United States

Environmental Protection

Agency

Office of Prevention, Pesticides

And Toxic Substances

(7510P)

EPA 712-C-07-056

Draft November 5, 

2009

Product Performance 

Test Guidelines

OPPTS 810.2100:

Sterilants—Efficacy Data 

Recommendations

Public Review Draft
1

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-HQ-OPPT-2009-0576.

DRAFT DOCUMENT DISCLAIMER: This draft document is distributed solely for the 

purpose of external review. It has not been formally disseminated by the EPA and should not 

be construed to represent any Agency determination or policy. The information correction 

process under the Agency’s Information Quality Guidelines does not apply until this 

document is formally disseminated by the EPA in its final form. This draft document should 

only be cited or quoted in the context of providing comments. 
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OPPTS 810.2100: Sterilants—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 U.S.C. 346a). It addresses testing to demonstrate 

effectiveness of products which are sterilants. 

(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, inanimate, environmental surfaces, and, which bear label claims as 

sterilants.

(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. 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 Chemicals 

(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) Validation testing approaches, which may be needed to augment the full range of 

efficacy tests in special circumstances, are also described.

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

(1) Test procedure. The Agency recommends use of the Official Methods of Analysis 

of AOAC International, Official Method 966.04 Sporicidal Activity of Disinfectants test, Method 

II (Ref. 1) to demonstrate the sterilant efficacy of products. Sixty carriers representing each of 

two types of surfaces (porcelain penicylinders and silk suture loops) should be tested against 

spores of both Bacillus subtilis (B. subtilis) (American Type Culture Collection (ATCC) 19659) 

and Clostridium sporogenes (C. sporogenes) (ATCC 3584) on three samples representing three 

different batches of the product, one of which should be at least 60 days old (240 carriers per 

sample, or a total of 720 carriers). The inoculum employed should provide a count of 1 x 105– 1 

x 106colony forming units per carrier. Any sterilant which is a vapor or gas and is 
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recommended for use in a specific device should be tested using the AOAC International 

Sporicidal Activity of Disinfectants test in that specific device and according to the directions for 

use of that specific device. Modifications to the AOAC Sporicidal Activity of Disinfectants test 

to address this use should be submitted to the Agency for review and approval prior to 

conducting the tests. 

(2) Evaluation of sterilant success. The product should kill the test spores on all of the 

720 carriers without any failures.

(e) Validation testing for all products with sterilant claims. Data submitted to 

support sterilant claims are subject to independent validation testing in a second laboratory.

(1) Test procedure. The Agency recommends use of the Official Methods of Analysis of 

AOAC International, Official Method 966.04 Sporicidal Activity of Disinfectants test, Method II

(Ref. 1) to demonstrate the sterilant efficacy of products. Thirty carriers representing each of the 

two types of surfaces (porcelain penicylinders and silk suture loops), should be tested against the 

spores of both B. subtilis and C. sporogenes on one sample of the product. The inoculum 

employed should provide a count of 1 x 105– 1 x 106colony forming units per carrier.

(2) Evaluation of sterilant success. The product should kill the test spores on all 120 

carriers without any failures.

(f) Sprays, gases, and foams. (Reserved.)

(g) Additional spore formers, Clostridium difficile (C. difficile) claims. This section 

addresses interim efficacy tests for products with claims to inactivate C. difficile spores on hard, 

non-porous, inanimate surfaces. The Agency recommends three possible options, as described in 

paragraphs (g)(1)(i) through (g)(1)(iii) of this guideline.

(1) Water-soluble powders and liquid products, qualitative testing—(i) Test 

procedure for sterilant/sporicide plus C. difficile claim. The Agency recommends use of the 

Official Methods of Analysis of AOAC International, Official Method 966.04 Sporicidal 

Activity of Disinfectants test, Method II (Ref. 1) to demonstrate the sterilant efficacy of 

products, as described in (d)(1). In addition, conduct a confirmatory test using the Official 

Methods of Analysis of AOAC International, Official Method 966.04 Sporicidal Activity of 

Disinfectants test, Method I. Until the Agency identifies a representative toxigenic strain or 

suitable surrogate(s) to be used in testing against C. difficile, one of the following toxigenic 

strains should be used for testing: ATCC 700792, ATCC 43598 or ATCC 43599. C. difficile

spores are inoculated on thirty carriers (porcelain penicylinders) for two samples, representing 

two different batches of the product (a total of 60 carriers).

(A) Evaluation of sporicidal success. The product should kill all of the test spores on 

all of the 780 carriers without any failures.

(B) Reserved.

(ii) Test procedure for C. difficile sporicides--qualitative testing. The Agency 
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recommends use of the Official Methods of Analysis of AOAC International, Official Method 

966.04 Sporicidal Activity of Disinfectants test, Method I (Ref. 1) using C. difficile ATCC 

700792, ATCC 43598 or ATCC 43599. Sixty carriers (porcelain penicylinders) should be tested 

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

60 days old (a total of 180 carriers). The inoculum employed should provide a target count of 1 

x 105– approximately 1 x 106colony forming units per carrier. 

(A) Evaluation of sporicidal success. The product should kill all of the test spores on 

all of the 180 carriers without any failures.

(B) Reserved.

(iii) Test procedure for C. difficile sporicides--quantitative testing. The Agency 

recommends the use AOAC Method 2008.05: Quantitative Three Step Method (Efficacy of 

Liquid Sporicides Against Spores of Bacillus subtilis on a Hard Nonporous Surface) (Ref. 2) or 

ASTM E 2197-02: Standard Quantitative Carrier Test Method to Evaluate the Bactericidal, 

Fungicidal, Mycobactericidal, and Sporicidal Potencies of Liquid Chemical Germicides (Ref. 3). 

Until the Agency identifies a representative toxigenic strain or suitable surrogate(s) to be used in 

testing against C. difficile, one of the following toxigenic strains should be used for testing: 

ATCC 700792, ATCC 43598 or ATCC 43599. The inoculum employed should provide a target 

count of > 106colony forming units per carrier. The product should be tested on three samples 

representing three different batches of product, one of which should be at least 60 days old. The 

number of carriers will vary depending on the test method.

(A) Evaluation of sporicidal success. The product should achieve a log reduction of at 

least 6 logs based on recoverable spores.

(h) Sprays, gases, and foams. (Reserved.)

(i) Bacillus anthracis (B. anthracis) claims. This section addresses efficacy tests for all 

products with claims to inactivate B. anthracis spores on inanimate surfaces. The Agency

recommends three possible approaches, as described in paragraphs (h)(1)(i) through (h)(1)(iii) of 

this guideline.

(1) Water-soluble powders, liquid products, gases and vapors—(i) Test procedure 

for sterilant/sporicide plus B. anthracis claim. The Agency recommends use of the Official 

Methods of Analysis of AOAC International, Official Method 966.04 Sporicidal Activity of 

Disinfectants test (Ref. 1) to demonstrate the sterilant efficacy of products. Sixty carriers 

representing each of two types of surfaces (porcelain penicylinders and silk suture loops) should 

be tested against spores of both B. subtilis (ATCC 19659) and C. sporogenes (ATCC 3584) on 

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

60 days old (240 carriers per sample, or a total of 720 carriers). The inoculum employed should 

provide a target count of 1 x 105– approximately 1 x 106colony forming units per carrier. In 

addition, conduct a confirmatory test using virulent B. anthracis spores (or a surrogate 

acceptable to EPA) inoculated on thirty carriers representing each of two types of surfaces 

(porcelain penicylinders and silk suture loops) on two samples, representing two different 
5

batches of the product (a total of 120 carriers).

(A) Evaluation of sporicidal success. The product should kill all of the test spores on 

all of the 840 carriers without any failures.

(B) Reserved.

(ii) Test procedure for sporicidal decontaminants--qualitative testing. The Agency 

recommends use of the Official Methods of Analysis of AOAC International, Official Method 

966.04 Sporicidal Activity of Disinfectants test (Ref. 1) using virulent B. anthracis spores (or a 

surrogate acceptable to EPA). Sixty carriers representing either or both of two types of surfaces 

(porcelain penicylinders and/or silk suture loops) should be tested on three samples representing 

three different batches of product, one of which should be at least 60 days old. The inoculum 

employed should provide a target count of 1 x 105– approximately 1 x 10

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colony forming units 

per carrier. If one surface type is tested, then there are 60 carriers per sample, or a total of 180 

carriers; if both surfaces types are tested, then the total number of carriers is 360.

(A) Evaluation of sporicidal success. The product should kill all of the test spores on 

all of the 180 (or 360) carriers without any failures.

(B) Reserved.

(iii) Test procedure for sporicidal decontaminants--quantitative testing. The 

Agency recommends the use of a well developed, quantitative sporicidal test method acceptable 

to EPA using virulent B. anthracis spores (or a surrogate acceptable to EPA) on porous and/or 

nonporous surfaces acceptable to EPA. The inoculum employed should provide a target count of 

approximately 1 X 10

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colony forming units per carrier. The product should be tested on three 

samples representing three different batches of product, one of which should be at least 60 days 

old. The number of carriers will vary depending on the test method. The coupon material(s) 

should be representative of those found at the site(s) that appear on the product’s labeling, and be 

acceptable to EPA.

(A) Evaluation of sporicidal success. The product should achieve a log reduction of at 

least 6 logs based on recoverable spores.

(B) Reserved.

(2) Simulated use testing for gas and vapor products—(i) Test procedure. In

addition to conducting one of the three laboratory studies in paragraphs (k)(1)(i) through 

(h)(1)(iii) of this guideline, simulated-use testing should also be conducted for vapor and gas 

products. Protocols for the simulated-use test should be submitted to the Agency for review and 

approval prior to conducting the test. The testing should be conducted under conditions that are 

representative of the uses specified on the product’s labeling, and in a setting that is 

representative of the label use site(s). For example, a product intended for use in a room or a 

large warehouse should be tested in an empty room or large chamber. The purpose of the test 

would be to assure that key parameters for efficacy (chemical concentration, temperature, 
6

relative humidity and contact time) are accurately monitored and maintained throughout the 

enclosed space, and establish product generation rate (lbs/hr) and rate/volume (lbs/hr/ft3).

(ii) Additional considerations. Important issues to consider in developing the protocol 

for this test include:

(A) The test should be set up in a sealed enclosure at least the size of a typical office and 

designed to measure the distribution of the product and conditions needed to meet the measure of 

success in the laboratory efficacy test. Items that might be treated (e.g., dressers, upholstered 

furniture, carpet, etc,) during an actual fumigation, should be included in this test. 

(B) The protocol should specify the dimensions of the enclosure, number and location of

monitoring devices (e.g., for gas or vapor concentration, total mass of gas or vapor injected into 

the enclosure, temperature, relative humidity), product application equipment, heaters and fans, 

contact time, etc. The equipment used to monitor and maintain these test parameters should be 

described. 

(C) All recorded test results pertaining to the test conditions/parameters should be 

submitted to the Agency. The maximum volume of space that can be treated with a particular 

unit should be reported to the Agency. The minimum total mass of gas or vapor required to 

maintain the required concentration and contact time per cubic foot of space to be 

decontaminated should be reported.

(D) Appropriate positive and negative controls should be employed.

(E) This test must be conducted either in accordance with Good Laboratory Practices 

(GLP) per 40 CFR Part 160 or in a federal laboratory with an appropriate Quality Assurance 

Project Plan (QAPP).

(iii) Evaluation of sporicidal success. Measurements should show that the same 

concentration, temperature, and relative humidity, can be maintained for the required contact 

time needed to achieve 100% kill (i.e., no growth on any of the carriers) in the qualitative 

laboratory test, or a 6 log reduction in the quantitative test is demonstrated in the simulated-use 

test. In addition, measurements of the fumigant mass injection/generation rate (e.g., 

pounds/hour), divided by the volume of the simulated use test bed, that was used to arrive at the 

required generation rate/volume (e.g., pounds per hour/cubic foot) for the fumigation, should be 

included with the data, and listed on the product label.

(j) 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

material in paragraphs (j)(1)(i) through (j)(1)(xiv) in this guideline:

(i) Study title;

(ii) Product Identity;
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(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 

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

(2) Data Report 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 (Ref. 3); 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 material in paragraphs 

(j)(2)(i) through (j)(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;
8

(vi) Manufacture date of each product batch; 

(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 is 

encouraged to 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 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.

(k) 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, 

Official Method 966.04 Sporicidal Activity of Disinfectants, 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,

Official Method 2008.05 Quantitative Three Step Method (Efficacy of Liquid Sporicides Against 

Spores of Bacillus subtilis on a Hard Nonporous Surface), Eighteenth edition. AOAC 

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

(3) Annual Book of ASTM Standards, Standard Quantitative Carrier Test Method to 
9

Evaluate the Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal Potencies of Liquid 

Chemical Germicides, Designation E 2197-02. American Society for Testing and Materials, 100 

Barr Harbor Drive, West Conshohocken, PA 19428, current edition.