United States

Environmental Protection Agency Office of Chemical Safety

and Pollution Prevention (7510P) EPA 712-C-07-074 Draft: May 11, 2011



# Product Performance
Test Guidelines

OCSPP 810.2400:
Disinfectants and
Sanitizers for Use on
Fabrics and Textiles— Efficacy Data
Recommendations2 NOTICE

This guideline is one of a series of test gui delines established by the Office of Chemical Safety and Pollution Prevention (OCS PP) (formerly the Offi ce of Prevention,

Pesticides and Toxic Substanc es (OPPTS) prior to Apr il 22, 2010), United States Environmental Protection Agency for us e 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 int ended 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 r equirements 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 m eans 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 circumstanc es warrant and without prio r notice. The methods contained in this guideline are strongly re commended 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 al ternatives to the methods recommended in these guidelines, with your supporting ra tionale. 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 si de 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-OPPT2009-0576.

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 Agen cy’s Information Quality Guidelines does not apply until this document is formally dissemi nated by the EPA in its final form. This draft document should only be cited or quoted in the context of pr oviding comments.3 OCSPP 810.2400: Fabrics and Textil es – efficacy data recommendations.

(a) Scope.—

(1) Applicability

. This guideline describes test me thods that EPA believes will generally satisfy testing r equirements 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 bear ing 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 effi cacy testing for antimicrobial pesticides intended to be used on fabrics and te xtiles, 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 pe rmit any data from a study which was not conducted in accordance with this par t.” 40 CFR §160.12(b) requires with any submitted research data“[a] statement describing in deta il all differences between the practices used in the study and those required by this part.” Note: The Association of Official Analytical Chemis ts (AOAC) recommended tests are designed to be conducted as written. For deviations (e.g., cultures grown with shaking inste ad of static, dilution of culture prior to drying on carriers) propos ed 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 A gency may consult with the AOAC prior to accepting modifications to their standar dized methods. Refer to OCSPP Test Guideline 810.2000 for general testing reco mmendations prior to initiating tests.

(d) Laundry additives (Non-residual). This section addresses efficacy testing for antimicrobial products which bear label recommendations for use in the treatment of laundry (as a pre-soak treatment) or in household and commercial laundry4 operations to provide various levels of antim icrobial activity including disinfection,

sanitization or residual sanitization.

(1) Disinfecting pr e-soak treatments. Effectiveness of products for “onestep” cleaning and disinfecting of hard surfaces (in the presence of m oderate amounts of organic soil) may be extrapolated to disinfec tion of soiled fabrics by total immersion in the use solution prior to routine laundry operations.

(i) Limited disinfectants .—(A) Test procedure. The Agency recommends the

use of the AOAC Inter national Use-Dilution Methods 1 (Ref. 1) modified to include 5% organic soil. Sixty carriers for each of thr ee samples, representing three different batches, one of which should be at leas t 60 days old, should be tested against

Salmonella enterica ( S. enterica ) American Type Culture Co llection (ATCC 10708) (formerly designated as Salmonella choleraesuis ) for effectiveness against Gramnegative bacteria, or Staphylococcus aureus ( S. aureus ) (ATCC 6538) for effectiveness against Gram-positive bacteria.

(B) Evaluation of success for limited di sinfectant pre-soak treatments. The product should kill all test microorganism s 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 Di sinfectants.—(A) Test procedure. The Agency recommends the use of the AOAC Inte rnational Use-Diluti on Methods (Ref.1), modified to include 5% organic soil. Sixt y carriers for each of three samples, representing three different batches, one of which shoul d 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 presoak treatments. The product should kill all test microorganisms on 59 out of each

set of 60 carriers in ≤ 10 minutes. In addition, per t he 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 Internati onal Use-Dilution Methods (Ref.1) modified to include 5% organic soil. Sixty carrier s 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).

1 Per Pesticide Registration Notice 2001-4, the ph enol resistance assay is no longer expected to be

conducted and submitted for all carrier based tests.5 (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 UseDilution 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 pr oducts 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 laundry operations.

(i) Test Procedure . The Agency recommends t he following test procedure.

(A) The American Society for Testing and Materials (ASTM) Test Method for Efficacy of Sanitizers Re commended for Inanimate Non-Food Contact Surfaces (E1153-03) modified to include 5% organic so il, (Ref.2). Three product samples, representing three different batches, one of which shoul d 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 contro l 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 10 5 organisms must have surviv ed 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-log 10 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 Laun dry Sanitizers and Disinfectants (E 2274- 03) (Ref. 3) or Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Wa shing Operations (E 2406-04) (Ref. 4). Alternatively, an actual in-use study utiliz ing washing machines may be used. The following elements should be used in either t he 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 fa cilities, it should also be tested against Pseudomonas aeruginosa (ATCC 15442).

(B) Tests should be conducted with three samples representing three different6 batches, one of which is at least 60 days ol d. 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 incl ude testing of bot h the fabric and laundry water (5 mL from the automatic was her, or 0.5 mL from the simulated washing device) in individual wide-mouth jars cont aining 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 mach ine should be taken into c onsideration when conducting efficacy tests. The water to fabric ratio in common top loading machines is approximately 10:1 (wash volume to fabric weight), whil e 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 machi nes and therefore, th e fabric to water ratios noted above are to be considered as examples.

(F) Growth or no-growth should be reco rded 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 gr owth should be demonstrated against each test microorganism within the contac t 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 fo r Evaluation of Laundry Sanitizers and Disinfectants (E 2274-03) (Ref. 3) or St andard Test Method for Evaluation of Laundry

Sanitizers and Disinfectants for Use in Hi gh Efficiency Washing Operations (E 2406- 04) (Ref. 4). Alternatively, an actual in-u se study utilizing washing machines may be used. The following elements should be used in either the simulated-use or actual inuse study.

(A) Tests should be conducted with three samples representing three different batches, one of which is at least 60 days ol d. 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 incl ude testing of bot h the fabric and laundry water (5 mL from the automatic was her, or 0.5 mL from the simulated washing7 device) in individual wide-mouth jars cont aining subculture media and neutralizers. The laundry water-to-media volu me 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 mach ine should be taken into c onsideration when conducting efficacy tests. The water to fabric ratio in common top loading machines is approximately 10:1 (wash volume to fabric weight), whil e 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 machi nes and therefore, th e fabric to water ratios noted above are to be considered as examples.

(E) Quantitative bacteriological assays should be conducted and plate counts reported for the cloth sw atches and laundry water

(ii) Evaluation of sanitization success. The results should demonstrate a

reduction of ≥ 99.9 percent (a 3-log 10 reduction ) in bacteria over the control count for both laundry water and fabric. This reduction should be demonstrated against each test microorganism within the cont act time claimed on the label.

(e) Laundry additives (Residual). This section addresses efficacy testing for antimicrobial products which bear claims to prov ide residual self-sanitizing activity (i.e.,

reduction in the number of microorganisms which may c ontaminate the items) on treated fabrics when used in automatic washing ma chines (usually in the final rinse).

(1) Self-sanitizing additives. -(i) Test Procedure. The Agency recommends

use of the American Association of Text ile Chemists and Colorists (AATCC) Test
Method 100-2004 Quantitative Pr ocedure: Assessment of Antibacterial Finishes on Textile Materials (Ref. 5), or the Standard Test Method for Determining the Antimicrobial Activity of Immobilized An timicrobial 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 bacteriol ogical assays should be per formed at the following time intervals: 0, 30 min., 1-hr, 2-hr, 3-hr, 6-hr, and 24-hr. Cons ideration 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-log 10 reduction ) in bacteria over the control count for both laundry water and fabric. This reduction should be demonstrated against each test microorganism within the cont act time claimed on the label.8 (6) Impregnated self-sanit izing fabrics and textiles.

[Reserved.] A protocol should be submitted for Agency approval prior to conducting any tests.

(f) Carpet sanitizers. This section addresses effica cy testing for antimicrobial products which bear label recommendations 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 ag enda 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 t he 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, fact ories) should be Staphylococcus aureus (S. aureus) (ATCC 6538) and Enterobacter aerogenes (E. aerogenes) (ATCC 13048). Applicants who intend to market their product to healthcare facilitie s (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, and additi onal 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 ba cking do not interfere wit h the test results.

(2) Evaluation of sanitizing success. The results should demonstrate a

reduction of ≥ 99.9 percent (a 3 log 10 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 uphol stered furniture treatments. This section addresses efficacy testing for antimicrobial products which bear a label9 recommendation for use as a sterilant, disi nfectant 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 ma ttresses, upholstered furniture, pillows, and similar objects to sterilize, disinfect, or sanitize against pathogenic microorganisms. Simulated-use studies, in which artificially contaminated articl es (e.g., mattresses, upholstered furniture, pillow s) are employed, should be performed to demonstrate the level of effectiveness intended (sterilization, disinfection, or sanitization). Potential applicants should contact the Agency and sc hedule 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 dev elopment product performance data the applicant may have generated in support of t he application. The following items

should be taken into consideration when devel oping protocols for generating data for mattresses, pillows and upholstered furniture.

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

(ii) Samples should be taken or withdr awn 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 sterilizati on, 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 thr ee samples representing three different batches of the product, one of wh ich 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 Material s (ASTM) Test Method for Efficacy of

Sanitizers Recommended for Inanimate Non-Food Contact Surfaces (ASTM E1153- 03) (Ref. 2). Three product samples, repr esenting 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 Enterobacter10 aerogenes (ATCC 13048). The ASTM method states “an average of at least 7.5 x 10 5
organisms must have survived the inoculated control squares for the test to be valid.” Two different fabric types should be test ed. The fabrics shoul d represent natural fabrics, such as cotton, and synthetic f abrics, such as polyester or rayon.

(2) Evaluation of sanitizing success. The results should demonstrate a

reduction of ≥ 99.9 percent (a 3-log 10 reduction) in the number of each test microorganisms over the scrubbed c ontrol count within 5 minutes.

(i) Data collection a nd reporting—(1) General. To assist in the proper review and evaluation of product performanc e, complete descriptions of the test employed and the results obtain ed should be submitted to the Agency. All test reports should include, at the leas t, the following information:

(i) Study title;

(ii) Product Identity;

(iii) Guideline number/Data Requirement;

(iv) Identification of the test ing laboratory or organization;

(v) Location where the test was performed;

(vi) Name(s) of the person(s) responsible for the test;

(vii) Statement of Co nfidentiality 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, incl uding 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;11 (xv) Number of samples, batches and replicates tested;

(xvi) Manufacture date of each product batch;

(xvii) Identification of all ma terial or procedural optio ns employed, where such choice is provided for or recommended in the test method selected (e.g., growth media, drying time for inocul ated carriers, neutralization conf irmation and/or subculture media, secondary subculturing);

(xviii) Test exposure conditions (e.g., cont act time, temperature, and relative humidity);

(xix) Complete reports of result s obtained for each replication;

(xx) Any control data essential to es tablish the validit y of the test.

(xxi) Carrier counts;

(xxii) Statistical treat ment of the data;

(xxiii) Conclusions;

(xxiv) References;

(xxv) Appendices, including st udy protocol and all ra w 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/e fficacystudystandards.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 pr otocol, identifying and describing each modification, should be provided with the st udy report. The applicant is encouraged to submit the proposed modifica tion 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 materi als and procedures employed in testing should be described in a manner consistent with orig inal research reports publish ed in technical or scientific journals. Where references to published reports or papers are made, copies or reprints of such references should be prov ided with the test reports. The applicant should submit the proposed test ing protocols for in-use or simulated-use studies (with12 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 additional background information.

(1) Official Methods of Analysis of the Association of Official Analytical

Chemists , Use-Dilution Methods, Chapter 6, Disinfectants; Current edition.

Association of Official Anal ytical Chemists (AOAC), 2200 Wilson Boulevard Arlington,

VA 22201.

( 2) Annual Book of Standards , Test Method for Efficacy of Sanitizers

Recommended for Inanimate Non-Food Cont act Surfaces, Designation E1153-03. American Society for Testing and Mate rials, 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). Am erican 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 Hi gh Efficiency Washing Operations (E 2406- 04). American Society for Testing and Ma terials, 100 Barr Harbor Drive, West Conshohocken, PA 19428.

(5) American Association of Textile Ch emists and Colorists (AATCC) Test
Method 100-2004 Quantitative Pr ocedure: 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 Mate rials, 100 Barr Harbor Drive, West Conshohocken, PA 1 9428.