United States

Environmental Protection

Agency

	Office of Prevention, Pesticides

And Toxic Substances

(7510P)		EPA 712-C-07-056

 	Product Performance Test Guidelines

		

		OPPTS 810.2100:

Sterilants—Efficacy Data Recommendations

Public Review Draft

NOTICE

          This guideline is one of a series of test guidelines
established by the Office of Prevention, Pesticides and Toxic Substances
(OPPTS), United States Environmental Protection Agency for use in
testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA)
(15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the
Federal Food, Drug, and Cosmetic (FFDCA) (21 U.S.C. 346a).

	The OPPTS 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.  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 OPPTS harmonized test guidelines and
to access the guidelines electronically, please go to
http://www.epa.gov/oppts and select “Test Methods & Guidelines” on
the left side navigation menu.  You may also access the guidelines in  
HYPERLINK "http://www.regulations.gov"  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:   Sterilants—efficacy data recommendations.  

(a) Scope

(1) Applicability.  This guideline is intended to meet 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
effectiveness of products which are sterilants.  

(2) Background.  The source materials used in developing this OPPTS test
guideline are OPP guidelines 91-2: Products for use on hard surfaces and
91-30: Acceptable methods (Pesticide Assessment Guidelines, Subdivision
G, Product Performance.  EPA report 540/9-82-026, October 1982

(b) Purpose.  This guideline addresses efficacy testing for
antimicrobial pesticides intended to be used on hard, inanimate,
environmental surfaces, and, which bear label claims as sterilants.

(c)  General considerations

(1)  This guideline recommends tests to be conducted and data to be
submitted which the Agency believes will generally satisfy the
requirements for pesticide registration.  Studies conducted under this
guideline should be completed under EPA’s Good Laboratory Practice
regulations (40 CFR Part 160).  Note:  The Association of Official
Analytical Chemicals (AOAC) recommended tests are expected 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 OPPTS Test Guideline 810.2000 for general testing recommendations
prior to initiating tests.

(2)  Validation testing approaches, which may be needed to augment the
full range of efficacy tests in special circumstances, are also
described.

(d)  Water-soluble powders and non-volatile liquid products

(1)  Test procedure.  The Agency recommends use of the Official Methods
of Analysis of AOAC International, Official Method 966.04 Sporicidal
Activity of Disinfectants test, Method II (Ref. 1) to demonstrate the
sterilant efficacy of products. Sixty carriers representing each of two
types of surfaces (porcelain penicylinders and silk suture loops) should
be tested against spores of both Bacillus subtilis (B. subtilis)
(American Type Culture Collection (ATCC) 19659) and Clostridium
sporogenes (C. sporogenes) (ATCC 3584) on three samples representing
three different batches of the product, one of which should be at least
60 days old (240 carriers per sample, or a total of 720 carriers).  The
inoculum employed should provide a count of 1 x 105 – 1 x 106 colony
forming units per carrier.   Any sterilant which is a vapor or gas and
is recommended for use in a specific device should be tested using the
AOAC International Sporicidal Activity of Disinfectants test in that
specific device and according to the directions for use of that specific
device.  Modifications to the AOAC Sporicidal Activity of Disinfectants
test to address this use should be submitted to the Agency for review
and approval prior to conducting the tests.  

(2)  Evaluation of sterilant success.  The product should kill the test
spores on all of the 720 carriers without any failures.

(e)  Validation testing for all products with sterilant claims.  Data
submitted to support sterilant claims are subject to independent
validation testing in a second laboratory.

(1) Test procedure. The Agency recommends use of the Official Methods of
Analysis of AOAC International, Official Method 966.04 Sporicidal
Activity of Disinfectants test, Method II (Ref. 1) to demonstrate the
sterilant efficacy of products.  Thirty carriers representing each of
the two types of surfaces (porcelain penicylinders and silk suture
loops), should be tested against the spores of both B. subtilis and C.
sporogenes on one sample of the product.  The inoculum employed should
provide a count of 1 x 105 – 1 x 106 colony forming units per carrier.

(2)  Evaluation of sterilant success.  The product should kill the test
spores on all 120 carriers without any failures.

(f)  Sprays, gases, and foams.  (Reserved.)

(g) Additional spore formers, Clostridium difficile (C. difficile)
claims.  This section addresses interim efficacy tests for products with
claims to inactivate C. difficile spores on hard, non-porous, inanimate
surfaces.  The Agency recommends three possible options, as described in
paragraphs (g)(1)(i) through (g)(1)(iii) of this guideline.

(1)  Water-soluble powders and liquid products, qualitative
testing—(i) Test procedure for sterilant/sporicide plus C. difficile
claim.  The Agency recommends use of the Official Methods of Analysis of
AOAC International, Official Method 966.04 Sporicidal Activity of
Disinfectants test, Method II (Ref. 1) to demonstrate the sterilant
efficacy of products, as described in (d)(1).  In addition, conduct a
confirmatory test using the Official Methods of Analysis of AOAC
International, Official Method 966.04 Sporicidal Activity of
Disinfectants test, Method I.  Until the Agency identifies a
representative toxigenic strain or suitable surrogate(s) to be used in
testing against C. difficile, one of the following toxigenic strains
should be used for testing:  ATCC 700792, ATCC 43598 or ATCC 43599.  C.
difficile spores are inoculated on thirty carriers (porcelain
penicylinders) for two samples, representing two different batches of
the product (a total of 60 carriers).

(A) Evaluation of sporicidal success.  The product should kill all of
the test spores on all of the 780 carriers without any failures. 

(B) Reserved.

(ii) Test procedure for C. difficile sporicides--qualitative testing. 
The Agency recommends use of the Official Methods of Analysis of AOAC
International, Official Method 966.04 Sporicidal Activity of
Disinfectants test, Method I (Ref. 1) using C. difficile ATCC 700792,
ATCC 43598 or ATCC 43599.  Sixty carriers (porcelain penicylinders)
should be tested on three samples representing three different batches
of product, one of which should be at least 60 days old (a total of 180
carriers).  The inoculum employed should provide a target count of 1 x
105 – approximately 1 x 106 colony forming units per carrier.    

(A) Evaluation of sporicidal success.  The product should kill all of
the test spores on all of the 180 carriers without any failures.

(B) Reserved.

(iii)  Test procedure for C. difficile sporicides--quantitative testing.
 The Agency recommends the use AOAC Method 2008.05: Quantitative Three
Step Method (Efficacy of Liquid Sporicides Against Spores of Bacillus
subtilis on a Hard Nonporous Surface) (Ref. 2) or ASTM E 2197-02:
Standard Quantitative Carrier Test Method to Evaluate the Bactericidal,
Fungicidal, Mycobactericidal, and Sporicidal Potencies of Liquid
Chemical Germicides (Ref. 3).  Until the Agency identifies a
representative toxigenic strain or suitable surrogate(s) to be used in
testing against C. difficile, one of the following toxigenic strains
should be used for testing:  ATCC 700792, ATCC 43598 or ATCC 43599.  The
inoculum employed should provide a target count of > 106 colony forming
units per carrier. The product should be tested on three samples
representing three different batches of product, one of which should be
at least 60 days old. The number of carriers will vary depending on the
test method.

(A) Evaluation of sporicidal success.  The product should achieve a log
reduction of at least 6 logs based on recoverable spores. 

(h)  Sprays, gases, and foams.  (Reserved.)

(i)  Bacillus anthracis (B. anthracis) claims.  This section addresses
efficacy tests for all products with claims to inactivate B. anthracis
spores on inanimate surfaces.  The Agency recommends three possible
approaches, as described in paragraphs (h)(1)(i) through (h)(1)(iii) of
this guideline.

(1)  Water-soluble powders, liquid products, gases and vapors—(i) Test
procedure for sterilant/sporicide plus B. anthracis claim.  The Agency
recommends use of the Official Methods of Analysis of AOAC
International, Official Method 966.04 Sporicidal Activity of
Disinfectants test (Ref. 1) to demonstrate the sterilant efficacy of
products.  Sixty carriers representing each of two types of surfaces
(porcelain penicylinders and silk suture loops) should be tested against
spores of both B. subtilis (ATCC 19659) and C. sporogenes (ATCC 3584) on
three samples representing three different batches of the product, one
of which should be at least 60 days old (240 carriers per sample, or a
total of 720 carriers).  The inoculum employed should provide a target
count of 1 x 105 – approximately 1 x 106 colony forming units per
carrier.  In addition, conduct a confirmatory test using virulent B.
anthracis spores (or a surrogate acceptable to EPA) inoculated on thirty
carriers representing each of two types of surfaces (porcelain
penicylinders and silk suture loops) on two samples, representing two
different batches of the product (a total of 120 carriers).

(A) Evaluation of sporicidal success.  The product should kill all of
the test spores on all of the 840 carriers without any failures.

(B) Reserved.

(ii) Test procedure for sporicidal decontaminants--qualitative testing. 
The Agency recommends use of the Official Methods of Analysis of AOAC
International, Official Method 966.04 Sporicidal Activity of
Disinfectants test (Ref. 1) using virulent B. anthracis spores (or a
surrogate acceptable to EPA).   Sixty carriers representing either or
both of two types of surfaces (porcelain penicylinders and/or silk
suture loops) should be tested on three samples representing three
different batches of product, one of which should be at least 60 days
old.  The inoculum employed should provide a target count of 1 x 105 –
approximately 1 x 106 colony forming units 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.

(A) Evaluation of sporicidal success.  The product should kill all of
the test spores on all of the 180 (or 360) carriers without any
failures.

(B) Reserved.

(iii)  Test procedure for sporicidal decontaminants--quantitative
testing.  The Agency recommends the use of a well developed,
quantitative sporicidal test method acceptable to EPA using virulent B.
anthracis spores (or a surrogate acceptable to EPA) on porous and/or
nonporous surfaces acceptable to EPA.  The inoculum employed should
provide a target count of approximately 1 X 107 colony forming units per
carrier. The product should be tested on three samples representing
three different batches of product, one of which should be at least 60
days old. The number of carriers will vary depending on the test method.
 The coupon material(s) should be representative of those found at the
site(s) that appear on the product’s labeling, and be acceptable to
EPA.

(A) Evaluation of sporicidal success.  The product should achieve a log
reduction of at least 6 logs based on recoverable spores.

(B) Reserved.

(2) Simulated use testing for gas and vapor products—(i) Test
procedure.  In addition to conducting one of the three laboratory
studies in paragraphs (k)(1)(i) through (h)(1)(iii) of this guideline,
simulated-use testing should also be conducted for vapor and gas
products.  Protocols for the simulated-use test should be submitted to
the Agency for review and approval prior to conducting the test.  The
testing should be conducted under conditions that are representative of
the uses specified on the product’s labeling, and in a setting that is
representative of the label use site(s).   For example, a product
intended for use in a room or a large warehouse should be tested in an
empty room or large chamber.  The purpose of the test would be to assure
that key parameters for efficacy (chemical concentration, temperature,
relative humidity and contact time) are accurately monitored and
maintained throughout the enclosed space, and establish product
generation rate (lbs/hr) and rate/volume (lbs/hr/ft3).

(ii)  Additional considerations.  Important issues to consider in
developing the protocol for this test include:

	(A)  The test should be set up in a sealed enclosure at least the size
of a typical office and designed to measure the distribution of the
product and conditions needed to meet the measure of success in the
laboratory efficacy test.  Items that might be treated (e.g., dressers,
upholstered furniture, carpet, etc,) during an actual fumigation, should
be included in this test.  

	(B)  The protocol should specify the dimensions of the enclosure,
number and location of monitoring devices (e.g., for gas or vapor
concentration, total mass of gas or vapor injected into the enclosure,
temperature, relative humidity), product application equipment, heaters
and fans, contact time, etc.  The equipment used to monitor and maintain
these test parameters should be described.  

	(C)  All recorded test results pertaining to the test
conditions/parameters should be submitted to the Agency.  The maximum
volume of space that can be treated with a particular unit should be
reported to the Agency.  The minimum total mass of gas or vapor required
to maintain the required concentration and contact time per cubic foot
of space to be decontaminated should be reported.

	(D)  Appropriate positive and negative controls should be employed.

	(E)  This test must be conducted either in accordance with Good
Laboratory Practices (GLP) per 40 CFR Part 160 or in a federal
laboratory with an appropriate Quality Assurance Project Plan (QAPP). 

	(iii)  Evaluation of sporicidal success.  Measurements should show that
the same concentration, temperature, and relative humidity, can be
maintained for the required contact time needed to achieve 100% kill
(i.e., no growth on any of the carriers) in the qualitative laboratory
test, or a 6 log reduction in the quantitative test is demonstrated in
the simulated-use test.  In addition, measurements of the fumigant mass
injection/generation rate (e.g., pounds/hour), divided by the volume of
the simulated use test bed, that was used to arrive at the required
generation rate/volume (e.g., pounds per hour/cubic foot) for the
fumigation, should be included with the data, and listed on the product
label.

(j)  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 material in paragraphs (j)(1)(i) through (j)(1)(xiv) in this
guideline:

(i) Study title;

(ii)  Product Identity;

(iii)  Guideline number; 

(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)  40 CFR Part 160 Good Laboratory Practice compliance;

(viii)  Purpose of the study;

(ix)  Date and time of the start and end of the test;

(x)  Statistical treatment of the data;

(xi)  Conclusions;

(xii)  References;

(xiii)  Appendices;

(xiv)  Certification

The applicant is encouraged to use the EPA’s standard efficacy report
format, which may be found at   HYPERLINK
"http://www.epa.gov/oppad001/efficacystudystandards.htm" 
http://www.epa.gov/oppad001/efficacystudystandards.htm .

(2)  Data Report for recommended methods.  When recommended methods from
the Official Methods of Analysis of AOAC International; the Annual Book
of Standards of the American Society for Testing and Materials (Ref. 3);
or, EPA methods are used to develop efficacy data, certain minimal
information, in addition to that described in this guideline, should be
provided in the test report.  The report should include, at the least,
the material in paragraphs (j)(2)(i) through (j)(2)(xii) of this
guideline;

(i)  Test employed, and any significant modifications thereto (e.g.,
organic load, hard water);

(ii)  Test microorganisms employed, including identification of the
specific strain (ATCC or other);

(iii)  Description of the test substance, including the percent of
active ingredient;

(iv)  Concentration or dilution of the product tested and how prepared;

(v)  Number of samples, batches and replicates tested;

(vi)  Manufacture date of each product batch; 

(vii)  Identification of all material or procedural options employed,
where such choice is permitted or recommended in the test method
selected (e.g., growth media, drying time for inoculated carriers,
neutralization confirmation and/or subculture media, secondary
subculturing);

(viii)  Test exposure conditions (e.g., contact time, temperature, and
relative humidity);

(ix)  Complete reports of results obtained for each replication;

(x)  Any control data essential to establish the validity of the test.

(xi)  Carrier counts;

(xii) Any additional data pertinent for specific tests described in this
guideline.

(3)  Data for modifications of recommended methods.  When recommended
methods are modified 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.

(4)  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.

(k)  References:  The references in this paragraph may be consulted for
additional background information.

(1)  Official Methods of Analysis of the AOAC International, Chapter 6,
Disinfectants, Official Method 966.04 Sporicidal Activity of
Disinfectants, Eighteenth edition.  AOAC International, Suite 500, 481
North Frederick Avenue, Gaithersburg, MD 20877-2417.

(2)  Official Methods of Analysis of the AOAC International, Chapter 6,
Disinfectants, Official Method 2008.05 Quantitative Three Step Method
(Efficacy of Liquid Sporicides Against Spores of Bacillus subtilis on a
Hard Nonporous Surface), Eighteenth edition.  AOAC International, Suite
500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.

(3)  Annual Book of ASTM Standards, Standard Quantitative Carrier Test
Method to Evaluate the Bactericidal, Fungicidal, Mycobactericidal, and
Sporicidal Potencies of Liquid Chemical Germicides, Designation E
2197-02.  American Society for Testing and Materials, 100 Barr Harbor
Drive, West Conshohocken, PA 19428, current edition.

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