
United States
Environmental Protection
Agency

Office of Chemical Safety and Pollution Prevention
(7510P)
	EPA 712-C-07-056
                                                   	January (insert date), 2011
	Product Performance Test Guidelines
		
		OCSPP 810.2100:
            Sterilants -- Efficacy Data Recommendations




                                       


                                       
                                       
                                        
                                     NOTICE
  
            This guideline is one of a series of test guidelines established by the Office of Chemical Safety and Pollution Prevention (OCSPP) (formerly the Office of Prevention, Pesticides and Toxic Substances (OPPTS) prior to April 22, 2010), United States Environmental Protection Agency for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug, and Cosmetic (FFDCA) (21 U.S.C. 346a), referred to hereinafter as the harmonized test guidelines.
  
  	The harmonized test guidelines serve as a compendium of accepted scientific methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA.  This document provides guidance for conducting appropriate tests, and is also used by EPA, the public, and the companies that are subject to data submission requirements under TSCA, FIFRA and/or the FFDCA.  At places in this guidance, the Agency uses the word "should."  In this guidance, use of "should" with regard to an action means that the action is recommended rather than mandatory.  As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances warrant and without prior notice.  The methods contained in this guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. You may propose alternatives to the methods recommended in these guidelines, with your supporting rationale. The Agency will assess such proposals on a case-by-case basis.  
  
  	For additional information about OCSPP harmonized test guidelines and to access the guidelines electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & Guidelines" on the left side navigation menu.  You may also access the guidelines in http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
  
  


OPPTS 810.2100:   Sterilants -- 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 sterilants.  
      
      (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 sterilants.
      
      (c)  General considerations
      
      (1) This guideline recommends methods for use in tests to be conducted to address the data requirements for pesticide registration. Good Laboratory Practice Standards (GLP) as defined in 40 CFR Part 160 apply to studies to support sterilants used on hard surfaces.  According to 40 CFR §160.17: "EPA may refuse to consider reliable for purposes of supporting an application for a research or marketing permit any data from a study which was not conducted in accordance with this part." 40 CFR §160.12 (b) requires with any submitted research data "[a] A statement that the study was conducted in accordance with this part; [b] A statement describing in detail all differences between the practices used in the study and those required by this part; or [c] A statement that the person was not a sponsor of the study, did not conduct the study, and does not know whether the study was conducted in accordance with this part." Note: The Association of Official Analytical Chemicals (AOAC) recommended tests are designed to be conducted as written. For deviations (e.g., cultures grown with shaking instead of static, dilution of culture prior to drying on carriers) proposed to be used in the conduct of these tests, obtain written approval from the Agency and document such deviations in the study reports submitted to the Agency. The Agency may consult with the AOAC prior to accepting modifications to their standardized methods. Refer to OCSPP Test Guideline 810.2000 for general testing recommendations prior to initiating tests.
      
      (2) Validation testing approaches, which may be needed to augment the full range of efficacy tests in special circumstances, are also described.
      
      (3) Type of surface. When an antimicrobial product is intended to be effective in treating a hard, porous surface, some of the recommended methods may be modified to simulate this more stringent condition by substitution of a hard, porous surface carrier (e.g., porcelain penicylinder or unglazed ceramic tile) for the hard, nonporous surface carrier (stainless steel cylinder or glass slide) specified in the method.  In addition, control data (e.g., quantitation of dried carrier, neutralization confirmation, sterility controls) should be developed to assure the validity of the test results when this modification of the method is employed. Since the use of a hard, porous surface would simulate the more stringent test condition, demonstrated efficacy on hard, porous surfaces would suffice to support an analogous claim for efficacy of the product on hard, non-porous surfaces as well.
      
      (4)  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 [MRID]) to the supporting data developed for the original product are cited by the applicant.
      
      (ii)  Minor Formulation Change in a Registered Product. In this situation, the change in the formulation is relatively minor, e.g., a change of an inert ingredient. The label claims and directions for use are unchanged from those accepted for the registered formulation, and specific references (MRID) to the supporting data developed for the original formulation are cited by the applicant. If the only change in the formulation is the addition of a fragrance or dye, confirmatory data do not need to be submitted. However, when the product is an aerosol formulation, confirmatory data should be submitted for all formulation changes, including the addition of fragrances and dyes.
      
      (iii) The confirmatory data are to be developed from testing the applicant's own finished product. When the test methodology utilized in deriving the original supporting efficacy data were modified to include additional elements not specified in the recommended method, such as organic soil, hard water, longer or shorter contact time, etc., the confirmatory data should be produced under similarly modified conditions.
      
      (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 I (Ref. 1) to demonstrate the sterilant efficacy of products. For Bacillus subtilis, Method II should be used. 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 >=60 days old (240 carriers per sample, or a total of 720 carriers). The inoculum employed should provide a count of 1 x 10[5]  -  1 x 10[6] spores per carrier. Any sterilant which is a vapor or gas and is 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.g., growth of test organism after carrier treatment).
      
      (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, or can be tested in the same laboratory with separate study director, Quality Assurance Unit and staff.
      
      (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 I (Ref. 1) to demonstrate the sterilant efficacy of products. For Bacillus subtilis, Method II should be used. 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 10[5]  -  1 x 10[6] spores per carrier.
      
      (2) Evaluation of sterilant success. The product should kill the test spores on all 120 carriers without any failures (e.g., growth of test organism after carrier treatment).
      
      (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 four possible options, as described in paragraphs (g)(1)(i) through (g)(1)(iv) 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 I (Ref. 1) to demonstrate the sterilant efficacy of products, as described in (d)(1). For Bacillus subtilis, Method II should be used. In addition, conduct a confirmatory test using the Official Methods of Analysis of AOAC International, Official Method 966.04 Sporicidal Activity of Disinfectants test, modified for C. difficile testing. 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). The inoculum employed should provide a target count of 1 x 10[5]  -  1 x 10[6] spores per carrier.
      
      (ii) Evaluation of sporicidal success. The product should kill all of the test spores on all of the initial and confirmatory (780 carriers) without any failures (e.g., growth of test organism after carrier treatment). 
      
      (ii) Test procedure for C. difficile sporicides--qualitative testing.  The Agency recommends use of the Official Methods of Analysis of AOAC International, Official Method 966.04 Sporicidal Activity of Disinfectants test, modified for C. difficile testing (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 >=60 days old (a total of 180 carriers). The inoculum employed should provide a target count of 1 x 10[5]  - 1 x 10[6] spores 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 (e.g., growth of test organism after carrier treatment).
      
      (iii) Test procedure for C. difficile sporicides--quantitative testing. The Agency recommends use of the AOAC Method 2008.05: Quantitative Three Step Method (TSM) (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 > 10[6] spores per carrier. The product should be tested on three samples representing three different batches of product, one of which should be >=60 days old. For the AOAC TSM use 10 carriers per lot. For the ASTM E2197, use 10 carriers per test lot and 3 carriers for the control. 
      
      (A) Evaluation of sporicidal success. The product should achieve a log reduction of >=6 logs based on recoverable spores. 
      
      (iv) Test procedure for C. difficile sporicides  -  Towelettes. The Agency recommends use of the AOAC Sporicidal Activity Test, modified for towelettes. 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 > 10[6] spores per carrier. The product should be tested on three samples representing three different batches of product, one of which should be >=60 days old. One towelette will be used to wipe 10 carriers.  In addition to the efficacy testing, a determination for the amount of time the carrier remains wet should be made. This wetness determination will be used to generate the contact time to be used in the efficacy test.
      
      (A) Evaluation of sporicidal success. The product should achieve a log reduction of >=6 logs based on recoverable spores within the contact time measured in the wetness determination.
      
      (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 >=60 days old (240 carriers per sample, or a total of 720 carriers). The inoculum employed should provide a target count of 1 x 10[5]  - 1 x 10[6] spores 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 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 initial and confirmatory carriers (840 carriers) without any failures (e.g. growth of test organism after carrier treatment).
      
      (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 >=60 days old. The inoculum employed should provide a target count of 1 x 10[5]  - 1 x 10[6] 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 sporicidal success. The product should kill all of the test spores on all of the 180 (or 360) carriers without any failures (e.g., growth of test organism after carrier treatment).
      
      (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 1 X 10[7] spores per carrier. The product should be tested on three samples representing three different batches of product, one of which should be >=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 >=6 logs based on recoverable spores.
      
      (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, 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 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.  
      
	(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 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.
      
	(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 of the test organism on any of the carriers) in the qualitative laboratory test, or a >=610 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) 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 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 commercial sterilants for aseptic packaging of low acid foods. 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, one of which should be >= 60 days old (180 carriers per organism). The inoculum employed should provide a count of 1 x 105  -  1 x 106 spore forming units per carrier. Other 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. The type of spore former to be used is dependent on the type of chemical sterilant as follows:

      (2) Hydrogen peroxide sterilants should be tested against Bacillus subtilis (ATCC 19659) and Clostridium sporogenes (ATCC 3584).
      
      (3) Peroxyacid based sterilants should be tested against Bacillus subtilis (ATCC 19649), Clostridium sporogenes (ATCC 3584) and Bacillus cereus (ATSS 14579).
      
      (4) 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.

	(i) Evaluation of sterilant success. The product should kill the test spores on all of the carriers without any failures. 
      
      (k)  Data collection and reporting -- (1) General. 
      To assist in the proper review and evaluation of product performance, complete descriptions of the test employed and the results obtained should be submitted to the Agency.  All test reports should include, at the least, the following information:
      
      (i) Study title;
      
      (ii) Product Identity;
      
      (iii) Guideline number/Data Requirement; 
      
      (iv) Identification of the testing laboratory or organization;
      
      (v) Location where the test was performed;
      
      (vi) Name(s) of the person(s) responsible for the test;
      
      (vii) Statement of Confidentiality Claims;
      
      (viii) Statement of 40 CFR Part 160 Good Laboratory Practice compliance and Quality Assurance Statement;
      
      (ix) Purpose of the study;
      
      (x) Date and time of the start and end of the test;
      
      (xi) Test employed and any modifications (e.g., organic soil, hard water, etc), when using standard tests (e.g., AOAC, ASTM, etc) all deviations to the test methods should be reported;
      
      (xii) Test microorganisms employed, including identification of the specific strain (ATCC or other);
      
      (xiii) Description of the test substance, including the percent of active ingredient;
      
      (xiv) Concentration or dilution of the product tested and how prepared;
      
      (xv) Number of samples, batches and replicates tested;
      
      (xvi) Manufacture date of each product batch; 
      
      (xvii) Identification of all material or procedural options employed, where such choice is provided for or recommended in the test method selected (e.g., growth media, drying time for inoculated carriers, neutralization confirmation and/or subculture media, secondary subculturing);
      
      (xviii) Test exposure conditions (e.g., contact time, temperature, and relative humidity);
      
      (xix) Complete reports of results obtained for each replication;
      
      (xx) Any control data essential to establish the validity of the test.
      
      (xxi) Carrier counts;
      
      (xxii) Statistical treatment of the data;
      
      (xxiii) Conclusions;
      
      (xxiv) References;
      
      (xxv) Appendices, including study protocol and all raw data reports associated with the conduct of the study.
      
      The applicant is encouraged to use the EPA's standard efficacy report format, which may be found at http://www.epa.gov/oppad001/efficacystudystandards.htm.
      
      (2) Data for modifications of recommended methods. Where recommended methods are modified for testing conducted 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.
      
      (3) Data for other methods. When recommended methods, or modifications thereto, are not employed to develop efficacy data (such as actual in-use or many kinds of simulated-use testing), complete testing protocols should be submitted with the test reports. All materials and procedures employed in testing should be described in a manner consistent with original research reports published in technical or scientific journals. Where references to published reports or papers are made, copies or reprints of such references should be provided with the test reports. The applicant should submit the proposed testing protocols for in-use or simulated-use studies (with a proposed label to show the claims to be supported by the protocol) to the Agency for review and evaluation prior to initiation of the test.
      
      (l) References: The references in this paragraph may be consulted for addi - tional background information.
      
      (1) Official Methods of Analysis of the AOAC International, Chapter 6, Disinfectants, Official Method 966.04 Sporicidal Activity of Disinfectants, Current edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.
      
      (2) Official Methods of Analysis of the AOAC International, Chapter 6, Disinfectants, Official Method 2008.05 Quantitative Three Step Method (Efficacy of Liquid Sporicides Against Spores of Bacillus subtilis on a Hard Nonporous Surface), Current 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 Evaluate the Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal Potencies of Liquid Chemical Germicides, Designation E 2197. American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition.
