United States

Environmental Protection

Agency

Office of Prevention, Pesticides

And Toxic Substances

(7510P)

EPA 712-C-07-074

Draft November 5, 

2009

Product Performance 

Test Guidelines

OPPTS 810.2200:

Disinfectants for Use on 

Hard Surfaces—Efficacy 

Data Recommendations

Public Review Draft
NOTICE

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

Prevention, Pesticides and Toxic Substances (OPPTS), 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 (FFDCA) (21 U.S.C. 346a).

The OPPTS 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 the test, 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. 

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 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 OPPTS harmonized test guidelines and to 

access the guidelines electronically, please go to http://www.epa.gov/oppts 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.

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. 
1

OPPTS 810.2200: Disinfectants for use on hard surfaces - efficacy data 

recommendations

(a) Scope.

(1) Applicability. This guideline is intended to meet 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 I/S/C. 346a). It addresses testing to demonstrate 

effectiveness of products which are disinfectants, fungicides, virucides, and tuberculocides. 

(2) Background. The source materials used in developing this OPPTS 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 tests to be conducted and data to be submitted which the 

Agency believes will generally satisfy the requirements for pesticide registration. All studies 

conducted under this guideline should be completed under EPA’s Good Laboratory Practice 

regulations (40 CFR Part 160). Note: The Association of Official Analytical Chemists (AOAC)

recommended tests are expected 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 OPPTS Test 

Guideline 810.2000 for general testing recommendations prior to initiating tests.

(2) [Reserved]

(3) 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 

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 [MRIDs]) to the supporting data developed for the original product 
2

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 (MRIDs) 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 does 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 must be developed on 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 

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 

Germicidal 

Spray Test

Broad-spectrum 

disinfectant/hard 

non-porous 

surfaces.

Water soluble 

powders/liquids

AOAC UseDilution 

Method or

AOAC Hard 

Surface 

Carrier Test 

(distilled water 

only)

Staphylococcus 

aureus and 

Salmonella enterica

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 for each 

batch.

Spray products AOAC 

Germicidal 

Spray 

Products Test

Towelettes Modified 

Germicidal 

Spray Test
3

Level of 

Efficacy

Test Methods Test 

Organisms

No. of 

Batches/Carriers

Evaluation 

of Success

Hospital 

disinfectant/hard 

non-porous 

surfaces.

Water soluble 

powders/liquids

AOAC UseDilution 

Method or

AOAC Hard 

Surface 

Carrier Test

(distilled water 

only)

Staphylococcus 

aureus 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.

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-

97

Virus claimed on 

the label or 

approved surrogate.

Two batches. Complete 

inactivation of the 

virus. Where 

cytotoxicity is 

present, 

demonstrate a 

Spray products AOAC 3log10 reduction.

Germicidal 

Spray 

Products Test 

modified for 

viruses or 

ASTM E1053-

97

Towelettes Modified 

Germicidal 

Spray Test

Tuberculocidal 

disinfectant/hard 

non-porous 

surfaces.

Water soluble 

powders/liquids

AOAC 

Tuberculocidal 

Activity of 

Disinfectants, 

Quantitative 

Tuberculocidal 

Activity Test Mycobacterium 

bovis BCG

Two batches, ten carriers 

per batch

10/10 carriers are 

negative for 

growth and there 

is no growth in the 

additional test 

media.

Spray products AOAC 

Germicidal 

Spray 

Products Test 

modified for 

tuberculocidal 

activity
4

Level of 

Efficacy

Test Methods Test 

Organisms

No. of 

Batches/Carriers

Evaluation 

of Success

Towelettes Modified 

Germicidal 

Spray Test

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.

Two batches, ten carriers 

for each batch.

All bacteria are 

killed

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 (see reference 

(i)(1) of this guideline) 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 at least 60 days old, should be tested against Salmonella enterica (S. 

enterica)(formerly designated as Salmonella choleraesuis)(ATCC 10708) for effectiveness 

against Gram-negative 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.

(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 at least 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. Sixty carriers for each of three 

samples, representing three different batches, one of which should be at least 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 
5

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

inoculum. Instead of spraying the inoculated surface of the glass slide, the product should be 

tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing 

the slides after the specified holding time. The towelette should be removed from its container 

and subsequently handled with sterile gloves. One towelette should be used to wipe a minimum 

of 10 inoculated slides. 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 applicant should submit their towelette protocol to the Agency

for review and approval prior to conducting the test.

(iv) Evaluation of limited disinfectant success. For the AOAC International UseDilution 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 within 

ten minutes or less. In addition, per the 2009 revisions for the Use-Dilution Method, the mean 

log density for S. aureus must 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 within ten minutes or less.

(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 against either S. aureus or S. enterica (depending on whether the product is 

claimed to be effective against Gram-negative or 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.

(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 or S. enterica 

(depending on whether the product is claimed to be effective against Gram-negative or Grampositive 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.
6

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

modified AOAC Germicidal Spray Products as Disinfectants test. Ten carriers for each of two 

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

either S. aureus or S. enterica (depending on whether the product is claimed to be effective 

against Gram-negative or 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 glass slide, the product should be tested by wiping the surface of the 

glass slide with the saturated towelette, and then subculturing the slides after the specified 

holding time. The towelette should be removed from its container and subsequently handled 

with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. 

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 applicant should submit their towelette protocol to the Agency for review and 

approval prior to conducting the test.

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

all the test microorganisms on all carriers/slides within ten minutes or less. In addition, per the 

2009 revisions for the Use-Dilution Method, the mean log density for S. aureus must 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. 

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

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 at 

least 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 at least 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 
7

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. Sixty carriers for each of three 

samples, representing three different batches, one of which should be at least 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. Instead of spraying the inoculated surface of the glass slide, the product should be 

tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing 

the slides after the specified holding time. The towelette should be removed from its container 

and subsequently handled with sterile gloves. One towelette should be used to wipe a minimum 

of 10 inoculated slides. 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 applicant should submit their towelette protocol to the Agency

for review and approval prior to conducting the test.

 

(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 within ten minutes or less. In addition, per the 2009 revisions for the Use-Dilution 

Method, the mean log density for S. aureus must 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 within ten minutes or less.

(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 and S. enterica. 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 and S. enterica. 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
8

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. Ten carriers for each of two 

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

S. aureus and S. enterica. 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 

glass slide, the product should be tested by wiping the surface of the glass slide with the 

saturated towelette, and then subculturing the slides after the specified holding time. The 

towelette should be removed from its container and subsequently handled with sterile gloves. 

One towelette should be used to wipe a minimum of 10 inoculated slides. 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 

applicant should submit their towelette protocol to the Agency for review and approval prior to 

conducting the test.

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

product should kill all the test microorganisms on all carriers/slides within ten minutes or less. 

In addition, per the 2009 revisions for the Use-Dilution Method, the mean log density for S. 

aureus must 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. 

(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 at 

least 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 
9

three different batches, one of which should be at least 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. Sixty carriers for each of three 

samples, representing three different batches, one of which should be at least 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 glass slide, the product should be 

tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing 

the slides after the specified holding time. The towelette should be removed from its container 

and subsequently handled with sterile gloves. One towelette should be used to wipe a minimum 

of 10 inoculated slides. 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 applicant should submit their towelette protocol to the Agency

for review and approval prior to conducting the test.

 

(iv) Evaluation of hospital or healthcare 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 within ten minutes or less. In addition, per the 2009 revisions for the Use-Dilution 

Method, the mean log density for S. aureus and P. aeruginosa must 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. 

(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 
10

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. 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 glass slide, the product should be tested by wiping the 

surface of the glass slide with the saturated towelette, and then subculturing the slides after the 

specified holding time. The towelette should be removed from its container and subsequently 

handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated 

slides. 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 applicant should submit their towelette protocol to the Agency for review and 

approval prior to conducting the test.

 

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

product should kill all the test microorganisms on all carriers/slides within ten minutes or less. 

In addition, per the 2009 revisions for the Use-Dilution Method, the mean log density for S. 

aureus and P. aeruginosa must 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.

(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 
11

towelette containers can not 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. The 

applicant should submit their towelette protocol to the Agency for review and approval prior to 

conducting the test. 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 glass slide, the product should be tested by 

wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides 

after the specified holding time. One towel will 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 

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 towel 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 (see reference (i)(1) of 

this guideline) modified to include a 5% organic soil challenge. Sixty carriers for each of three 

samples, representing three different batches, one of which is at least 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) should be 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 
12

set of 60 carriers/slides within ten minutes or less. In addition, per the 2009 revisions for the 

Use-Dilution Method, the mean log density for S. aureus and P. aeruginosa must 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 broadspectrum 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 

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. 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 (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 glass slide, the product 

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

subculturing the slides after the specified holding time. The towelette should be removed from

its container and subsequently handled with sterile gloves. One towelette should be used to wipe 

a minimum of 10 inoculated slides. 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 applicant should submit their towelette 

protocol to the Agency for review and approval prior to conducting the test.

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

should kill all the test microorganisms on all carriers/slides within ten minutes or less. 
13

(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. 4) Two samples representing two different 

batches of the product should be evaluated for efficacy against Trichophyton mentagrophytes (T. 

mentagrophytes)(ATCC 9533). 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.

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

of Disinfectants test, all fungal spores in all replicates for each batch should be killed. For the 

AOAC International Use-Dilution Methods, all fungal spores on all 10 carriers should be killed 

within ten minutes or less.

(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 (see reference (i)(3) of this guideline). 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/slides should 

be killed within ten minutes or less.

(3) Single-Use Towelettes

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

Spray Products as 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 105

conidia per carrier. If the product is intended to be represented as fungicidal in the presence of 
14

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 glass slide, 

the product should be tested by wiping the surface of the glass slide with the saturated towelette, 

and then subculturing the slides after the specified holding time. The towelette should be 

removed from its container and subsequently handled with sterile gloves. One towelette should 

be used to wipe a minimum of 10 inoculated slides. 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 applicant should submit their 

towelette protocol to the Agency for review and approval prior to conducting the test.

 

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

carriers/slides should be killed within ten minutes or less. 

(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 

form, the studies in paragraphs (e)(4)(1) 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 can not 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. 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. The applicant should submit their towelette 

protocol to the Agency for review and approval prior to conducting the test. 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 
15

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 

glass slide, the product should be tested by wiping the surface of the glass slide with the 

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

towelette should be used to wipe a minimum of 10 inoculated slides. 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. 

(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 towel 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. For 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. 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 slides, stainless steel penicylinders, 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 (see 

reference (i)(1) of this guideline) modified for virucidal testing or the ASTM E1053-97 Virucidal 

Test Method (Ref. 5). One surface for each of two samples, representing two different batches 

of disinfectant, should be tested against a recoverable virus titer of at least 104viable viral 

particles from the test surface for a specified exposure period 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 E1053-97 

Virucidal Test Method (Ref. 5). One surface for each of two samples, representing two different 

batches of disinfectant, should be tested against a recoverable virus titer of at least 104viable 
16

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. One surface for each of two 

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

virus titer of at least 104viable viral particles from the test surface for a specified exposure 

period 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 glass slide, the 

product should be tested by wiping the surface of the glass slide with the saturated towelette, and 

then subculturing the slides after the specified holding time. The towelette should be removed 

from its container and subsequently handled with sterile gloves. One towelette should be used to 

wipe a minimum of 10 inoculated slides. 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 applicant should submit their 

towelette protocol to the Agency for review and approval prior to conducting the test.

 

(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 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.

(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 (see reference 

(i)(6)) and should include a minimum of four determinations per each dilution.
17

(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). 

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

If cytotoxicity is present, at least a 3-log10 reduction in viral titer should be demonstrated 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 typical laboratory report of a single test with one virus (recovered from a treated 

surface) involving a tissue culture assay system would include the details of the methods 

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

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
18

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

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
19

(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)(1) 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 can not 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. 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. The 

applicant should submit their towelette protocol to the Agency for review and approval prior to 

conducting the test.

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

International Germicidal Spray Products as Disinfectants (see reference (i)(3) of this guideline) 

modified for virucidal towelette testing or the ASTM E1053 / current edition Virucidal Test 

Method (see reference (i)(5)) modified for virucidal towelette testing. 

(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 
20

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., 

herpesvirus), 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 titer of at least 104viral particles from the test 

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

surface of the glass slide, the product should be tested by wiping the surface of the glass slide 

with the saturated towelette, and then subculturing the slides after the specified holding time. 

One towelette should be used to wipe a minimum of 10 inoculated slides. 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 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.

(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 (see reference 

(i)(6)) and should include a minimum of four determinations per each dilution.

(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).

(C) Evaluation of virucidal success. The product should demonstrate complete 

inactivation of the virus at all dilutions. If cytotoxicity is present, at least a 3-log reduction in 

viral titer should be demonstrated beyond the cytotoxic level recovered from the carrier surface.
21

(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 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 (see reference (i)(8) of this guideline) 

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

included with the bacterial inoculum.

(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 (see reference (i)(7) of this guideline). 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.

(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, using the same test 
22

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 that when the survivor curve intersects the one survivor line.

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

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.

(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. 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 glass slide, the product should be 

tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing 

the slides after the specified holding time. The towelette should be removed from its container 
23

and subsequently handled with sterile gloves. One towelette should be used to wipe a minimum 

of 10 inoculated slides. 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 applicant should submit their towelette protocol to the Agency

for review and approval prior to conducting the test.

 

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

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 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 can not 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. 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. The applicant should submit their 

towelette protocol to the Agency for review and approval prior to conducting the test.

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

Products as Disinfectants test, modified for towelette testing. 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 glass slide, the product should be tested by wiping the surface of the 

glass slide with the saturated towelette, and then subculturing the slides after the specified 

holding time. One towelette should be used to wipe a minimum of 10 inoculated slides. 
24

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 tuberculocidal towelette success. All organisms on all carriers/slides 

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 towel 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 information in paragraphs (h)(1)(i) 

through (h)(i)(xiv) of this guideline:

(i) Study title;

(ii) Product identity;

(iii) Guideline number; 

(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) 40 CFR Part 160 Good Laboratory Practice compliance;

(viii) Purpose of the study;

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

(x) Statistical treatment of the data;

(xi) Conclusions;

(xii) References;

(xiii) Appendices;

(xiv) Certification

The applicant is encouraged to use the EPA’s standard efficacy report format, which may 
25

be found at http://www.epa.gov/oppad001/efficacystudystandards.htm.

(2) Data for recommended methods. When recommended methods from the Official 

Methods of Analysis of AOAC International; the Annual Book of Standards of the American 

Society for Testing and Materials; or, EPA methods are used to develop efficacy data, certain 

minimal information, in addition to that described in this guideline, should be provided in the test 

report. The report should include, at the least, the information in paragraphs (h)(2)(i) through 

(h)(2)(xii) of this guideline:

(i) Test employed, and any significant modifications thereto (e.g., organic load, hard 

water);

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

or other);

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

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

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

(vi) Preparation dates of each product batch (individually formulated preparation of the 

product);

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

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

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

subculturing);

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

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

(x) Any control data essential to establish the validity of the test;

(xi) Carrier counts;

(xii) Any additional data pertinent for specific tests described in this guideline.

(3) 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.

(4) Data for other methods. When recommended methods, or modifications thereto, are 
26

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), Eighteenth 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, Eighteenth 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, Eighteenth edition. AOAC 

International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417. 

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

Official Method 955.17 Fungicidal Activity of Disinfectants. Eighteenth edition. AOAC 

International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.

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

Intended for Inanimate Environmental Surfaces, Designation E1053-97. American Society for 

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

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

Virucidal Agents in Virucidal Efficacy Evaluations, Designation E1483-04. American Society 

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

edition.

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

Official Method 965.12 Tuberculocidal Activity of Disinfectants. Eighteenth edition. AOAC 

International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.

(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).