United States

Environmental Protection

Agency

Office of Chemical Safety 

and Pollution Prevention

(7503P)

EPA XXX-X-XX-XXX

April 2015

Product Performance 

Test Guidelines

OCSPP 810.2100:

Sterilants & Sporicides

Recommendations for 

Efficacy Testing 
NOTICE

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

Environmental Protection Agency’s Office of Chemical Safety and Pollution Prevention 

(OCSPP) for use in testing pesticides and chemical substances to develop data for 

submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, 

et seq.), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et 

seq.), and section 408 of the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 

346a). Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides 

and Toxic Substances (OPPTS). To distinguish these guidelines from guidelines issued by 

other organizations, the numbering convention adopted in 1994 specifically included 

OPPTS as part of the guideline’s number. Any test guideline developed after April 22, 2010 

will use the new acronym (OCSPP) in their title. 

The OCSPP test guidelines serve as a compendium of accepted standardized 

methodologies and protocols that are intended to provide data to inform regulatory decisions 

under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the

tests, and is also used by EPA, the public, and the companies that are subject to data 

submission requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, 

these guidelines are not binding on either EPA or any outside parties, and the EPA may 

depart from the guidelines where circumstances warrant and without prior notice. At places 

in this guidance, the Agency uses the word “should.” In this guidance, use of “should” with 

regard to an action means that the action is recommended rather than mandatory. The

procedures contained in this guideline are strongly recommended for generating the data that 

are the subject of the guideline, but EPA recognizes that departures may be appropriate in 

specific situations. You may propose alternatives to the recommendations described in these 

guidelines, and the Agency will assess them for appropriateness on a case-by-case basis. 

For additional information about these test guidelines and to access the guidelines 

electronically, please go to http://www.epa.gov/ocspp and select the topic entitled, “Test 

Methods and Guidelines.” You may also access the guidelines in 

http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-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: Sterilant/Sporicide—Efficacy Data Recommendations for Public Health 

Uses

(a) Scope

(1) Applicability. This guideline describes test methods that EPA believes will generally 

satisfy testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) 

(7U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a). 

It addresses testing to demonstrate the effectiveness of antimicrobial pesticides bearing claims for 

use as a sterilant/sporicide. Refer to OCSPP 810.2000: General Considerations for Testing

Antimicrobial Agents for additional guidance. 

(2) Background. The source materials used in developing this OCSPP test guideline are 

OPP guidelines 91-2: Products for use on hard surfaces and 91-30: Acceptable methods (Pesticide 

Assessment Guidelines, Subdivision G, Product Performance. EPA report 540/9-82-026, October 

1982).

(b) Purpose. This guideline addresses efficacy testing for antimicrobial pesticides intended to be 

used on hard, inanimate, environmental surfaces, and, which bear label claims as a sterilant and/or 

sporicide.

(c) General Considerations

(1) Claims. This guideline provides guidance for developing data solely for the purpose of 

supporting a basic sterilant/sporicide claim with or without an additional sporicidal 

claim (e.g., Bacillus anthracis, Clostridium difficile). Furthermore, guidance is 

provided for applicants seeking only a sporicidal claim for specific spore-forming 

bacteria such as B. anthracis and C. difficile. 

(2) Verification Tests. Data submitted to support sterilant/sporicide claims are subject to 

independent verification testing in a second laboratory, or can be tested in the same 

laboratory with a new set of inoculated carriers, separate study director, Quality 

Assurance Unit and technical staff.

(3) Alternate Materials. When an antimicrobial product is intended to be effective in 

treating a specific hard porous surface, a method modification may be necessary which 

specifies the use of the porous material (as a carrier) in the test protocol. In addition, 

control carrier count data (mean log10 spores per carrier), neutralization confirmation

data, and sterility controls should be developed to assure the validity of the test results 

when these types of method modifications are employed. Method modifications should

be submitted to the Agency in writing for approval prior to data generation and 

collection. 

(4) Entire Room or Large Chamber Application. A simulated use test is needed when 

an antimicrobial product is intended for treating an entire room or large chamber. For 

general guidance, see section (g)(2) of this document; however, a testing protocol 

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specific to the product should be developed and submitted to the Agency for approval 

prior to data generation and collection.

(d) Sterilant/Sporicide Claim. This section provides testing guidance for products with 

sterilant/sporicide claims to inactivate spores of Bacillus subtilis and Clostridium sporogenes

on hard inanimate surfaces.

(1) Liquid Products (ready-to-use, dilutable concentrates and water soluble powder

formulations) and Gases.

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

(AOAC) Official Method 966.04 Sporicidal Activity of Disinfectants test

(revised 2013, see Ref. 1). To demonstrate efficacy of a product, testing against 

spores of Bacillus subtilis [American Type Culture Collection (ATCC) 19659]

and Clostridium sporogenes (ATCC 3584), each inoculated on two types of 

carriers (porcelain penicylinders and silk suture loops), should be conducted. 

Furthermore, neutralizer confirmation should be demonstrated using AOAC 

966.04, method II, section h using growth media and incubation conditions 

suitable for each microbe.

(ii) Batches. Three batches of the product at or below the lower certified limit(s) 

(LCL) listed on the confidential statement of formula (CSF) of the product 

should be tested. 

(iii) Number of Carriers and Related Requirements. Sixty carriers of each type 

should be tested against spores of both B. subtilis and C. sporogenes (240 

carriers per batch, or a total of 720 carriers for all three batches). For a valid 

test, a mean control count of 1 × 105 – 1 × 106 spores per carrier should be 

attained for both microbes on both carrier types. The inoculated carriers should

meet the level of acid resistance outlined in the AOAC 966.04 method.

(iv) Spore Production. Use AOAC 966.04 (Method I) for testing spores of C. 

sporogenes. For C. sporogenes, use cooked meat medium amended with 

MnSO4 for spore generation and inoculation of porcelain penicylinders and silk 

suture loops. Follow AOAC 966.04 (Method II) for testing spores of B. subtilis

on porcelain penicylinders and silk suture loops. For B. subtilis, use nutrient 

agar amended with MnSO4 for spore generation. 

(v) Claims with Use of a Device. Any sterilant which is a vapor or gas and is 

recommended for use in a specific device should be tested using the AOAC 

Method 966.04 in that specific device and according to the directions for use of 

that specific device. To address this situation, modifications to the AOAC 

Method 966.04 may be necessary; method modifications should be submitted 

to the Agency for review and approval prior to conducting the tests. 

(vi) Dacron Loops. Method 966.04 also provides for the use of Dacron loops (also 

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termed braided polyester), instead of silk suture loops, for testing peracetic acid 

containing products.

(vii) Evaluation of Success. The product should kill the test spores on all of the 720 

carriers without any positives (i.e., no growth of test organism in the subculture 

medium). No contamination of the culture tubes is allowed.

(viii) Verification Testing. For verification tests, follow the above testing guidance 

except 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 at the lower certified 

limit(s) (LCL). In addition, the inoculated carriers should meet the methodrecommended levels of acid resistance, control carrier counts and confirmation 

of neutralization should be conducted. For verification testing, the product 

should kill the test spores on all 120 carriers without any positives (i.e., no 

growth of test organism in the subculture medium). No contamination of the 

culture tubes is allowed.

 

(2) Sprays, mists and foams. A liquid product applied as a spray using a pump or trigger 

sprayer should be tested as a liquid as described above. For all other spray formulations 

types (aerosols) and other formulations such as mists, foams and gels, the applicant 

should submit a testing protocol consistent with the anticipated application and use 

pattern of the product for Agency review and approval prior to testing. Use AOAC 

method 966.04 for spore production, control counts, and neutralization testing.

(e) Sporicidal products with Clostridium difficile claims. Refer to the “Guidance for Efficacy 

Evaluation of Products with Sporicidal Claims Against Clostridium difficile” issued June 2014

(http://www.epa.gov/oppad001/cdif-guidance.html). A collaborative study is underway to 

evaluate the recommended test methods and issue revised guidance. 

(f) Sporicidal products with 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 

(f)(1)(i) through (f)(1)(iii) of this guideline.

(1) Liquids (ready to use, dilutable concentrates and water soluble powder 

formulations), gases and vapors.

(i) Test Procedure for Sterilant/Sporicide plus a B. anthracis Claim. The 

Agency recommends use of the AOAC Method 966.04 (Method II) to 

demonstrate the sterilant/sporicidal efficacy of products as described in 

(d)(1)(i)-Test Procedure. In addition, conduct a confirmatory test using 

virulent B. anthracis spores (or a surrogate species acceptable to EPA) 

inoculated on thirty carriers representing each of two types of surfaces 

(porcelain penicylinders and silk suture loops) on two product samples, 

representing two different batches of the product (a total of 120 carriers).

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Control carrier counts should be 1 x 105 to 1 x 106 spores per carrier. 

(A) Evaluation of Success. The product should kill all of the test spores 

on all of the initial and confirmatory carriers (840 carriers) without 

any positives (i.e., no growth of test organism in the subculture 

medium). No contamination of the culture tubes is allowed.

(ii) Test Procedure for Sporicidal Decontaminants--qualitative testing. The 

Agency recommends use of the AOAC International Official Method 966.04 

Sporicidal Activity of Disinfectants test (Method II, revised 2013, see 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, tested at or below certified 

limits (LCL) of the product. The inoculum employed should provide a target 

count of 1 × 105 –1 x 106 spores 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. Media sterility controls and 

system controls (check for aseptic technique during carrier transfer process) are 

recommended per the method.

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

all of the 180 (or 360) carriers without any positives (i.e., no growth of 

test organism in the subculture medium). No contamination of the 

culture tubes is allowed.

(iii) Test Procedure for Sporicidal Decontaminants--quantitative testing. The 

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

method acceptable to EPA such as AOAC Method 2008.05 or ASTM method 

E2197-11 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 1 × 107 spores per carrier. The 

product should be tested on three samples representing three different batches 

of product, one batch tested at or below certified limits (LCL). 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 Success. The product should achieve a mean log 

reduction of ≥6 logs per lot tested based on the number of viable spores

recovered.

(g) Simulated Use Testing for Gas and Vapor Products

(1) Test procedure. In addition to conducting one of the three laboratory studies in section 

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

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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, 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).

(2) Additional Considerations. Important issues to consider in developing the protocol 

for this test include:

(i) The test should be conducted in a sealed enclosure at least the size of a typical 

office or other room that simulates the intended use pattern 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. 

(ii) 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. 

(iii) 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.

(iv) Appropriate controls should be employed to assess both the sterility of the test 

system and viability of the spore inoculum. Uninoculated carriers and/or 

uninoculated biological indicators should be placed in the test chamber to assess 

sterility of the test environment. Unexposed inoculated carriers and/or 

biological indicators should be used to determine the suitability of the growth 

medium designed for the recovery of viable spores.

(v) 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). 

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

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

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required contact time needed to achieve 100% kill (i.e., no growth of the test 

organism on any of the carriers) in the qualitative laboratory test, or a ≥6 log10

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.

(h) Water-soluble powders and non-volatile liquid products for Commercial Sterilants for 

Aseptic Packaging of Low Acid Food. This section addresses efficacy tests for products with 

claims to inactivate spores on hard, non-porous, inanimate surfaces for aseptic packaging.

(1) Test procedure. The Agency recommends modifications of the AOAC International 

Official Method 966.04 as described above to demonstrate the sterilant efficacy of 

commercial sterilants for aseptic packaging of low acid foods. However, sixty carriers 

representing one type of surface (stainless steel penicylinders) should be tested against

spores on three samples representing three different batches of the product (180 carriers 

per organism). Products should demonstrate effectiveness in 20 seconds or less at 

temperatures less than 80 ̊C. If a longer contact time or a different temperature is 

required for the product, the applicant should contact the Agency prior to testing. The 

inoculum employed should provide a count of 1 × 105 – 1 x 106 spore forming units 

per carrier. Other modifications to the AOAC Method 966.04 to address this use should 

be submitted to the Agency for review and approval prior to conducting the tests. The 

type of spore former to be used is dependent on the type of chemical sterilant as follows:

(i) Hydrogen peroxide based sterilants should be tested against B. subtilis

(ATCC 19659), C. sporogenes (ATCC 3584), and B. cereus (ATCC 14579).

(ii) Peroxyacid based sterilants should be tested against B. subtilis (ATCC 

19659), C. sporogenes (ATCC 3584) and B. cereus (ATCC 14579).

(iii) For sterilant classes other than those listed above, consultation with EPA 

and FDA is recommended prior to generation of product data to identify the 

organisms for supporting aseptic packaging label claims.

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

carriers without any failures. No contamination of the culture tubes is allowed.

(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, 

Official Method 966.04 Sporicidal Activity of Disinfectants, 2013. AOAC 

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

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(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), 2013. 

AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 

20877-2417.

(3) Annual Book of ASTM Standards, Standard Quantitative Disk Carrier Test Method for 

Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal 

Activities of Chemicals, Designation E 2197-11. ASTM International, 100 Barr Harbor 

Drive, West Conshohocken, PA 19428-2959.

(4) Annual Book of ASTM Standards, Standard Test Method for Production of Clostridium 

difficile Spores for Use in Efficacy Evaluation of Antimicrobial Agents, Designation 

E2839-11. ASTM International, 100 Barr Harbor Drive, West Conshohocken, PA 

19428-2959.

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