registered towelette 

product.

(h) Data collection and reporting—(1) General. To assist in the proper review and 

evaluation of product performance, complete descriptions of the test employed and the results 

obtained should be submitted to the Agency. All test reports should include, at the least, the 

following information:

(i) Study title;

(ii) Product Identity;
25

(iii) Guideline number/Data Requirement; 

(iv) Identification of the testing laboratory or organization;

(v) Location where the test was performed;

(vi) Name(s) of the person(s) responsible for the test;

(vii) Statement of Confidentiality Claims;

(viii) Statement of 40 CFR Part 160 Good Laboratory Practice compliance and Quality 

Assurance Statement;

(ix) Purpose of the study;

(x) Date and time of the start and end of the test;

(xi) Test employed and any modifications (e.g., organic soil, hard water, etc.), when 

using standard tests (e.g., AOAC, ASTM, etc.) all deviations to the test methods should be 

reported;

(xii) Test microorganisms employed, including identification of the specific strain 

(ATCC or other);

(xiii) Description of the test substance, including the percent of active ingredient;

(xiv) Concentration or dilution of the product tested and how prepared;

(xv) Number of samples, batches and replicates tested;

(xvi) Manufacture date of each product batch; 

(xvii) Identification of all material or procedural options employed, where such choice is 

provided for or recommended in the test method selected (e.g., growth media, drying time for 

inoculated carriers, neutralization confirmation and/or subculture media, secondary 

subculturing);

(xviii) Test exposure conditions (e.g., contact time, temperature, and relative humidity);

(xix) Complete reports of results obtained for each replication;

(xx) Any control data essential to establish the validity of the test.

(xxi) Carrier counts;
26

(xxii) Statistical treatment of the data;

(xxiii) Conclusions;

(xxiv) References;

(xxv) Appendices, including study protocol and all raw data reports (per 40 CFR Part 

160.185) associated with the conduct of the study.

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.

(2) Data for modifications of recommended methods. When recommended methods 

are modified to support specific claims and/or use patterns for a product, the protocol, identifying 

and describing each modification, should be provided with the study report. The applicant should

submit the proposed modification to the Agency for review and evaluation prior to initiation of 

the test.

(3) Data for other methods. When recommended methods, or modifications thereto, are 

not employed to develop efficacy data (such as actual in-use or many kinds of simulated-use 

testing), complete testing protocols should be submitted with the test reports. All materials and 

procedures employed in testing should be described in a manner consistent with original research 

reports published in technical or scientific journals. Where references to published reports or 

papers are made, copies or reprints of such references should be provided with the test reports. 

The applicant should submit the proposed testing protocols for in-use or simulated-use studies 

(with a proposed label to show the claims to be supported by the protocol) to the Agency for 

review and evaluation prior to initiation of the test.

(i) 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, 

Hard Surface Carrier Test Methods, Current edition. AOAC International, Suite 500, 481 North 

Frederick Avenue, Gaithersburg, MD 20877-2417. 

(3) 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. 
27

(4) Annual Book of ASTM Standards, Standard Practice for Evaluation of Pre-saturated 

or Impregnated Towelettes for Hard Surface Disinfection, Designation E2362. American Society 

for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current 

edition.

(5) 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.

(6) Annual Book of ASTM Standards, Test Method for Efficacy of Virucidal Agents 

Intended for Inanimate Environmental Surfaces, Designation E1053. American Society for 

Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition.

(7) Annual Book of ASTM Standards, Standard Test Method for Neutralization of 

Virucidal Agents in Virucidal Efficacy Evaluations, Designation E1483. American Society for 

Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition.

(8) 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).

(9) 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.