Document ID: EPA-HQ-OPP-2009-0681-0002
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2010-01-27T05:00Z

United States

Environmental Protection

Agency

	Office of Prevention, Pesticides

And Toxic Substances

(7510P)		EPA 712-C-07-005

 	Product Performance Test Guidelines 

		

		OPPTS 810.2000:

General Considerations for Public Health Uses of Antimicrobial Agents

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 810.2000: General considerations for public health uses of
antimicrobial agents.

(a)  Scope.

     (1)  Applicability.  This guideline is intended to meet 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) (21U.S.C. 346a).

     (2) Background.  The source material used in developing this OPPTS
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 158, the Agency has
waived all requirements to submit efficacy data unless the pesticide
product bears a claim to control 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 his 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 (e.g., zoonotic
microorganisms) submission of efficacy data for any pesticide product,
registered or proposed for registration.

     (4)  Series organization.  Table 1 outlines the organization of the
OPPTS Test Guideline Series 810.2000.

Table 1.  Organization of the OPPTS Test Guideline Series 810.2000.

Pesticide Type	Guideline Number	Previous Subdivision –G Guideline
Number(s)

Antimicrobials for use as Sterilants	810.2100 	91-2(a)

Antimicrobials for use as Disinfectants, Fungicides, Virucides, &
Tuberculocides	810.2200 	91-2(b)(c)(d)(e)(f)(g)(i)

91-7(a)(1)

91-3

Antimicrobials for use as Sanitizers – Food & Non-Food Contact
Surfaces, Residual	810.2300 	91-2(j)(k)(l)(m)

91-3

Antimicrobials for use on Textiles	810.2400	91-4(a)(1)(2)(3)(4)

91-4(b)(c)(d)

Antimicrobials for use in the Air 	810.2500	91-5

Antimicrobials for use in Water (Swimming pool, Drinking water)	810.2600
91-8

     (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
intends to update these guidelines periodically.  However, the use of
new methods may be approved, on a case-by-case basis, prior to guideline
updates. 

(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 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 OPPTS Test Guideline
Series 810.2000 address 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 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)  Products bearing claims to control organisms that may pose a
threat to human health, either directly or through transmission of
disease-causing organisms on environmental surfaces or the environment,
are considered public health related antimicrobials, and require
specific efficacy data to support labeling claims and patterns of use. 
Unqualified and non-specific claims for products as sterilants,
disinfectants, or sanitizers are considered to include or imply
effectiveness against microorganisms infectious to man.  Antimicrobial
products recommended for use in hospital or medical environments,
including but not limited to; sickrooms in public or private dwellings,
are similarly considered as human health-related.  Such claims or
recommendations need to be expressly qualified or deleted in order to
remove implications of human health significance.  

(iii) Products of human health significance.  The types of products in
paragraphs (c)(1)(iii)(A) and (c)(1)(iii)(B) in this guideline are
considered to be of human health significance.

(A) Products bearing labeling claims to control specific microorganisms
that are infectious for man on/in environmental surfaces or the
environment, such as Staphylococcus aureus, Mycobacterium tuberculosis,
and Pseudomonas aeruginosa, are considered to be directly related to
human health.	 

(B) All sterilants, disinfectants, swimming pool water
disinfectants/sanitizers, human drinking water disinfectants and
purifiers, and food-contact surface sanitizers are considered to be
human health-related, whether control of infectious microorganisms is
specifically claimed.	

(2)  Antimicrobial products with nonpublic 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.  The types of products in
paragraphs (c)(2)(A) through (c)(2)(D) in this guideline are considered
to be non-public health related products.

(A)  Slime, odor control and other non-public health agents.  Slime 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 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 labeling
claims for effectiveness at the bacteriostatic (inhibition of growth)
level are not appropriate for such uses.  Bacteriostatic claims are
generally only acceptable 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 (see reference (i)(1) of this
guideline).  The exemption (40 CFR 152.25 (a)) covers qualifying
articles and substances bearing claims to merely protect the article or
substance itself, if 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 this 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 required 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 is requesting 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. 

(d)  Definitions.  Because of the variety of microorganisms to be
controlled and the different claims and many use patterns of
antimicrobial products, uniform product terminology and a common
understanding of a few key words are important to a program for
evaluating product performance.  Even though the OPPTS 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 in the regulations of
antimicrobial pesticides.  The terms in the OPPTS Test Guideline Series
810.2000  are generally used with the meanings set forth in this
paragraph.

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 fouling of underwater structures or objects.

Antiseptic means a drug product applied topically to the skin to help
prevent infection or to help prevent cross contamination.  Antiseptic
products are applied on or in the living body of man or other animals. 
Antiseptic products are not identified as pesticides and are regulated
by the Food and Drug Administration.

Aseptic means free of microbial contamination.

Bacteriostat means a substance, or mixture of substances that inhibits
the growth of bacteria in the inanimate environment.

Biocide/ Microbiocide mean any substance, or mixture of substances, that
kills a number of living microorganisms (e.g., virucide-virus,
mycobactericide-mycobacteria, algicide-algae; bactericide-bacteria;
fungicide-fungi; slimicide-slime-forming microorganisms).  Note:  The
terms bactericide and fungicide, as used in conjunction with the term
microbiocide, are only related to industrial uses.

Biofilm means a community of bacteria or other microorganisms encased in
an extracellular polysaccharide substance that attach to a variety of
substrates (such as hard surfaces and liquids).  

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.

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.  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 eliminates a specific species of infectious or  public health
microorganism, 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 inhibit the
growth of fungi in the inanimate environment.

Microbiological water purifier means any unit, water treatment product
or system that removes, kills, or inactivates 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 inhibit
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.

One-Step Disinfectant means a substance, or mixture of substances that
has been tested and found to be effective in the presence of a light to
moderate bioburden, 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, but does not destroy or eliminate all bacteria or other
microorganisms.

Slimicide means a substance, or mixture of substances that reduces the
number of slime-forming microorganisms in industrial water systems
(e.g., paper mills).  For the purposes of these guidelines, slimicide
claims are reserved for non-public health industrial label claims.

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.

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 a bioburden.  The sanitizer or
disinfectant use directions should state the need to pre-clean surfaces
prior to sanitizing or disinfecting.

Virucide means a substance, or mixture of substances that destroys or
irreversibly inactivates viruses in the inanimate environment.

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.  

(ii) Identification should be made of the test substance and
quantitative description of its chemical composition should be reported.

(iii) Manufacturer and production batch numbers of the test substance
should be reported.  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).  

(2)  General considerations

(i) Good Laboratory Practice Standards.  Antimicrobial products should
be tested in accordance with the Good Laboratory Practice Standards
outlined in 40 CFR Part 160 and following the proposed directions for
use.  

(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 or reused.

(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 information in paragraphs
(e)(3)(i) through (e)(3)(iv) in this guideline should be submitted from
the Antibiotic Resistance Confirmation testing.

	

(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).

(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 a standard National Clinical Laboratory Standards (NCCLS)
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, and/or distilled water), 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 is a component of 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.

(ii)  The duck hepatitis B virus test (DHBV) has been adopted as a
surrogate for the chimpanzee test used in testing efficacy of
disinfectants against human hepatitis B virus (Ref. 6). 

(iii) The bovine viral diarrhea virus (BVDV) has been adopted as a
surrogate for the hepatitis C virus (Ref. 7).

(iv) The feline calicivirus has been adopted as a surrogate for the
Noroviruses (Ref.8).

(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 fruits and
vegetables.  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.  (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)(1) 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 generally 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
disinfectant 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 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 include 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 solution (e.g., 4.75 mL test solution +
0.25 mL blood serum) before adding 0.5 mL of the test organism.

(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 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, the method
should be modified in a manner acceptable to the Agency, to reflect the
deviation in exposure intended.  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 carrier
after drying should be conducted in order to determine the validity of
the test results obtained with the treated carriers.  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 a 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.  A specific medium capable of
neutralizing the antimicrobial effects of a product should be employed
prior to the microbiological assay.  In addition, data should be
submitted to demonstrate that the neutralizer employed inactivates the
active ingredients and does not possess any antimicrobial activity
itself.  Some of the recommended methods described in this section rely
solely upon the selection of an appropriate subculture medium to
neutralize the antimicrobial effects of certain general types of
chemical compounds (active ingredients).  In lieu of chemical
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 procedures in paragraphs
(h)(3)(i) through (h)(3)(ii)(C) in this guideline should be followed.

(i) Efficacy final reports should describe the neutralization techniques
employed during the study. In addition, evidence should be submitted to
demonstrate that the neutralizer identified inactivates the
antimicrobial ingredient and that the neutralization process itself does
not possess any antimicrobial activity. These controls are termed
Neutralization Confirmation. This confirmation is conducted by
demonstrating the growth of an inoculum of 5-100 CFU test organism/mL
growth media into a parallel test (including the neutralization process)
conducted without the test organism or following incubation of the
actual test. In addition, the Neutralization Confirmation inoculum is
inoculated into the parallel test without the neutralization process to
confirm lack of antimicrobial activity of the process (Ref. 15). 

(ii) Examples of neutralization techniques:  (A)  In carrier-based test
methods, the carrier is initially deposited in a tube of growth media
(i.e., primary subculture). The carrier may then be transferred to a
second tube of growth media (i.e., secondary subculture). The primary
and/or the secondary subculture may include a chemical agent to achieve
neutralization. Secondary subcultures may be helpful in achieving
neutralization either through dilution or incorporation of chemical
agents in the growth media.  A neutralization confirmation for
carrier-based test methods may be conducted by demonstrating the growth
an inoculum of 5-100 CFU of test organism/ml growth media, into a
parallel test with the neutralization process conducted without the test
organism or following incubation of the actual test. 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. 

(B) 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.  Follow by
inoculation of a low level of organism (5-100 CFU/mL) and subsequent
plating.  Plate counts should be within 1.0 log of a parallel population
control.  

(C) 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 for validation of efficacy of products selected on a
case-by-case basis.

(6)  Test failure.  Failure of a product to meet the specified testing
or evaluation of success, 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, Eighteenth 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, Eighteenth 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, Official Method 960.09 - Germicidal and Detergent
Sanitizing Action of Disinfectants, Eighteenth 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 E1053-97.  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. Eighteenth 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/oppad001/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, Eighteenth 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 E1054-02.  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 E1482-04.  American Society for Testing and Materials, 100
Barr Harbor Drive, West Conshohocken, PA 19428, current edition.

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