&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety and
Pollution Prevention
(751 OP)
EPA712-C-07-005
September 4,2012
Product Performance
Test Guidelines
OCSPP 810.2000:
General Considerations
for Uses of Antimicrobial
Agents
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NOTICE
This guideline is one of a series of test guidelines established by the Office of
Chemical Safety and Pollution Prevention (OCSPP) (formerly the Office of Prevention,
Pesticides and Toxic Substances (OPPTS) prior to April 22, 2010), 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 Act
(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 intended 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 requirements 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 means 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 circumstances warrant and without prior notice. The
methods 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 methods recommended in these
guidelines, with your supporting rationale. 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 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 810.2000: General considerations for testing antimicrobial agents.
(a) Scope.
(1) Applicability. This guideline describes test methods that EPA believes will
generally satisfy testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA)(7U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21
U.S.C. 346a).
(2) Background. The source material used in developing this OCSPP test guideline is
OPP guideline 91-1: General Requirements for Antimicrobial Agents (Pesticide Assessment
Guidelines, Subdivision G, Product Performance, EPA report 540/9-82-026, October 1982).
(b) Overview—Product performance.
(1) General concepts. Any evaluation of product performance is conducted in light of
expressed and implied labeling claims or recommendations concerning pests, sites, methods of
application, application equipment, dosage rates, timing and number of applications, use
situations, nature and level of pest control, duration of pest control, compatibility with other
chemicals, benefits and/or adverse effects of product use, compatibility of common practices
associated with the sites, active ingredient status of chemicals in the formulation, and equipment.
(i) Laboratory and/or simulated-use testing is conducted to determine the effectiveness of
a substance, or mixture of substances, to control or kill specific pest organisms, and in some
cases to determine whether the substance has sufficient pesticide potential to warrant larger scale
testing (e.g., swimming pool disinfectants).
(ii) In some cases, effectiveness and usefulness of the proposed product is further proven
through advanced large-scale laboratory tests, field tests, in-use tests, or simulated-use tests by
procedures which closely approximate actual use and which employ typically used application
equipment (e.g. fumigant sterilants).
(2) [Reserved]
(3) Waiver policy. As outlined in 40 CFR Part 161, the Agency has waived all
requirements to submit efficacy data unless the pesticide product bears a claim to control one or
more pest microorganisms that pose a threat to human health and whose presence cannot readily
be observed by the user, including but not limited to, microorganisms infectious to man in any
area of the inanimate environment. However, pursuant to FIFRA, each registrant must ensure
through testing that its products are efficacious when used in accordance with label directions
and commonly accepted pest control practices. The registrant must develop and maintain the
relevant data upon which the determination of efficacy is based. The agency reserves the right to
require, on a case-by-case basis submission of efficacy data for any pesticide product, registered
or proposed for registration.
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(4) Series organization. Table 1 outlines the organization of the OCSPP Test
Guideline Series 810.2000.
Table 1. Organization of the OCSPP Test Guideline Series 810.2000.
Guideline Name
Sterilants - Efficacy Data
Recommendations
Disinfectants for Use on Hard
Surfaces - Efficacy Data
Recommendations
Sanitizers for Use on Hard
Surfaces - Efficacy Data
Recommendations
Disinfectants and Sanitizers for
Use on Textiles - Efficacy Data
Recommendations
Air Sanitizers - Efficacy Data
Recommendations
Disinfectants for Use in Water -
Efficacy Data Recommendations
Prions - Efficacy Data
Recommendations
Guideline Number
810.2100
810.2200
810.2300
810.2400
810.2500
810.2600
810-2700
Previous Subdivision -G
Guideline Number(s)
91-2(a)
91-2(b)(c)(d)(e)(f)(g)(i)
91-7(a)(l)
91-3
91-2G)(k)(l)(m)
91-3
91-4(a)(l)(2)(3)(4)
91-4(b)(c)(d)
91-5
91-8
Not Applicable
(5) Future guidelines. The Agency recognizes the fact that novel technologies
associated with antimicrobial products may evolve over time and would potentially involve test
methods that are not referenced in this current guideline. In addition, the Agency is considering
adopting the use of quantitative test methods as a possible replacement for current qualitative
methods [e.g., Association of Official Analytical Chemists (AOAC) Use-Dilution Methods] in
the future. The Agency expects to update the guidelines periodically. However, the use of new
methods may occur, on a case-by-case basis, prior to guideline updates and these new methods
will be published on the EPA Antimicrobial Policy and Guidance Documents website until such
time that they can be added to the guidelines.
(c) Public health and nonpublic health uses of antimicrobial products
(1) Antimicrobial products with public health uses, (i) Health-related
considerations. Microbial pests can be categorized into two basic types: Those that present
potential public health hazards because of their infectious nature to humans and those that cause
economic or aesthetic problems such as spoilage, fouling, or production of offensive odors in the
substrate in which they grow. The OCSPP Test Guideline Series 810.2000 addresses
antimicrobial pesticide products with public health uses for which efficacy test data are required
to be submitted to support registration. These include all antimicrobial products intended to
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control microorganisms infectious to man in any area of the inanimate environment where these
microorganisms may present a hazard to human health. The label claims for an antimicrobial
product determine whether it is considered to be related to human health.
(ii) Public health claim. An antimicrobial pesticide is considered to make a public health
claim if the pesticide product bears a claim to control one or more pest microorganisms that pose
a threat to human health, and whose presence cannot readily be observed by the user, including
but not limited to, microorganisms infectious to man in any area of the environment. A product
makes a public health claim if one or more of the following apply:
(A) A claim is made for control of one or more specific microorganisms that are directly
or indirectly infectious or pathogenic to man (or both man and animals). Examples of specific
microorganisms include, but are not limited to, Mycobacterium tuberculosis, Pseudomonas
aeruginosa, Escherichia coli (E. coli\ human immunodeficiency virus (HIV), Streptococcus and
Staphylococcus aureus. Claims for control of microorganisms infectious or pathogenic only to
animals (such as canine distemper virus or hog cholera virus) are not considered public health
claims.
(B) A claim is made for the pesticide product as a sterilant, disinfectant, virucide,
sanitizer or tuberculocide against one or more microorganisms that are infectious or pathogenic
to man.
(C) A claim is made for the pesticide product as a fungicide against one or more fungi
infectious or pathogenic to man, or the product does not clearly state that it is intended for use
only against non-public health fungi.
(D) A claim is made for the pesticide product as a microbiological water purifier or
microbial purification system or swimming pool disinfectant/sanitizer.
(E) A non-specific claim is made that the pesticide product will beneficially impact or
affect public health at the site of use on in the environments in which it is applied.
(2) Antimicrobial products with non-public health uses. Registrants who propose non-
health related claims for their product (e.g., control of odor-causing bacteria) should be aware
that generally the Agency does not require submission of efficacy data to support such claims.
However, the registrant is still responsible for ensuring that these products perform as intended
by developing efficacy data which should be kept on file. The Agency still has the responsibility
of making sure that the use directions proposed for non-public health related claims are
appropriate and adequate. Therefore, the Agency retains the option of requiring the submission
of efficacy data for non-public health related claims on a case-by-case basis.
(i) Non-public health claim. An antimicrobial pesticide is considered to make a non-
public health claim if the pesticide product bears a claim to control
microorganisms of economic or aesthetic significance, where the presence of the
microorganism would not normally lead to infection or disease in humans.
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Examples of non-public health claims would include, but are not limited to:
(A) Slimicides, odor control and other non-public health agents. Slimicides and odor
control agents, preservatives, algicides, and other products expressly claiming control of
microorganisms of economic or aesthetic significance are not considered to be human health-
related, but nevertheless must bear accurate labeling claims and adequate dosage
recommendations, and complete directions for use.
(B) Bacteriostatic/Fungistatic products. Since elimination or significant reduction in the
number of microorganisms (sterilization, disinfection, sanitization) should be demonstrated
before a product is considered acceptable for use against microorganisms infectious for humans,
or for use in medical or sickroom environments, products bearing lower level labeling claims for
effectiveness at the bacteriostatic/fungistatic (inhibition of growth) level would generally not be
appropriate for such uses. Bacteriostatic/fungistatic claims are generally acceptable only for
products expressly recommended for control of microorganisms of aesthetic significance (e.g.,
spoilage bacteria, odor-causing bacteria).
(C) Treated articles. The Agency has clarified its policy on applicability of the treated
articles exemption to antimicrobial pesticides and provided guidance on appropriate language or
label claims in Pesticide Registration Notice 2000-1 (Ref. 1). The treated articles exemption (40
CFR 152.25(a)) covers qualifying articles and substances bearing claims to merely protect the
article or substance itself, if, among other things, the pesticide is registered for such use. This
exemption does not include articles or substances bearing implied or explicit public health claims
against human pathogens. Applicants who intend to market products with claims (such as public
health claims) that go beyond the scope of the treated articles exemption should contact the
Antimicrobials Division prior to conducting testing to support such a use.
(D) Animal disease pathogens and zoonotic microorganisms. For products labeled for
public health and/or non-public health uses, submission of studies to EPA on certain animal
disease pathogens and zoonotic microorganisms may be appropriate prior to approval of the label
claim. For example, although label claims against foot and mouth disease virus, Newcastle
disease virus, and avian influenza A virus are not considered to be human health related, the
Agency requires, on a case-by-case basis, the submission of efficacy data to support these claims
because these pathogens have animal health significance or the potential to infect humans.
Applicants should consult the Agency for a current listing of organisms which meet these
criteria. These organisms are also listed on the World Organization for Animal Health's website,
see, http://www.oie.int/animal -health-in-the-world/oie-listed-diseases-2011/.
(d) Definitions. Because of the variety of microorganisms to be controlled and the
different claims and many use patterns of antimicrobial products, consistent product terminology
and a common understanding of a few key words are important to a program for evaluating
product performance. Even though the OCSPP Test Guideline Series 810.2000 guidelines cover
only public health uses, terms covering non-public health use patterns and/or organisms are
included in order to support consistency and clarity. The terms in the OCSPP Test Guideline
Series 810.2000, are generally used with the meanings set forth in this paragraph.
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Algicide means any substance, or mixture of substances, which kills or effectively
reduces the number of living algae in water.
Algistat means any substance, or mixture of substances, that inhibits the growth of algae
in water.
Antibacterial means any substance, or mixture of substances, that destroys or eliminates
bacteria in the inanimate environment.
Antibiotic resistant means the ability of a bacterial cell to resist the effects of antibiotics.
Antifoulant means any substance, or mixture of substances, that is used to prevent the
biological fouling of underwater structures or objects.
Bacteriostat means a substance, or mixture of substances, that inhibits the growth of
bacteria in the inanimate environment.
Biocide/Microbicide means any substance, or mixture of substances, that kills a number
of living microorganisms (e.g., virucide—virus, mycobactericide—mycobacteria, algicide—algae;
bactedcide-bacteria; fungicide—fungi; slimicide—slime-forming microorganisms).
Biofilm means a dynamic, self-organized accumulation of microorganisms and
environmental by-products immobilized on a substrate and embedded in an organic polymer
mix. This organic polymer mix is also known by the term "glycocalyx."
Confirmatory data is a reduced set of data which may be used to support an application
or amendment for registration of a product, or a minor formulation change of a registered
product. Each guideline further addresses confirmatory data
Deodorizers means a substance, or mixture of substances, that are of two basic types: (1)
Those that prevent or delay the formation of bacterial odors by killing microorganisms which
produce them, and (2) those that mask, chemically destroy or neutralize odors (refer to 40 CFR
152.10). Products that claim deodorization by antimicrobial means are subject to registration as
pesticides under FIFRA.
Disinfectant means a substance, or mixture of substances that destroys or irreversibly
inactivates bacteria, fungi and viruses, but not necessarily bacterial spores, in the inanimate
environment.
Fungicide means a substance, or mixture of substances, that destroys fungi (including
yeasts) and/or fungal spores pathogenic to man or other animals in the inanimate environment.
Fungistat means a substance, or mixture of substances, that inhibits the growth of fungi
in the inanimate environment.
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Microbiological water purifier means any unit, water treatment product or system that
removes, kills or inactivates disease-causing microorganisms from the water, including bacteria,
viruses and protozoan cysts so as to render the treated water safe for drinking.
Microbiostat means a substance, or mixture of substances, that inhibits the growth of
microorganisms (e.g., bacteriostat, fungistat, algistat).
Mycobactericide means a substance, or mixture of substances, that destroys or
irreversibly inactivates mycobacteria in the inanimate environment.
Non-volatile means a substance that does not evaporate readily.
One-Step Disinfectant means a substance, or mixture of substances, that has been tested
and found to be effective in the presence of light to moderate soil, and, therefore, may be used
without a pre-cleaning step in the use directions.
Preservative means a substance, or mixture of substances that inhibits the growth of
microorganisms capable of causing biological deterioration of a material(s).
Product performance refers to all pesticidal aspects of a product's effectiveness and
usefulness.
Sanitizer means a substance, or mixture of substances, that reduces the bacterial
population in the inanimate environment by significant numbers, (e.g., 3 log 10 reduction) or
more, but does not destroy or eliminate all bacteria. Sanitizers meeting Public Health Ordinances
are used on food contact surfaces and are termed sanitizing rinses.
Slime means a dynamic, self-organized accumulation of microorganisms and
environmental by-products immobilized on a substrate and embedded in an organic polymer
mix. This organic polymer mix is also known by the term "glycocalyx."
Slimicide means a substance, or mixture of substances, that reduces the number of slime-
forming microorganisms in industrial water systems (e.g., paper mills).
Sterilant means a substance, or mixture of substances, that destroys or eliminates all
forms of microbial life in the inanimate environment, including all forms of vegetative bacteria,
bacterial spores, fungi, fungal spores, and viruses.
Sporicide means a substance, or mixture of substances, that irreversibly inactivates
bacterial spores in the inanimate environment.
Tuberculocide means a substance, or mixture of substances, that destroys or irreversibly
inactivates tubercle bacilli in the inanimate environment.
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Two-Step Sanitizer or Two-Step Disinfectant means a substance or mixture of substances
that has not been registered for effectiveness against microorganisms in the presence of light to
moderate soil, and, therefore, need a pre-cleaning step for surfaces prior to sanitizing or
disinfecting.
Virucide means a substance, or mixture of substances, that destroys or irreversibly
inactivates viruses in the inanimate environment.
Volatile means a substance that evaporates readily.
Zoonotic microorganism means an infectious agent that can be transmitted between
animals and humans.
(e) General testing considerations
(1) Test substance.
(i) Unless otherwise specified, antimicrobial pesticides should be tested on the
formulation with the lowest certified limit(s) of the active ingredient(s) and, in some cases (e.g.,
pressurized sprays, towelettes) with the product in the same packaging intended to be marketed.
The following information should be provided:
(ii) Identification of the test substance and quantitative description of its chemical
composition should be reported.
(iii) Manufacturer and production batch numbers of the test substance. If a product is
diluted, the report should specify the quantities and identification of each diluent.
(iv) The manufacturer should also submit effectiveness data to show that they can
consistently reproduce the formulation (batch replication), as well as to show that the product
will retain its effectiveness for a minimal period of storage under average conditions to which it
is likely to be exposed (shelf-life stability)(Ref. 2).
(v) In situations where it may not be technically feasible to test at the exact Lower
Certified Limits (LCL) (e.g., reactive chemistries with a degradation profile, multiple active
ingredients, formulations with inversely related active ingredients), a rationale should be
provided to the Agency for approval of the testing if testing cannot be performed at the LCL.
Efforts should be made to test as close to the LCL as possible.
(2) General considerations
(i) Good Laboratory Practice Standards. Good Laboratory Practice Standards (GLP) as
defined in 40 CFR Part 160 apply to studies to support all antimicrobial products used on hard
surfaces. 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
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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."
(ii) Use pattern. Depending upon the type of antimicrobial agent, target microorganisms,
and the site to be treated, all tests should address those factors that would normally be expected
to be encountered in the use pattern intended for the product, such as the method of application,
the nature of the surface, item or substrate to be treated, the presence or absence of soil or other
interfering conditions, temperature, exposure period, and the number of times or duration of time
that the use solution can be used. Representative surfaces for testing are identified in each 810
Guideline.
(iii) Additional factors. The actual test procedure to be employed will vary according to
the characteristics of the product, the target pests and the pattern of use intended. A specification
of methods in these guidelines for all conceivable public health uses is not feasible, and the
applicant should be responsible for the validity of the test method selected to substantiate a
product's efficacy. The applicants should ensure themselves that the selected method is current
and applicable to the product and uses proposed for registration.
(iv) New methods. If applicants believe they have alternative protocols for demonstrating
the efficacy of a product, such protocols should be submitted to the Agency for review. In
addition to modifying the standard methods, registrants may, in consultation with the Agency,
develop and submit protocols for claims where no standard test methods have been developed.
(3) Use of Antibiotic Resistant Test Organisms. Organisms to be labeled as antibiotic
resistant should be accompanied by scientific data that substantiates the antibiotic resistance.
The antibiotic resistance confirmation should be conducted using the organism(s) listed on the
label, and, if possible, should be performed at the same time as the efficacy testing. The
confirmation may also be conducted within the usual transfer cycle or other appropriate transfer
depending upon organism's growth requirements. The following information identified in
paragraphs (e)(3)(i) through (e)(3)(iv) in this guideline should be submitted with the Antibiotic
Resistance Confirmation testing or included in the final report.
(i) Test organisms should be characterized according to paragraphs (e)(3)(i)(A) through
(e)(3)(i)(D) of this guideline:
(A) The source and identity (e.g. ATCC, private source, other).
(B) The method of preparation prior to testing (e.g. transfer history).
(C) The method used to confirm the identity (e.g. biochemical test, Gram stain,
morphology).
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(D) The method of preservation/storage (e.g. refrigerated agar slants, cryogenic beads,
other).
(ii) Results of the testing including the numerical values of all antibiotics tested. An
example of values would be Minimum Inhibitory Concentration (MIC) s for automated test, zone
sizes for manual tests, and comparison to a standard Clinical and National Standards Institute
interpretation of such tests.
(iii) The scientific method used to obtain the results (Kirby-Bauer, disc agar diffusion, or
gradient agar diffusion; automated MIC procedures or equivalent). If automated procedures are
used, the manufacturer of such automation should be specified.
(iv) Quality control procedures used to verify results.
(f) Special considerations
(1) Hard Surface Carrier Test vs. Use-Dilution Methods The AOAC International
Hard Surface Carrier Test Method (Ref. 3) has only been validated for use with distilled water.
For other conditions (hard water, organic soil), the AOAC International Use-Dilution Method
(Ref. 4) is the recommended method.
(2) Elimination of Phenol Resistance Testing. As described in Pesticide Registration
Notice 2001-4 (Ref. 5) the Agency is no longer recommending the use of the phenol resistance
assay when conducting carrier-based efficacy tests. The phenol resistance assay was a
component of the AOAC Use-Dilution Test methods, as well as the Tuberculocidal Activity of
Disinfectants method.
(3) Surrogate microorganisms. The Agency has approved the use of several surrogate
organisms to be used as replacements for microorganisms that cannot be tested because of
biohazards or unavailability of scientifically accepted methods. Applicants should consult with
the Agency for guidance on additional surrogates. Examples of surrogate organisms are as
follows in paragraphs (f)(3)(i) through (f)(3)(iv) of this guideline.
(i) Mycobacterium bovis BCG has been adopted as a surrogate for human Mycobacterium
tuberculosis. See Guideline 810.2200.
(ii) The duck hepatitis B virus (DHBV) has been adopted as a surrogate for the
chimpanzee test used in testing efficacy of disinfectants against human hepatitis B virus (Ref. 6).
The data consistency control (method validation) stated in the referenced protocol utilizing two
dilutions of ETC 835 has been eliminated.
(iii) The bovine viral diarrhea virus (BVDV) has been adopted as a surrogate for the
hepatitis C virus (Ref. 7). The data consistency control (method validation) stated in the
referenced protocol utilizing two dilutions of BARD AC 2280 has been eliminated.
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(iv) The feline calicivirus has been adopted as a surrogate for the Noroviruses (Ref.8).
The data consistency control (method validation) stated in the referenced protocol utilizing two
dilutions of bleach has been eliminated.
(4) Antimicrobial rinses for fruits and vegetables. To support label claims for
consumer-use products as antimicrobial rinses for fruits and vegetables, products should meet a
two log reduction of five outbreak strains of Salmonella spp., Listeria monocytogenes, and
Escherichia coli O157:H7. Currently there is no standard method for assessing the efficacy of
antimicrobial rinses for pathogen reduction on the surfaces of fruits and vegetables (raw
agricultural commodities). Applicants should consult with the Agency prior to conducting testing
to support this use.
(5) Use of Dacron Loops. The AOAC International has accepted the use of Dacron loops
(also termed braided polyester), instead of silk suture loops, for peracetic acid containing
products, as a method modification to the AOAC Sporicidal Activity Test. Consult the Agency
for other chemistries. (Ref. 9).
(g) Special situations. When it is intended that an antimicrobial product be used in a
manner that is not reflected by the test conditions specified in the recommended AOAC methods
(e.g., inclusion of organic soil or hard water), one or more test conditions specified in the method
should be modified and/or supplementary data developed in order to provide meaningful results
relative to the conditions of use of the product. The information in paragraphs (g)(l) through
(g)(4) in this guideline is critical to the development and submission of the appropriate
supportive efficacy data.
(1) Type of surface. When an antimicrobial product is intended to be effective in treating
a hard, porous surface, some of the recommended methods may be modified to simulate this
more stringent condition by substitution of a hard, porous surface carrier (e.g., porcelain
penicylinder or unglazed ceramic tile) for the hard, nonporous surface carrier (stainless steel
cylinder or glass slide) specified in the method. In addition, control data (e.g., quantitation of
dried carrier, neutralization confirmation, sterility controls) should be developed to assure the
validity of the test results when this modification of the method is employed. Since the use of a
hard, porous surface would simulate the more stringent test condition, demonstrated efficacy on
hard, porous surfaces would suffice to support an analogous claim for efficacy of the product on
hard, non-porous surfaces as well.
(2) Hard water claim. Any product that bears label claims for effectiveness in hard
water should be tested by the appropriate method which has been modified to demonstrate
effectiveness of the product in synthetic hard water at the level claimed. The hard water tolerance
level may differ with the level of antimicrobial activity claimed (e.g., sterilization, disinfection,
or sanitization). To establish efficacy in hard water, all microorganisms (bacteria, viruses, and
fungi) claimed to be controlled by the product should be tested by the appropriate recommended
method at the same hard water tolerance level. Refer to the AOAC International Germicidal and
Detergent Sanitizing Action of Disinfectants test (Official Method 960.09) for guidance on the
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preparation of synthetic hard water (Ref. 10).
(3) Organic burden.
(i) An antimicrobial substance identified as a one-step cleaner-disinfectant or cleaner-
sanitizer, or intended to be effective in the presence of light to moderate amounts of organic
burden, should be tested for efficacy by the appropriate methods which have been modified to in-
clude a minimum of a 5% representative organic soil such as blood serum or scientifically
accepted equivalent as serum may be inhibitory to some viruses (Ref. 11). Registrants should
check with the Agency to determine the acceptability of an organic burden other than blood
serum.
(ii) A suggested procedure to simulate in-use conditions where the antimicrobial agent is
intended to treat dry inanimate surfaces contaminated with an organic soil load involves
contamination of the appropriate carrier surface with each test microorganisms culture
containing 5% v/v blood serum (e.g., 19 mL test microorganism culture +1 mL blood serum)
prior to the specified carrier-drying step in the method. Additional organic material need not be
incorporated into those procedures where at least 5 percent blood serum is already present in the
microbial inoculum to be dried on the surface. Control data (e.g., quantitation of dried carrier
counts, neutralization confirmation, sterility controls) should also be developed to assure the
validity of the test results when this modification is incorporated into the method. The organic
soil level suggested is considered appropriate for simulating lightly or moderately soiled surface
conditions. When the surface to be treated has heavy soil deposits, a cleaning step should be
recommended on the label prior to the application of the antimicrobial agent. The effectiveness
of antimicrobial agents should be demonstrated in the presence of a specific organic soil at an
appropriate concentration level when specifically claimed and/or indicated by the pattern of use.
(iii) A suggested procedure for incorporating a light to moderate organic soil load where
the antimicrobial agent is not tested against a dry inanimate surface, such as the AOAC
International Fungicidal Activity of Disinfectants test (Ref. 12) and the Quantitative
Tuberculocidal Test (Ref. 13) involves adding a minimum of 5% (v/v) blood serum directly to
the test organism (e.g., 4.75 mL test organism + 0.25 mL blood serum) before inoculating the
test solution.
(iv) When a product is recommended for certain patterns of use where the organic soil
claimed is of a specific type, such as soap film residue, the product should be tested in the
presence of that specific organic soil. Registrants should provide specific information in the data
report regarding the way in which the organic soil, such as soap film residue was prepared (e.g.,
percentages of ingredients).
(4) Exposure period. The exposure period required for an antimicrobial product to be
effective may be shorter than the exposure period specified in the recommended method. A
modification to provide a shorter exposure period is restricted by the manipulative limitations
inherent in the test procedure. A modification to provide a longer exposure period is restricted by
the practical considerations of the use patterns (e.g., an exposure period of >10 min cannot be
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recommended for a product that will effectively evaporate from the treated surface in <10 min).
If the product is to be represented in labeling for use at exposure periods shorter than those
specified in the method this deviation in the method should be included in the test protocol
submitted with the data. For liquid products containing volatile active ingredients where the
product is applied to an environmental surface, the exposure period should be determined by the
AOAC International Germicidal Spray Products As Disinfectants test (Ref. 14). Use of methods
that immerse contaminated carriers in the disinfectant fluid would not closely simulate the way
in which the volatile disinfectants perform on environmental surfaces.
(h) Microbiological technique considerations
(1) Microorganism survival after drying on a hard surface. Quantitative
determinations of the microbial counts on the untreated control carriers after drying should be
conducted in order to determine the validity of the test results obtained with the treated carriers.
The control carrier count evaluation should be performed at the completion of the test. These
quantitative determinations should be performed for all carrier-based assays, whether or not
modifications are made to the method being used. The test results should include the individual
dried carrier counts obtained by the method. The detailed final report for this testing should
include information and descriptions regarding: preparation of the inoculum; application of the
inoculum to the carrier; the time/temperature and relative humidity conditions for drying the
microorganisms on the carrier; the technique for removal of the microorganisms from the carrier;
and the specific assay procedure indicating such details as replication, subculture media,
diluents, and the incubation time/temperature conditions for the enumeration procedure
employed.
(2) Microorganism survival for suspension tests. Quantitative determination of the
microbial count of the inoculum in a parallel untreated diluent should be conducted in order to
determine the level of microbial challenge in the test (Numbers Control). These quantitative
determinations should be performed for all suspension assays, whether or not modifications are
made to the method being used. The test results should include the individual counts obtained by
the recovery method. The detailed final report for suspension testing should include information
and descriptions regarding: preparation of the inoculum, the volumes used for inoculation, and
the specific assay procedure indicating such details as replication, subculture media, diluents,
and the incubation time/temperature conditions for the enumeration procedure employed.
(3) Neutralization. Neutralization is the process for inactivating or quenching
antimicrobial activity during efficacy testing. This may be achieved through physical (e.g.,
filtration, dilution, secondary subculture) and/or chemical (e.g., addition of sodium thiosulfate to
the diluent) means. For each efficacy test, neutralization procedures should be employed, at the
completion of the contact time, in order to preclude residual effects of the active ingredients in
the subculture medium. If neutralization is not properly employed, the results of efficacy testing
may be exaggerated. The method of neutralization, whether that be physical or chemical, should
be confirmed. Data should be submitted (Neutralization Confirmation) in the final report to
demonstrate that the neutralization method sufficiently inactivated the active ingredient(s) and
that any neutralizer medium used did not possess any antimicrobial activity. In lieu of chemical
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neutralization, it should be documented that appropriate subculture techniques have been
employed that preclude residual carryover of active ingredients. To document the absence of
residual effects of the active ingredients in the subculture medium, the examples of neutralization
techniques procedures in paragraphs (h)(3)(i) through (h)(3)(ii) in this guideline should be
followed.
(i) In qualitative test methods, after treatment, the inoculated carrier is initially deposited
in a tube of growth media (i.e., primary subculture). If the primary subculture tube is not
sufficient to achieve neutralization by the use of a chemical agent and/or through dilution of the
test substance, the carrier may then be transferred to a second tube of growth media (i.e.,
secondary subculture). Secondary subculture may be helpful in achieving neutralization. The
neutralization procedure employed should be confirmed and reported by running a parallel test
with uninoculated carriers being added to growth media containing a low level (i.e., 10 - 100
CFU/mL) of bacteria. Both the primary cultures and secondary subcultures should be incubated
and checked for growth in the test and the neutralization confirmation. Dried test carriers should
not be used to test the ability of a subculture medium to support organism growth, as this would
provide too large a bioburden and may lead to an inaccurate evaluation of the presence of any
bacteriostasis that may result from the carry-over of the antimicrobial substance on the carrier to
the subculture medium. Growth results for both primary and secondary subcultures should be
reported for the test and neutralization confirmation in the final report.
(ii) A neutralization confirmation for suspension based test methods should be conducted
for all neutralization/recovery methods employed in testing. Neutralization confirmation may be
conducted by neutralizing the test substance, without the organism, as in the test. After
neutralization, inoculation of a low level of organism (10-100 CFU/mL) and subsequent plating
should follow. Plate counts should be within 1.0 log of a parallel population control:
(A) For virucidal tests, scientifically accepted controls, including proper neutralization
controls should be performed (e.g., ASTM E1482) (Ref. 16).
(4) Batch replication for modified tests. Where batch replication has already been
performed and accepted for a product registration with unmodified tests by the recommended
methods, additional testing at the same use concentration under modified conditions (e.g.,
different exposure period, presence of organic soil or hard water, porous surface carriers, etc.)
may be conducted with reduced batch replications as in paragraphs (h)(4)(i) and (h)(4)(ii) in this
guideline.
(i) For basic efficacy claims (e.g., sterilants, disinfectants, sanitizers), two samples,
representing two different batches, instead of three.
(ii) For supplemental efficacy claims (e.g., fungicides, virucides, and tuberculocides), one
sample instead of two.
(5) Validation of efficacy. The Agency reserves the option to perform its own tests, on a
case-by-case basis, following the protocol submitted with the registration if that protocol meets
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current agency standards. In all other cases, the Agency will conduct the validation studies using the
current agency protocol."
(6) Test failure. Failure of a product formulation to meet the specified testing or
evaluation of success claimed on the product label, constitutes evidence that the product is
unlikely to be effective as claimed in actual use and is reportable under FIFRA section 6(a)(2).
(i) References. The references in this paragraph may be consulted for additional
background information:
(1) Environmental Protection Agency, Pesticide Registration Notice PR 2000-1,
Applicability of the Treated Articles Exemption to Antimicrobial Pesticides, March 6, 2000.
Office of Pesticide Programs, Antimicrobials Division. See http://www.epa.gov/PR_Notices/.
(2) Environmental Protection Agency, Pesticide Registration Notice PR 91-2, Accuracy
of Stated Percentages for Ingredients Statement, May 2, 1991. Office of Pesticide Programs,
Antimicrobials Division. See http://www.epa.gov/PR_Notices/.
(3) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants, Hard
Surface Carrier Test Methods, Current edition. AOAC International, Suite 500, 481 North
Frederick Avenue, Gaithersburg, MD 20877-2417.
(4) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants, Use-
Dilution Methods, Current edition. AOAC International, Suite 500, 481 North Frederick Avenue,
Gaithersburg, MD 20877-2417.
(5) Environmental Protection Agency, Pesticide Registration Notice 2001-4, Elimination
of Phenol Resistance Testing for Antimicrobial Disinfectant and Sanitizer Pesticides. Office of
Pesticide Programs, Antimicrobials Division. See http://www.epa.gov/PR_Notices/.
(6) Protocols for Testing the Efficacy of Disinfectants Against Hepatitis B Virus (HBV).
Office of Pesticide Programs, Antimicrobials Division. See
http://www.epa.gov/oppad001/regpolicy.htm.
(7) Virucidal Effectiveness Test Using Bovine Viral Diarrhea Virus (BVDV) as a
Surrogate for Human Hepatitis C Virus. Office of Pesticide Programs, Antimicrobials Division.
See http://www.epa.gov/oppad001/regpolicy.htm.
(8) Virucidal Effectiveness Test Using Feline Calicivirus as a Surrogate for Norovirus.
Office of Pesticide Programs, Antimicrobials Division. See
http://www.epa.gov/oppad001/regpolicy.htm.
(9) McDonnell, G. (2003) J. AOAC Int. 86,407-411.
(10) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants,
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Official Method 960.09 - Germicidal and Detergent Sanitizing Action of Disinfectants, Current
edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-
2417.
(11) Annual Book of ASTM Standards, Test Method for Efficacy of Virucidal Agents
Intended for Inanimate Environmental Surfaces, Designation El053. American Society for
Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition. .
(12) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants,
Official Method 955.17 Fungicidal Activity of Disinfectants. Current edition. AOAC
International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.
(13) Environmental Protection Agency, Data Call-in Notice for Tuberculocidal
Effectiveness Data for All Antimicrobial Pesticides with Tuberculocidal Claims (Registration
Division, Office of Pesticide Programs, June 13, 1986). See
http://www.epa.gov/oppadOO l/dis_tss_docs/dis-06.htm.
(14) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants,
Official Method 961.02 - Germicidal Spray Products as Disinfectants, Current edition. AOAC
International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.
(15) Annual Book of 'ASTM Standards, Standard Test Methods, Evaluation of Inactivators
of Antimicrobial Agents, Designation E1054. American Society for Testing and Materials, 100
Barr Harbor Drive, West Conshohocken, PA 19428, current edition.
(16) Annual Book of ASTM Standards, Standard Test Method for Neutralization of
Virucidal Agents in Virucidal Efficacy Evaluations, Designation E1482. American Society for
Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition.
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