Document ID: EPA-HQ-OPPT-2009-0150-0016
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2012-06-21T04:00Z

United States
Environmental Protection Agency
   Office of Chemical Safety
   and Pollution Prevention
   (7101)
EPA 730-C-11-003
May 30, 2012

Product Performance
Test Guidelines

OCSPP 810.2400:

Disinfectants and Sanitizers for Use on Fabrics and Textiles  -  Efficacy Data Recommendations

                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                        
                                     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 guidelines developed after April 22, 2010 will use the new acronym (OCSPP) in their title.
  
  	 The OCSPP 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.  At places in this guidance, the Agency uses the word "should."  In this guidance, the 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 these guidelines electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & 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.
  

OCSPP 810.2400:  Fabrics and Textiles  -  efficacy data recommendations.
 				
	(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) (7 U.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 disinfectants or sanitizers on fabrics and textiles.

      (2) Background.  The source materials used in developing this OCSPP test guideline are OPP guidelines 91-4 Products for use on fabrics and textiles 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 fabrics and textiles, and which bear label claims as disinfectants or sanitizers. 
      
		(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 disinfection and sanitization of fabrics and textiles.  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 Chemists (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.

      (d)  Laundry additives (Non-residual).  This section addresses efficacy testing for antimicrobial products which bear label claims for use in the treatment of laundry (as a pre-soak treatment) or in household and commer - cial laundry operations to provide various levels of antimicrobial activity including disinfection, sanitization or residual sanitization.  

      (1)  Disinfecting pre-soak treatments.  Effectiveness of products for "one-step" cleaning and disinfecting of hard surfaces (in the presence of moderate amounts of organic soil) may be extrapolated to disinfection of soiled fabrics by total immersion in the use solution prior to routine laun - dry operations.

      (i) Limited disinfectants. -- (A) Test procedure.  The Agency recommends the use of the AOAC International Use-Dilution Methods (Ref. 1) modified to include 5% organic soil. Sixty carriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against Salmonella enterica (S. enterica) American Type Culture Collection (ATCC 10708) (formerly designated as Salmonella choleraesuis) for effectiveness against Gram-negative bacteria, or Staphylococcus aureus (S. aureus) (ATCC 6538) for effectiveness against Gram-positive bacteria.
            
      (B)  Evaluation of success for limited disinfectant pre-soak treatments.  The product should kill all test microorganisms on 59 out of each set of 60 carriers in <=10 minutes.  In addition, per the 2009 AOAC revisions for the Use-Dilution Method, "The mean log density for S. aureus must be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10[6]); a mean log density <6.0 invalidates the test."  
	
	(ii) General or Broad Spectrum Disinfectants. -- (A) Test procedure. The Agency recommends the use of the AOAC International Use-Dilution Methods (Ref.1), modified to include 5% organic soil.   Sixty carriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against both S. enterica (ATCC 10708) and S. aureus (ATCC 6538).  

      (B) Evaluation of success for general or broad spectrum disinfectant pre-soak treatments.  The product should kill all test microorganisms on 59 out of each set of 60 carriers in <=10 minutes.  In addition, per the 2009 AOAC revisions for the Use-Dilution Method, "the mean log density for S. aureus must be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10[6]); a mean log density <6.0 invalidates the test."  
.

	(iii)  Hospital or healthcare disinfectants.--(A)  Test procedure.  The Agency recommends the use of the AOAC International Use-Dilution Methods (Ref.1) modified to include 5% organic soil.   Sixty carriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against each of the following: S. aureus (ATCC 6538), and Pseudomonas aeruginosa (P. aeruginosa) (ATCC 15442).
            
      (B)  Evaluation of success for hospital or healthcare disinfectant pre-soak treatments.  The product should kill all test microorganisms on 59 out of each set of 60 carriers in <=10 minutes.  In addition, per the 2009 AOAC revisions for the Use-Dilution Method, "the mean log density for S. aureus and Pseudomonas aeruginosa must be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10[6]); a mean log density <6.0 invalidates the test."  
      .
      
	(2) Sanitizing pre-soak treatments.  Effectiveness of products for "one-step" cleaning and sanitizing of hard surfaces (in the presence of moderate amounts of organic soil) may be extrapolated to sanitization of soiled fabrics by total immersion in the use solution prior to routine laun - dry operations.  

       (i) Test Procedure.  The Agency recommends the following test procedure.
       	
      (A)    The American Society for Testing and Materials (ASTM) Test Method for Efficacy of Sanitizers Recommended for Inanimate Non-Food Contact Surfaces (E1153-03) modified to include 5% organic soil, (Ref.2).  Three product samples, representing three different batches, one of which should be at least 60 days old, should be tested against each test bacterium. Unglazed ceramic tile should be used for the test carrier.  Five (5) test carriers and 3 control carriers should be used for each test.  The test microorganisms should be: Staphylococcus aureus (ATCC 6538) and Klebsiella pneumoniae (K. pneumoniae) (ATCC 4352).  Enterobacter aerogenes (ATCC 13048) may be substituted for K. pneumoniae. The ASTM method states "an average of at least  7.5 x 105 organisms must have survived the inoculated control squares for the test to be valid." 
       
	
      (ii) Evaluation of success for sanitizing pre-soak treatments.  The results should demonstrate a reduction of  >=99.9 percent (a 3-log10 reduction) in the number of each test microorganism over the parallel control count in <=5 minutes.
      
	(3)  Disinfecting laundry. -- (i)  Test procedures.  The Agency recommends a simulated-use study using the American Society for Testing and Materials (ASTM) Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants (E 2274-03) (Ref. 3) or Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations (E 2406-04) (Ref. 4).  Alternatively, an actual in-use study utilizing washing machines may be used.  The following elements should be used in either the simulated-use or actual in-use study.

	(A) The test microorganisms should be Klebsiella pneumoniae (ATCC 4352) and Staphylococcus aureus (ATCC 6538) for broad-spectrum disinfection.  If the product is intended for use in healthcare facilities, it should also be tested against Pseudomonas aeruginosa (ATCC 15442) in addition to S. aureus and K. pneumoniae.
	(B) Tests should be conducted with three samples representing three different batches, one of which is at least 60 days old. Each sample should be tested with nine fabric swatches against each of the test bacteria.

	(C) The basic bacteriological procedures should be performed as described in ASTM Methods E-2274 and E-2406.

	(D) The method should be designed to include testing of both the fabric and laundry water (5 mL from the automatic washer, or 0.5 mL from the simulated washing device) in individual wide-mouth jars containing subculture media and neutralizers.  The laundry water-to-media volume ratio should not exceed 1:40.

	(E) The effectiveness of an antimicrobial laundry product may be altered by differences in laundry machine types (top loading vs. high efficiency machines).  Therefore, the type of machine should be taken into consideration when conducting efficacy tests.  The water to fabric ratio in common top loading machines is approximately 10:1 (wash volume to fabric weight), while with high efficiency laundry machines, it can be as low as approximately 2.5:1.  It should be noted that machine manufacturers are constantly upgrading machines and therefore, the fabric to water ratios noted above should be considered as examples.

	(F) Growth or no-growth should be recorded after a 48-hour incubation period.

	(ii)  Evaluation of disinfection success.  There should be no growth in the subculture from the fabric and no growth in the subculture from the laundry water for all test microorganisms.  This lack of growth should be demonstrated against each test microorganism within the contact time claimed on the label.  

	(4)  Sanitizing laundry additives. -- (i)  Test procedures.  The Agency recommends a simulated-use study using the American Society for Testing and Materials (ASTM) Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants (E 2274-03) (Ref. 3) or Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations (E 2406-04) (Ref. 4).  Alternatively, an actual in-use study utilizing washing machines may be used.  The following elements should be used in either the simulated-use or actual in-use study.

	(A)  Tests should be conducted with three samples representing three different batches, one of which is at least 60 days old.  Each sample should be tested using three cloth swatches for each target microorganism (Klebsiella pneumoniae (ATCC 4352) and Staphylococcus aureus (ATCC 6538)).	

	(B)  The basic bacteriological procedures should be performed as described in ASTM Methods E-2274 and E-2406.

	(C)  The method should be designed to include testing of both the fabric and laundry water (5 mL from the automatic washer, or 0.5 mL from the simulated washing device) in individual wide-mouth jars containing subculture media and neutralizers.  The laundry water-to-media volume ratio should not exceed 1:40.

	(D)  ) The effectiveness of an antimicrobial laundry product may be altered by differences in laundry machine types (top loading vs. high efficiency machines).  Therefore, the type of machine should be taken into consideration when conducting efficacy tests.  The water to fabric ratio in common top loading machines is approximately 10:1 (wash volume to fabric weight), while with high efficiency laundry machines, it can be as low as approximately 2.5:1.  It should be noted that machine manufacturers are constantly upgrading machines and therefore, the fabric to water ratios noted above should be considered as examples.

	(E)  Quantitative bacteriological assays should be conducted and plate counts reported for the cloth swatches and laundry water
  
	(ii)  Evaluation of sanitization success.  The results should demonstrate a reduction of >=99.9 percent (a 3-log10 reduction ) in bac - teria over the control count for both laundry water and fabric.  This reduction should be demonstrated against each test microorganism within the contact time claimed on the label.

	(e)  Laundry additives (Residual).  This section addresses efficacy testing for antimicrobial products which bear claims to provide residual self-sanitizing activity (i.e., reduction in the number of microorganisms which may contaminate the items) on treated fabrics when used in automatic washing machines (usually in the final rinse).  

	(1)  Self-sanitizing additives.-(i) Test Procedure.  The Agency recommends use of the American Association of Textile Chemists and Colorists (AATCC) Test Method 100-2004 Quantitative Procedure: Assessment of Antibacterial Finishes on Textile Materials (Ref. 5), or the Standard Test Method for Determining the Antimicrobial Activity of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions (ASTM E2149-01) (Ref. 6).

	(A)  Testing should be conducted on three samples, representing three different batches of the product, one of which should be at least 60 days old.  The test microorganisms should be Staphylococcus aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352).

	(B)  Quantitative bacteriological assays should be performed at the following time intervals: 0, 30 min., 1-hr, 2-hr, 3-hr, 6-hr, and 24-hr.  Consideration by the Agency can be given to fewer or different time intervals, depending on the label claims, on a case-by-case basis.

	(ii)  Evaluation of Self-sanitizing success.  The results should demonstrate a reduction of >=99.9 percent (a 3-log10 reduction ) in bac - teria over the control count for both laundry water and fabric.  This reduction should be demonstrated against each test microorganism within the contact time claimed on the label.

	(6)  Impregnated self-sanitizing fabrics and textiles.

	A protocol should be submitted for Agency approval prior to conducting any tests for impregnated self-sanitizing label claims.

	(f)  Carpet sanitizers.  This section addresses efficacy testing for antimicrobial products which bear label claims for use in sanitizing carpets.

	(1)  Test procedure.  There is no standard test method for evaluating the effectiveness of sanitizers for use on carpets.  Applicants should contact the Agency and schedule a pre-registration meeting to discuss protocol development.  During the meeting, the applicant should provide an agenda of items for discussion, a draft label with the claims to be supported by the proposed protocol, and any research and development product performance data the applicant may have generated in support of the application.  The following items should be taken into consideration when developing protocols and generating data for carpet sanitizers.

	(i) The test microorganisms for carpets that are used in residential, commercial, or industrial settings (including, but not limited to; homes, schools, office buildings, restaurants, factories) should be Staphylococcus aureus (S. aureus)(ATCC 6538) and Enterobacter aerogenes (E. aerogenes)(ATCC 13048).  Applicants who intend to market their product to healthcare facilities (e.g., hospitals, hospices, dental and medical clinics, nursing homes, etc) should also test against Pseudomonas aeruginosa (ATCC 15442), in addition to S. aureus and E. aerogenes.  

	(ii)  Three product samples representing three separate batches, one of which is at least 60 day old, should be tested against the microorganisms listed above, on 2 different types of representative carpet (e.g., nylon, olefin, polyester). If the product is intended to be used on wool carpet, an additional representative sample of wool carpet should be tested.

	(iii)  All carpet samples should be identified by the pile fiber, pile yarn weight of finished carpet, pile density, and tuft height.  The carpet should be cut into 8 x 12 inch pieces.

	(iv)  Controls should be incorporated into the test protocol to demonstrate that bacteriostatic agents in the carpet pile or backing do not interfere with the test results.

	(2)  Evaluation of sanitizing success.  The results should demonstrate a reduction of >=99.9 percent (a 3 log10 reduction) in the number of each test microorganism over the scrubbed control count within the contact time claimed on the label.

	(g)  Mattress, pillows and upholstered furniture treatments. This section addresses efficacy testing for antimicrobial products which bear label claims for use as a sterilant, disinfectant or sanitizer on mattresses and upholstered furniture.

	(1)  Test procedure.  The use of gases and vapors is currently the only effective and practical means of treating mattresses, upholstered furniture, pillows, and similar objects to sterilize, disinfect, or sanitize against pathogenic microorganisms.  Simulated-use studies, in which artificially contaminated articles (e.g., mattresses, upholstered furniture, pillows) are employed, should be performed to demonstrate the level of effectiveness intended (sterilization, disinfection, or sanitization). Potential applicants should contact the Agency and schedule a pre-registration meeting to discuss protocol development.  During the meeting, the applicant should provide an agenda of items for discussion, a draft label with the claims to be supported by the proposed protocol, and any research and development product performance data the applicant may have generated in support of the application.  The following items should be taken into consideration when developing protocols for generating data for mattresses, pillows and upholstered furniture.

	(i)  Each test article should be inoculated throughout the entire article.

	(ii)  Samples should be taken or withdrawn randomly from the entire article and cultured for microbial growth.

	(iii)  A control using a similar untreated article should be employed.

	(iv)  For claims of sterilization, the test microorganisms should be Bacillus subtilis (B. subtilis) (ATCC 19659) and Clostridium sporogenes (C. sporogenes) (ATCC 3584).  For claims of broad-spectrum disinfection, the test microorganisms should be Staphylococcus aureus (S. aureus) (ATCC 6538) and Salmonella enterica (S. enterica) (ATCC 10708).  If the product is used in hospitals, Pseudomonas aeruginosa (P. aeruginosa) (ATCC 15442) should be tested along with S. aureus. Label claims for sanitization should be tested against S. aureus and Klebsiella pneumoniae (K. pneumoniae) (ATCC 4352).  Enterobacter aerogenes (E. aerogenes) (ATCC 13048) can be substituted for K. pneumoniae.

	(v)  Testing should be conducted on three samples representing three different batches of the product, one of which is at least 60 days old.

	(h) Surface Sanitization of Fabrics and Textiles.  This section addresses efficacy testing for antimicrobial products that bear claims of sanitization to the surface of fabrics and textiles.
      	(1)  Test Procedure.  The Agency recommends the use of The American Society for Testing and Materials (ASTM) Test Method for Efficacy of Sanitizers Recommended for Inanimate Non-Food Contact Surfaces (ASTM E1153-03) (Ref. 2).  Three product samples, representing three different batches, one of which should be at least 60 days old, should be tested against Staphylococcus aureus (ATCC 6538) and either  Klebsiella pneumoniae (ATCC 4352) or Enterobacter aerogenes (ATCC 13048).  The ASTM method states "an average of at least  7.5 x 105 organisms must have survived the inoculated control squares for the test to be valid." 
 Two different fabric types should be tested.  The fabrics should represent natural fabrics, such as cotton, and synthetic fabrics, such as polyester or rayon.

	(2)  Evaluation of sanitizing success.  The results should demonstrate a reduction of >=99.9 percent (a 3-log10reduction) in the number of each test microorganisms over the scrubbed control count within 5 minutes.

      (i)  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.

	(h) References:  The references in this paragraph may be consulted for addi - tional background information.

      (1) Official Methods of Analysis of the Association of Official Analyt - ical Chemists, Use-Dilution Methods, Chapter 6, Disinfectants; Current edition.  Association of Official Analytical Chemists (AOAC), 2200 Wilson Boulevard Arlington, VA 22201.
      
      (2) Annual Book of Standards, Test Method for Efficacy of Sanitizers Recommended for Inanimate Non-Food Contact Surfaces, Designation E1153-03.  American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428.

      (3) Annual Book of Standards, Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants (E 2274-03).  American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428.
      
      (4) Annual Book of Standards, Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations (E 2406-04).  American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428.
      
      (5)  American Association of Textile Chemists and Colorists (AATCC) Test Method 100-2004 Quantitative Procedure: Assessment of Antibacterial Finishes on Textile Materials
      
(6)  Book of Standards, Standard Test Method for Determining the Antimicrobial Activity of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken,