SEPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA 730-C-11-003
December 21, 2012
Product Performance
Test Guidelines
OCSPP 810.2400:
Disinfectants and
Sanitizers for Use on
Fabrics and Textiles -
Efficacy Data
Recommendations

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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" 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.

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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 commercial 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
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organic soil) may be extrapolated to disinfection 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 International Use-Dilution Methods1 (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 106); 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 106); 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 106); a mean log density
<6.0 invalidates the test."
1 Per Pesticide Registration Notice 2001-4, the phenol resistance assay is no longer expected to be
conducted and submitted for all carrier based tests.
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(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 laundry 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)
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-log-|0 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)
(Ref. 3) or Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants
for Use in High Efficiency Washing Operations (E 2406) (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 (5mL from the automatic washer, or 0.5 mL from the simulated washing
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device) in individual wide-mouth jars containing subculture media and neutralizes. 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.
(G)	A minimum average of 1.0 x 104 CFU/carrier should be recovered for a valid
test. A minimum average of 1.0 x 104 CFU/mL in the wash water control should be
recovered for a valid test.
(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
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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.
(F)	A minimum average of 1.0 x 104 CFU/carrier should be recovered for a valid
test. A minimum average of 1.0 x 104 CFU/mL in the wash water control should be
recovered for a valid test.
(ii) Evaluation of sanitization success. The results should demonstrate a
reduction of >99.9 percent (a 3-log-io reduction ) in bacteria 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-log-|0 reduction ) in bacteria 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.
(2)	Impregnated self-sanitizing fabrics and textiles.
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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.
(1)	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 8x12 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 log-|0 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-
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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."
One or two different fabric types should be tested depending on the label claim. The
fabrics should represent natural fabrics, such as cotton, and synthetic fabrics, such as
polyester or rayon.
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(2) Evaluation of sanitizing success. The results should demonstrate a
reduction of >99.9 percent (a 3-log-|0reduction) in the number of each test
microorganisms over the parallel control count.
(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,
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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/efficacvstudvstandards.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.
(j) References: The references in this paragraph may be consulted for addi-
tional 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
Analytical Chemists (AOAC), 2200 Wilson Boulevard Arlington, VA 22201.
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(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,
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