Document ID: EPA-HQ-OPPT-2009-0150-0028
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2015-06-10T04:00Z

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. 
  

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 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 x 10[5]  -  1 x 10[6] 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 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 method-recommended 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). 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 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 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 x 10[7] 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 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 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 x 10[5]  -  1 x 10[6] 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 addi - tional 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.
         
      (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.