United States Office of Chemical Safety and EPA 712-C-17-002

Environmental Protection Pollution Prevention February 2018

Agency (7510P)

Product Performance Test

Guidelines
OCSPP 810.2000:
General Considerations for
Testing Public Health
Antimicrobial Pesticides
Guidance for Efficacy Testing
 NOTICE

This guideline is one of a series of test guidelines established by the United States Environmental
Protection Agency’s Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing
pesticides and chemical substances to develop data for submission to the agency under the Toxic
Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug, and
Cosmetic Act (FFDCA) (21 U.S.C. 346a), referred to hereinafter as the harmonized test guidelines. Prior
to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides, and Toxic Substances
(OPPTS). To distinguish these guidelines from guidelines issued by other organizations, the numbering
convention adopted in 1994 specifically included OPPTS as part of the guideline’s number. Any test
guideline developed after April 22, 2010 will use the new acronym (OCSPP) in its title.

The OCSPP test guidelines serve as a compendium of accepted standardized 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 tests, and are also used by EPA, the public,
and the companies that are subject to data submission requirements under TSCA, FIFRA and/or FFDCA.
As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from these guidelines where circumstances warrant and without prior notice. 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. The procedures contained in this
guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA
recognizes that departures may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the agency will assess them for appropriateness on
a case-by-case basis.

For additional information about these test guidelines and to access these guidelines electronically, see
EPA’s Test Guidelines for Pesticides and Toxic Substances site at https://www.epa.gov/test-guidelines-
pesticides-and-toxic-substances. You may also access the guidelines at http://www.regulations.gov and
searching by Docket ID #: EPA-HQ-OPP-2015-0276.

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 Table of Contents

(A) Scope ................................................................................................................................................... 1

(1) Applicability. ............................................................................................................................. 1
(2) Background. .............................................................................................................................. 1
(B) Purpose and Overview—Product Performance................................................................................... 1
(1) General Concepts. ..................................................................................................................... 1
(2) Series Organization. .................................................................................................................. 1
(3) Guideline Updates. .................................................................................................................... 2
(4) Good Laboratory Practice Standards. ........................................................................................ 2
(5) Procedural Modifications of Recommended Methods. ............................................................. 2
(6) Other Methods. .......................................................................................................................... 3
(7) Confirmatory Data. .................................................................................................................... 3
(8) Agency Verification of Efficacy. .............................................................................................. 4
(9) Failure of Performance. ............................................................................................................. 4
(C) Public Health and Non-Public Health Uses of Antimicrobial Products .............................................. 4
(1) Antimicrobial Products with Public Health Claims. ................................................................. 4
(2) Antimicrobial Products with Non-Public Health Claims. ......................................................... 5
(D) Definitions ........................................................................................................................................... 6
(E) General Testing Considerations .......................................................................................................... 8
(1) Test Substance ........................................................................................................................... 8
(2) Test Methods ........................................................................................................................... 10
(3) Test Organisms. ....................................................................................................................... 11
(4) Control Carrier Counts. ........................................................................................................... 12
(5) Porous Test Materials. ............................................................................................................. 12
(6) Dilution of Products for Testing – Hard Water Guidance. ...................................................... 12
(7) Soil Load (Organic Burden). ................................................................................................... 13
(8) Contact Time (Exposure Period). ............................................................................................ 13
(9) Neutralization Confirmation.................................................................................................... 14
(10) Batch Replication for Modified Testing. ................................................................................. 14
(11) Repeat Testing. ........................................................................................................................ 15
(F) Data Collection and Reporting .......................................................................................................... 16
(G) References ......................................................................................................................................... 17

List of Tables

Table 1. Organization of the OCSPP Test Guideline Series 810 for Antimicrobial Products ...................... 2
Table 2. Lower Certified Limit (LCL) Requirements for Efficacy Testing of New Registrations or
Amendments to a Registered Product......................................................................................... 9

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OCSPP 810.2000: General Considerations for Testing Public Health Antimicrobial Pesticides

(A) Scope

(1) Applicability. OCSPP Test Guideline Series 810.2100 through 810.2700 describe general
information regarding product performance testing of public health antimicrobial pesticides to
meet requirements of the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA)(7 U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA)
(21 U.S.C. 346a).

(2) Background. Microbial pests can be categorized into two basic types: 1) those that present
potential public health hazards because they pose a threat to human health, including but not
limited to, microorganisms infectious to humans in any area of the inanimate environment (public
health claims) and 2) those microorganisms of economic or aesthetic significance (such as
spoilage, fouling, or production of offensive odors in the substrate in which they grow, or causing
biological deterioration of a material(s), where the presence of the microorganism would not
normally lead to infection or disease in humans (non-public health claims)).

The OCSPP 810 Test Guideline Series addresses antimicrobial pesticide products with public
health uses or that bear public health claims for which product performance test data are required
to be submitted to the agency to support registration or amended registration, including the
requirements of 40 CFR § 158.2220(a)(2). This document provides an update to the 2012 OCSPP
guideline 810.2000 and supersedes previous guidance.

(B) Purpose and Overview—Product Performance

(1) General Concepts. Evaluation of product performance is conducted based on expressed and
implied labeling claims or recommendations which may include 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, and ingredient status of chemicals in the formulation.

(i) Laboratory and/or simulated-use testing is designed and intended to determine the
effectiveness of a substance, or mixture of substances, to control or kill specific pest
organisms, when those substances are used in accordance with label instructions.

(ii) In some cases, effectiveness and usefulness of the proposed product is further determined
through advanced large-scale laboratory tests, field tests, or simulated-use tests using test
methods which closely approximate actual use and which employ typically used
application equipment (e.g., fumigant sterilants).

(2) Series Organization. Table 1 outlines the organization of the OCSPP Test Guideline Series 810;
Group B — Antimicrobial Efficacy Test Guidelines:

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Table 1. Organization of the OCSPP Test Guideline Series 810 for Antimicrobial Products
OCSPP
Guideline Name Guideline Number
General Considerations for Testing Public Health Antimicrobial Pesticides – 810.2000
Guidance for Efficacy Testing
Sterilants, Sporicides, and Decontaminants – Guidance for Efficacy Testing 810.2100
Disinfectants for Use on Environmental Surfaces – Guidance for Efficacy 810.2200
Testing
Sanitizers for Use on Hard Surfaces – Efficacy Data Recommendations 810.2300
Disinfectants and Sanitizers for Use on Fabrics and Textiles – Efficacy Data 810.2400
Recommendations
Air Sanitizers – Efficacy Data Recommendations 810.2500
Disinfectants and Sanitizers for Use in Water – Efficacy Data 810.2600
Recommendations
Products with Prions-Related Claims – Efficacy Data Recommendations 810.2700

(3) Guideline Updates. The agency recognizes that novel technologies and claims associated with
antimicrobial products may evolve over time and would potentially involve test methods that are
not referenced in the guideline series. In addition, the agency is considering adopting the use of
quantitative test methods as an alternative or replacement to several current qualitative methods
[e.g., Association of Official Analytical Chemists (AOAC International) Use-Dilution Methods].
The agency expects to update the guidelines periodically to address such changes; however, the
use of new methods may occur prior to guideline updates. In these instances, new methods which
may also be presented as Standard Operating Procedures (SOPs) may be published on the EPA’s
webpage until such time that they can be added to the guidelines (Antimicrobial Testing Methods
& Procedures Developed by EPA's Microbiology Laboratory). Once a new method is finalized
and published on the agency webpage, an applicant may utilize the method for product
performance testing. Applicants should consult with the agency for any method modifications
prior to testing. Applicants also have the option to submit alternative protocols for agency review.

(4) Good Laboratory Practice Standards. Good Laboratory Practice (GLP) Standards as defined
in 40 CFR Part 160 apply to studies submitted to support the registration or amendment of
antimicrobial products with public health claims. According to 40 CFR §160.17: “EPA may
refuse to consider reliable for purposes of supporting an application for a research or marketing
permit any data from a study which was not conducted in accordance with this part.” 40 CFR
§160.12 requires any study submitted to EPA to support an application for a research or marketing
permit to include statements signed by the applicant that “[a] the study was conducted in
accordance with this part; [b] describing in detail all differences between the practices used in the
study and those required by this part; or [c] 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.”

(5) Procedural Modifications of Recommended Methods. When an applicant seeks to modify

recommended methods to support specific claims and/or use patterns for a product, the applicant
should submit the proposed testing protocol which clearly identifies, describes, and justifies each
modification to the agency for review and evaluation prior to initiation of the test. Validation of

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 the modification may be necessary prior to agency approval. The final study report should identify
and describe each modification.

(i) The agency recommends that the registrant consult with the agency prior to initiation of
testing to confirm that the proposed changes in methods do not require submission of a
formal protocol.

(ii) An applicant may not be required to submit a protocol for agency review prior to testing
when it uses:

(a) alternative test organisms in recommended methods, including modifications to

support the growth and recovery of the alternative strain (e.g., modification of
growth/recovery media, incubation temperature, recovery method), or

(b) uses alternative carrier types in recommended methods.

(6) Other Methods. When recommended methods, or modifications thereto, are not employed to
develop product performance efficacy data, complete testing protocols should be submitted to the
agency for review and evaluation prior to initiation of the test. All pertinent information,
including protocols, should be included with the final study reports. All materials and procedures
employed in testing should be fully described. Where references to published reports or papers
are made, copies or reprints of such references should be provided with the test reports.

(7) Confirmatory Data. In certain situations, an applicant may rely on previously submitted product
efficacy data to support a new application or amendment to a registration of a product and submit
only confirmatory efficacy data on the applicant's own finished product to verify the product’s
antimicrobial efficacy. In cases where confirmatory data are appropriate, the applicant should
provide confirmatory testing for each label claim, contact time, and organism class. If the test
methodology utilized in the supporting (cited) efficacy data was modified to include additional
elements not specified in the recommended method (e.g., organic soil, hard water), the
confirmatory data should be produced under similarly modified conditions.

Specifically, formulation changes do not need confirmatory efficacy data when:

(i) Only the concentration of a fragrance or dye is increased, substituted, or decreased in a

formulation, and

(ii) the concentration of fragrances does not exceed 1.0% (w/w) of the total formulation, or

(iii) the total percentage of changes in dyes does not exceed 1.0% (w/w) of the total formulation.

See OPP Pilot Fragrance Notification Program for additional information. For pressurized spray
products, certification should be furnished specifying that all parts and materials used in
manufacturing the container for pressurized spray disinfectants are identical to those in the
registered product.

Examples of situations which do require the submission of confirmatory data are provided below.

(i) Identical Product Formulations. In this situation, the applicant manufactures a

formulation which is identical to a registered product that has complete supporting efficacy
data, and the two products are not produced by the same manufacturer using the same

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 production process. The chemical composition, label claims, and directions for use are
identical in substance to those of the original registration, and specific references (including
Master Record ID [MRID] Numbers) to the supporting data developed for the identical
product are cited by the applicant. In this case, confirmatory data are needed to verify that
the change in manufacturer (production process) does not impact the efficacy of the final
product.

(ii) Minor Formulation Change in a Registered Product (Inerts Only). In this situation, the
formulation change does not affect the concentration of the active ingredient(s) (e.g.,
addition, substitution, increase, or decrease in an agency-approved inert ingredient). The
label claims and directions for use are unchanged from those accepted for the registered
formulation, and specific references (including MRID numbers) to the supporting data
developed for the original formulation are cited by the applicant. Confirmatory data are
needed to verify that the change in inert ingredient does not impact the efficacy of the
product.

For situations that are not addressed above, applicants are encouraged to consult with the agency
prior to the initiation of testing to inquire whether the submission of confirmatory data is
appropriate.

(8) Agency Verification of Efficacy. The agency reserves the option to perform its own testing, on
a case-by-case basis, following the product specific test conditions used to register or amend the
registration of the product (e.g., contact time, hard water, dilution rate, and product application
procedure). The current version of the standard test method will be used for the evaluation of
efficacy.

(9) Failure of Performance. Failure of performance of registered formulations and claims is

reportable in accordance with 40 CFR § 159.188(a)(2).

(C) Public Health and Non-Public Health Uses of Antimicrobial Products

(1) Antimicrobial Products with Public Health Claims. A product makes a public health claim if
one or more of the following apply:

(i) A claim is made for control of one or more specific microorganisms that are directly or
indirectly infectious or pathogenic to humans (or both humans and animals).

(ii) 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
humans (or both humans and animals).

(iii) A fungicidal claim is made against one or more fungi infectious or pathogenic to humans
(or both humans and animals) or where the product does not clearly state that it is intended
only for use against non-public health fungi.

(iv) A claim is made for the pesticide product as a microbiological water purifier or microbial
purification system or swimming pool disinfectant/sanitizer.

(v) A non-specific claim is made that the pesticide product will beneficially impact or affect
public health at the site of use on/or in the environments in which it may be applied.

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(2) Antimicrobial Products with Non-Public Health Claims. For products that bear non-public
health related claims, the agency generally does not require submission of efficacy data to support
such claims. However, the applicant is still responsible for ensuring that these products perform
as intended by developing efficacy data and keeping the data on file. The agency 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
(40 CFR § 158.2220(a)(3)).

(i) Non-Public Health Claim. Examples of non-public health claims would include, but are
not limited to:

(a) Slimicides and odor control claims: 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 public health related, but
nevertheless must bear accurate labeling claims, adequate dosage recommendations,
and complete directions for use.

(b) Bacteriostatic and Fungistatic claims: Claims to control microorganisms of aesthetic

significance (e.g., spoilage bacteria, odor-causing bacteria, stain causing
microorganisms) are not considered to be public health related, but nevertheless must
bear accurate labeling claims, adequate dosage recommendations, and complete
directions for use.

(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. The treated articles
exemption (40 CFR Part 152.25(a)) covers qualifying articles and substances bearing
claims to merely protect the article or substance itself, if, among other things, the
treating 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.

(d) Animal disease pathogens and zoonotic microorganisms: For products labeled for
public health and/or non-public health uses, submission of studies to EPA about
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 and/or the potential to infect humans. Applicants should
consult the World Organization for Animal Health’s website for the current list of
diseases, infections, and infestations.

(ii) Testing. A crosswalk table has been provided for non-public-health claims and the OCSPP
810 Test Guideline Series (Crosswalk Table for Non-Public Health Guidelines). In certain
instances, the methods used to support public health related label claims may also be used
to support non-public-health label claims. This crosswalk is intended to assist interested
parties in determining which 810 guidelines may be appropriate for generating data to

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 support non-public-health claims. In addition, other testing approaches also may be
acceptable to support non-public health label claims.

(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 certain terms
are important. Even though the OCSPP 810 Test Guideline Series focus on 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 810 Test Guideline Series are generally used with the meanings set forth
in this paragraph.

Algicide any substance, or mixture of substances, which kills or effectively reduces the
number of living algae in water.

Algistat any substance, or mixture of substances, that inhibits the growth of algae in water.

Antibacterial 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 any substance, or mixture of substances, that is used to prevent the biological
fouling of underwater structures or objects.

Aseptic Packaging the filling of a commercially sterilized cooled product into pre-sterilized containers,
followed by aseptic hermetical sealing, with a pre-sterilized closure, in an
atmosphere free of microorganisms.

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

Batch a discrete experimental or commercial lot/test substance generated for testing. A

batch may be produced in a laboratory or facility.

Biofilm a dynamic, self-organized accumulation of a microorganism or microorganisms

and extracellular polymeric substances (called the biofilm matrix or slime matrix).

Certified Limits the outer limits of the range of the product's ingredients, calculated based on the
nominal concentration (as described under 40 CFR 158.350). The upper and lower
certified limits should be indicated on the product’s Confidential Statement of
Formula (CSF).

Confirmatory Data a reduced set of data which may be used to support an application or amendment
of the registration of a product. Each guideline further addresses confirmatory data
(OCSPP guidelines 810.2100 – 2600). Consult section B(7) above for guidance on
when confirmatory data should be submitted.

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Decontaminant any substance, or mixture of substances, that destroys or irreversibly inactivates
Bacillus anthracis spores (or surrogate acceptable to EPA) in the inanimate
environment.

Deodorizers a substance, or mixture of substances, that mask, chemically eliminate, or neutralize

odors through non-pesticidal means. Deodorizers that mask odors are not subject
to registration under FIFRA. Note: Products that claim odor elimination by
controlling public health microbes are subject to registration as pesticides under
FIFRA. Products which qualify as minimum risk pesticides under FIFRA 25(b) and
devices are not subject to registration.

Disinfectant 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 a substance, or mixture of substances, that destroys fungi (including yeasts) and
fungal spores pathogenic to humans or other animals in the inanimate environment.

Fungistat a substance, or mixture of substances, that inhibits the growth of fungi in the
inanimate environment.

Low Acid Food any foods, other than alcoholic beverages, with a finished equilibrium pH greater
than 4.6 and a water activity (aw) greater than 0.85. (Water activity (aw) is a
measure of the free moisture in a product and is the quotient of the water vapor
pressure of the substance divided by the vapor pressure of pure water at the same
temperature.)

Microbiological any unit, water treatment product or system that removes, kills or inactivates
Water Purifier disease-causing microorganisms from the water, including bacteria, viruses and
protozoan cysts, so as to render the treated water safe for drinking.

Nominal the concentration that is expected to be present in a product as a result of the

Concentration production or formulation process (should be equivalent to the active ingredient
(A.I.) concentration(s) that appear on the label).

Non-volatile a substance that does not evaporate readily.

One-Step a substance, or mixture of substances, that has been tested and found to be effective
Disinfectant in the presence of a 5% organic soil load and therefore, may be used without a pre-
cleaning step for visibly clean surfaces.

Performance a set of test criteria used to measure product performance in the context of a
Standards particular study, such as the number of positive carriers or a minimum log reduction
of bacteria following treatment. The performance criteria may be different for each
standard method and test microbe; thus the applicant should consult the “Evaluation
of Success” sections under the appropriate guideline for this information.

Preservative a substance, or mixture of substances that inhibits the growth of microorganisms

capable of causing biological deterioration of a material(s).

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 Prion a proteinaceous infectious particle.

Sanitizer a substance, or mixture of substances, that reduces the bacterial population in the
inanimate environment by significant numbers, (e.g., 3 log10 reduction or more),
but does not destroy or eliminate all bacteria. Sanitizers meeting Public Health
Ordinances may be used on food contact surfaces and are termed sanitizing rinses.

Slimicide a substance, or mixture of substances, that reduces the number of non-public health
slime-forming microorganisms (typically found in paper mills, industrial water
systems).

Sterilant 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 a substance, or mixture of substances, that irreversibly inactivates bacterial spores

in the inanimate environment.

Surrogate Microbe an alternative microbe or strain of microbe used to represent a pathogenic public
health pathogen typically used due to biosafety concerns, ease of culturing, and/or
availability.

Tuberculocide a substance, or mixture of substances, that destroys or irreversibly inactivates

tubercle bacilli in the inanimate environment.

Verification replicated testing of product done in a different laboratory or in the same laboratory
with an independent and separate study director, technical staff, and Quality
Assurance auditor.

Virucide a substance, or mixture of substances, that destroys or irreversibly inactivates

viruses in the inanimate environment.

Volatile a substance that evaporates readily.

Zoonotic an infectious agent that can be transmitted between animals and humans.
Microorganism

(E) General Testing Considerations

(1) Test Substance

(i) Antimicrobial pesticides should be tested using the formulation with active ingredient(s)
concentrations at the Lower Certified Limit (LCL). Refer to (E)(1)(i)(d) for more
information about LCL testing. In some methods of application, such as pressurized sprays
or towelettes, products should also be tested in the same packaging typical of what is
intended to be sold and distributed. For all efficacy testing, the following information
should be provided:

(a) Identification of the test substance and quantitative description of its chemical
composition.

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 (b) Manufacturer and batch numbers of the test substance. If a product is diluted, the
report should specify the quantities and identification of each diluent.

(c) A Certificate of Analysis (CoA) for each batch tested. The elements/content of the
CoA are: testing laboratory, concentration of each active ingredient, manufacturing
date, date of analysis (reflecting a date before the efficacy testing), analysis method
(method description may be referenced), analysis results (concentration of active(s)),
and laboratory signature.

(d) Efficacy testing at the Lower Certified Limit (LCL), is generally necessary to
demonstrate an antimicrobial product’s ability to consistently perform as labeled.
Table 2 specifies the types or classes of efficacy data which should employ testing at
the LCL for new and amended registrations, as well as circumstances where testing at
or below the nominal concentration is considered appropriate.

Table 2. Lower Certified Limit (LCL) Requirements for Efficacy Testing of New
Registrations or Amendments to a Registered Product
Claim
(see appropriate 810 guideline) LCL Requirement
Sterilant and/or Sporicide:
Base Spore Claims and Additional Spores Test at LCL
Disinfectant - Bacteria (Hospital, Broad or Limited-Spectrum):
Base Claims Test at LCL
Additional Bacteria Test at or below nominal
Virucide:
Hardest to Kill strain(s) per 810.2200, section Test at LCL
(f)2
Additional Viruses Test at or below nominal
Fungicide:
Base: Trichophyton interdigitale (ATCC 9533) Test at LCL
(formerly Trichophyton mentagrophytes)
Additional Fungi Test at or below nominal
Tuberculocide:
Base: Mycobacterium bovis BCG Test at LCL
Non-Food Contact Sanitizer:
Base Claims Test at LCL
Additional Bacteria: (e.g., MRSA, E. coli) Test at or below nominal
Food Contact Sanitizer:
Base Claims and Additional Bacteria Test at LCL
Emerging Pathogens:
Consult with the agency for specific organism Reserved
claims
Note that the guidance concerning LCL testing applies to all batches tested in a study. Consultation with the agency
prior to the initiation of testing is recommended for efficacy data types that do not fall into the efficacy classes
identified in Table 2. This listing may be revised to address LCL testing for other organisms should the agency’s
concerns change regarding emerging pathogens. In addition, the agency is aware of the potential difficulties
associated with generating test samples exactly at the lower certified limit. To address this issue, the agency has
specified an active ingredient concentration range above and below the lower certified limit which may be used for
efficacy testing. Individual active ingredient concentrations within this range (above the LCL) are considered

9
representative of the actual lower certified limit for efficacy testing purposes. The test concentration range above
the LCL should be determined as follows:
• For active ingredients (A.I.s) with a nominal concentration less than or equal to 1.0%, the tested value for
that active ingredient may be up to 2.0% above or below the lower certified limit stated on the CSF.
• For A.I.s with a nominal concentration above 1.0% and less than or equal to 20.0%, the tested value for
that active ingredient may be up to 1.0% above or below the lower certified limit stated on the CSF.
• For A.I.s with a nominal concentration above 20.0% and less than or equal to 100.0%, the tested value for
that active ingredient may be up to 0.6% above or below the lower certified limit stated on the CSF.
Using this approach, a product that has an active ingredient with a nominal concentration of 7.00% and a lower
certified limit of 6.65% (based on 40 CFR 158.350), would have a testing range of 6.58% to 6.72%. In this example,
the nominal concentration is greater than 1.0% and less than 20%; therefore, the appropriate testing range would
be up to 1.0% above the LCL of 6.65% (6.65 + 0.0665 = 6.7165, rounded to 3 significant figures = 6.72%). Products
with multiple active ingredients should use this approach to determine the usable testing range for each active in
the product.
For products with more than one A.I., when analytical methods cannot differentiate between different A.I.s in a
formulation, add together the individual LCL values to determine the lower certified limit of the total.
Applicants are expected to develop formulated product samples for efficacy testing within the LCL range. In
situations where products cannot be measured within this range, consultation with the agency may be necessary to
determine an appropriate range. Alternatively, product samples may be diluted from a higher active ingredient
concentration to achieve the target LCL. Water is the preferred diluent in these situations. Products likely to be
reactive or less stable in the presence of water may be diluted with a primary solvent already present in the product
formulation. The use of emulsifiers or surfactants as diluents should be avoided even if they are already present in
the subject formulation, as these may alter product efficacy. Applicants should consult with the agency prior to
testing if an appropriate diluent cannot be found. When dilution or any other alteration is performed to achieve the
acceptable LCL range, the efficacy report should specify exactly how the product (test material) was modified prior
to testing.

(2) Test Methods

(i) Test Method Selection. The method used for efficacy testing should be relevant to the
pest(s), the use sites and the label claims, as well as the application process and product
formulation type. Since the identification of appropriate methods for all public health uses
is not feasible, the applicant is responsible for the applicability of the test method selected
to substantiate product efficacy. The applicant should also ensure that selected methods are
current and valid for the purpose of pesticide registration. See the appropriate 810 guideline
for recommended methods.

(ii) Use Conditions. Efficacy testing should address factors or conditions that would normally
be encountered with the use pattern(s) intended for the product. This could include the
nature of the surface to be treated, the presence or absence of organic burden (soil) or other
interfering materials, water hardness, and the number of times or duration of time that the
product could be used. Representative surfaces for testing are identified in each 810
guideline.

(iii) New or Modified Methods. If there is no existing agency-approved standard method,

registrants may, in consultation with the agency, develop and submit protocols for tests to
support desired claims. If a standard method must be modified for testing, the modified
protocol for demonstrating the efficacy of a product should be developed and submitted to
the agency for review prior to testing. All new or modified protocols should be submitted
to the agency with a justification for the need for a new or modified method for review and

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 approval prior to data generation and collection. An assessment of method repeatability
should also be contained in the submission.

When using standard methods for testing additional microorganisms, tests may be modified
to conform to appropriate elements (e.g., media, growth conditions, and survivor recovery
for that specific test organism). These changes should be detailed in the protocol and final
report. These modifications may not require an EPA protocol review. However,
consultation with the agency is recommended prior to testing.

(3) Test Organisms. Test microorganisms are specified in the subsequent 810 guidelines for base
efficacy claims.

(i) Surrogate Microorganisms. Agency-approved surrogate microbes may be employed

when applicable. Use of surrogates (alternative microbe species or strains) may be
necessary due to biosafety concerns, ease of culturing, and/or availability. Agency-
approved surrogate organisms are provided below.

(a) Mycobacterium tuberculosis: For tuberculocidal claims, Mycobacterium bovis (BCG)

is an agency-approved surrogate for human Mycobacterium tuberculosis. See
guideline 810.2200.

(b) Hepatitis B: The duck hepatitis B virus (DHBV) is an agency-approved surrogate for
the chimpanzee test used in testing efficacy of disinfectants against human hepatitis
B virus. The data consistency control (method validation) stated in the referenced
protocol utilizing two dilutions of BTC 835 has been eliminated.

(c) Hepatitis C: The bovine viral diarrhea virus (BVDV) is an agency-approved surrogate
for the hepatitis C virus. The data consistency control (method validation) stated in
the referenced protocol utilizing two dilutions of BARDAC 2280 has been eliminated.

(d) Human norovirus: Feline calicivirus is an agency-approved surrogate for Human

Noroviruses. The data consistency control (method validation) stated in the referenced
protocol utilizing two dilutions of bleach has been eliminated.

Applicants should consult with the agency for guidance on additional surrogates.

(ii) Use of Antibiotic Resistant Test Organisms. Organisms to be labeled as antibiotic

resistant should be accompanied by scientific data that substantiates the antibiotic
resistance. Demonstration of antibiotic resistance should be conducted and documented
using the organism(s) listed on the label, and 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 should be submitted with the Antibiotic Resistance Confirmation testing and
included in the final report:

(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., resistance verification, biochemical
test, Gram stain, morphology).

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 (d) The method of preservation/storage (e.g., refrigerated agar slants, cryogenic beads,
other).

(e) Results of the testing including the numerical values of all antibiotics tested. An
example of relevant values would be Minimum Inhibitory Concentrations (MIC) for
automated tests, zone sizes for manual tests, and comparison to a standard Clinical
and National Standards Institute interpretation of such tests.

(f) The scientific method used to obtain the results (e.g., Kirby-Bauer, disc agar diffusion,
gradient agar diffusion; automated MIC procedures or equivalent). If automated
procedures are used, the manufacturer of such automation should be specified.

(g) Quality control procedures used to verify results.

(h) GLP statement.

(iii) Additional Microorganisms. Label claims for additional microbes other than the
designated test microorganism(s) are permitted, provided that the target microbe is likely
to be present in or on the recommended use areas and surfaces, and each claim is supported
with appropriate efficacy testing.

(4) Control Carrier Counts.

(i) For carrier-based assays, quantitative determinations of the microbial counts on the
untreated control carriers should be conducted in order to determine the validity of the test
results obtained with the treated carriers as specified in the method. 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. Pooling of carriers for assessing
microbial load may be conducted per the method (e.g., Use Dilution Method).

(ii) 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.

(5) Porous Test Materials. When an antimicrobial product is intended to be effective in treating a
specific, porous surface (hard or soft), the porous material (as a carrier) is to be specified in the
test protocol for the existing standard method. Examples of hard porous surfaces include porcelain
penicylinders and unglazed ceramic tiles. Examples of soft porous surfaces include fabrics (e.g.,
cotton, polyester, etc.). 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. A new protocol review is not needed when replacing
the standard method hard non-porous carrier with a hard porous carrier (e.g., AOAC Use-Dilution
stainless steel carrier replaced with porcelain penicylinder). However, applicants should consult
with the agency to determine whether the proposed carrier type is representative of the desired
claim.

(6) Dilution of Products for Testing – Hard Water Guidance. Where the use-directions of a
product require the user to dilute the product, hard water should be used for the dilution prior to

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 testing. The use of distilled water may be acceptable in cases where adequate justification is
provided to the agency (e.g., devices sensitive to calcium carbonate build-up). For these products,
the label should specify dilutions using distilled water.

For all other products, dilutions for efficacy testing should be conducted using one of the three
hard water options below. The study report should include the hard water analysis results as
calcium carbonate concentration in parts per million (ppm):

1. Regular (un-softened) tap water with a minimum of 200 ppm calcium carbonate. Water
hardness may be adjusted if necessary
2. AOAC Synthetic hard water of 400 ppm calcium carbonate. (Refer to the most recent version
of the AOAC Germicidal and Detergent Sanitizing Action of Disinfectants test (Official
Method 960.09) for guidance on the preparation of synthetic hard water.)
3. OECD hard water formula of 375 ppm hard water. (Refer to Annex 3 of OECD Guidance
Document dated June 21, 2013)

The hard water tolerance level may differ with the level of antimicrobial activity claimed (e.g.,
sterilization, disinfection, or sanitization). To support an efficacy claim 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 level of hard water.

(7) Soil Load (Organic Burden). The effectiveness of antimicrobial agents should be demonstrated
in the presence of a specific organic soil at an appropriate concentration level when specifically
claimed on the product’s label and/or indicated by the product’s pattern of use.

(i) 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 carrier inoculation and specified carrier-drying step in the method.
Additional organic material need not be incorporated into those procedures where at least
5% 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 an organic
burden is used. An antimicrobial substance identified as a one-step cleaner-disinfectant or
cleaner-sanitizer, or intended to be effective in the presence of 5% organic burden on
visibly clean surfaces, should be tested for efficacy with a minimum of a 5% organic soil
added to the inoculum. Registrants should check with the agency to determine the
acceptability of an organic burden other than blood serum.

(ii) 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) and employed in the testing.

(8) Contact Time (Exposure Period). The contact time used in efficacy testing should be the same
or shorter than the contact time identified on the product label. If a contact time different from
the range identified in the test method or guideline is preferred, consultation with the agency prior
to testing is recommended and a modification to the standard approach may be needed. In most
cases, a modification to provide a longer exposure period is limited by the practical considerations

13
 of the use patterns (e.g., an exposure period of >10 min for a product that will likely evaporate
from the treated surface within 10 min). Clearly identify and justify all method modifications in
the test protocol. For liquid or spray products containing volatile active ingredients where the
product is applied to a hard non-porous surface, the maximum contact time may be determined
by visually inspecting evaporation over the proposed contact period. As identified in the AOAC
International Germicidal Spray Products as Disinfectants test method, this approach should
involve product application (using the proposed application method) to glass slide carriers. For
spray products, use of methods that immerse contaminated carriers in the disinfectant fluid do not
accurately simulate the way in which volatile disinfectants perform on environmental surfaces.
For towelette products, the maximum contact time may be determined by a wetness test (refer to
section I(1)(a) in 810.2100 for guidance on the wetness determination test).

(9) Neutralization Confirmation. Neutralization is the process of inactivating a test material’s

antimicrobial activity. Thus, effectively, the addition of the neutralizing agent serves to end the
contact time. 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 should be employed at the completion of the contact time in order to
preclude residual effects of the active ingredient(s) in the subculture medium. The method of
neutralization, whether physical or chemical, should be confirmed in the laboratory concurrently
with testing. Neutralization confirmation data should be submitted in the final report to
demonstrate that the neutralization process sufficiently inactivated the active ingredient(s) and
that the neutralizing agent does not possess antimicrobial activity. All subculture techniques
employed to preclude residual carryover of active ingredient should be described in detail in the
study report. To demonstrate the absence of residual effects of the active ingredient(s) in the
subculture media, the examples of neutralization techniques in the paragraphs below should be
followed.

(i) In qualitative test methods such as the AOAC Use-Dilution method, the neutralization
confirmation procedure should demonstrate the recovery of a low level (e.g., 10–100
colony-forming units (CFU)) of the test organism in the neutralizer/subculture media.
Dried inoculated test carriers should not be used to test the ability of a subculture medium
to support organism growth. Growth results for both primary and secondary subcultures
should be reported for the test and neutralization confirmation in the final report.

(a) Specialized neutralizer/subculture media such as Dey/Engley broth will not show
turbidity; rather, a color change to the medium (yellow for growth of S aureus and
S. enterica) or the presence of pellicle at the surface of the medium (for P. aeruginosa)
is used to assess the results as a positive or negative outcome.

(ii) A neutralization confirmation for quantitative 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 (e.g., 10-100 CFU/mL) and
subsequent plating should follow. Plate counts should be within 50% of a parallel
population control or as specified in the relevant method.

(a) For virucidal tests, scientifically accepted controls including proper neutralization
controls should be performed (e.g., ASTM E1482).

(10) Batch Replication for Modified Testing. For certain use modifications to a registered product,
a reduced number of product batches (lots) may be tested. In order to be eligible to use this

14
 approach however, the registered product should have acceptable efficacy data supporting the
product that were performed without modification and included the number of batches specified
in the relevant method/guideline. Under these conditions, additional data performed at the same
use concentration previously tested may be conducted with reduced batch replications to support
a single modified use condition such as organic soil load, hard water concentration, temperature,
or use of porous surface carriers. The batch number reductions are as follows:

(i) For testing strains of the base efficacy claims (e.g., sterilant, disinfectant, sanitizer), two
samples, representing two different batches may be tested, instead of three. The
performance standard for testing remains as stated in the respective 810 guidelines.

(ii) For supplemental efficacy claims (e.g., fungicidal, virucidal, and additional bacterial
disinfection claims), one sample instead of two may be tested.

(11) Repeat Testing. The agency defines repeat testing as retesting a product lot under the same
conditions as the original test (e.g., same temperature, contact time, soil load, dilution rate, etc.).
Repeat testing is appropriate only in certain cases. The following table provides a general retest
strategy for evaluation of antimicrobial product efficacy.

Outcome Passed/Failed1 Test Lot Retest?

Mean control carrier count level above acceptable range Failed Test Yes
Mean control carrier count level above acceptable range Passed Test Not necessary
Mean control carrier count level below acceptable range Failed Test No
Mean control carrier count level below acceptable range Passed Test Yes
Presence of contamination in subculture media2 Passed or Failed Test See conditions3
Documented control failure Passed or Failed Test Yes
Documented operator error or test system failure which Passed or Failed Test Yes
results in the study to be deemed invalid by the laboratory’s
Quality Assurance Officer or Unit
1
Failed tests are defined as test outcomes in which the product did not meet the performance standard for the
efficacy claim. Performance standards are outlined in the OCSPP 810.2000 series in the “Evaluation of Success”
sections for each efficacy claim.
2
Contaminants are defined as microorganisms which are not the test organism that are present in the subculture
medium. Use methods such as gram staining, colony morphology, and biochemical assays to identify the
contaminant and provide results to EPA.
3
Conditions:
• In the case where a contaminant and the test organism are both present in the subculture tube, the outcome
is considered a positive carrier.
• For a 60 carrier test
o One contaminated carrier (subculture medium tube) may occur per 60 carrier test, without a retest, if
the total number of positive carriers including the contaminant meets the performance standard.
o A test where the total number of positive carriers, including the contaminant, exceeds the performance
standard by one positive carrier is invalid and may be repeated.
o A test with more than one contaminated carrier is invalid and may be repeated in a second laboratory
or in the same laboratory with a different study director, technical staff, and Quality Assurance auditor.
o A test may be repeated only one time, (using 60 carriers) per test lot.
• For a 10 carrier test
o A test with one contaminated carrier is invalid and may be repeated.
o A test may be repeated only one time per test lot. No growth should be present in the retest.
• A test with more than one contaminated carrier is invalid and may be repeated in a second laboratory or
in the same laboratory with a different study director, technical staff, and Quality Assurance auditor.

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 Repeat Testing Criteria:
1. All product lots should be tested at the concentration required for the claim (i.e., LCL or nominal
concentration of the A.I.s).
2. Testing should be performed under the original testing conditions.
3. A valid rationale to support retesting should be provided in the report. In the case where the product sample
is determined to be the source of the contaminant, notify the agency of the finding.
4. In case of contamination, run all identification tests necessary to rule out the test system organism. Results
should be included in the final study report.
5. Report all passing and failing data.
In cases where repeat testing is not appropriate, the applicant should report the failure(s) and may choose to conduct
a different test by removing soil load or using an increased contact time and/or a higher concentration to support
the new efficacy label claim for the product.

(F) Data Collection and Reporting

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. The applicant is encouraged
to use the EPA’s Standardized Efficacy Study Report and Efficacy Study Summary as a template for the
study report. All test reports should include, at a minimum, the following information:

(1) Study title;

(2) Product identity: Formulation (Basic vs. Alternate), Formulation type (e.g., liquid, spray,
solid/powder, towelettes), and Batch numbers;
• Units of measurement used for testing should correlate with use- instructions on the label. For
example, in cases where ounces are used, measurement should specify weight or volume (fluid
ounces). Include conversion table or equations where appropriate.
(3) Testing laboratory analysis of AI concentration for each test batch (if not applicable, include
Certificate of Analysis in appendices). Include conversion table when ppm of concentration is
used;
(4) Guideline number/Data Requirement;
(5) Identification of the testing laboratory or organization;
(6) Location where the test was performed;
(7) Name(s) of the person(s) responsible for the test;
(8) Statement of Confidentiality Claims;
(9) Statement of 40 CFR Part 160 Good Laboratory Practice compliance and Quality Assurance
Statement;
(10) Purpose of the study;
(11) Date and time of the Experimental Start Date and Experimental Termination Date as defined by
40 CFR Part 160;
(12) Name of the test method and appropriate references; and any modifications (e.g., organic soil,
hard water, etc.), when using standard tests (e.g., AOAC, ASTM, etc.); All modifications to the
test methods should be reported and justified;
(13) Test microorganisms employed, including identification of the specific strain (ATCC or other),
preparation of the inoculum and application to the carrier, the time/temperature and relative
humidity conditions for drying the microorganisms on the carrier;

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 (14) Concentration or dilution of the product tested and how prepared;
(15) Number of samples, batches and replicates tested;
(16) Manufacture date of each product batch;
(17) Identification of all material or procedural options employed, where such choice is provided for
or recommended in the test method selected (e.g., growth media, drying time for inoculated
carriers, neutralization confirmation and/or subculture media, secondary subculturing);
(18) Test exposure conditions (e.g., contact time, temperature, and relative humidity);
(19) Recovery procedure (e.g., removal of the microorganisms from the carrier), including the specific
assay procedure indicating such details as replication, subculture media, diluents, and the
incubation time/temperature conditions for the enumeration procedure employed;
(20) All protocol deviations should be identified;
(21) Complete reports of results obtained for each replication;
(22) Any control data, including neutralization confirmation results, essential to establish the validity
of the test.
(23) Control carrier counts (i.e., microbial load);
(24) Formula used to calculate control counts and log reduction values (if applicable) as well as any
statistical treatment of the data;
(25) Conclusions;
(26) Any pictures or figures explaining the outcome or observations;
(27) References; and

(28) Appendices, including Certificate of Analysis, study protocol, invalid data, and all raw data
reports associated with the conduct of the study.

(G) References

The references in this paragraph may be consulted for additional background information:

(1) Environmental Protection Agency, Antimicrobials Division, Office of Pesticide Programs.

Antimicrobial Pesticide Registration. See
https://www.epa.gov/pesticide-registration/antimicrobial-pesticide-registration.

(2) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants, Current edition.
AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.

(3) Annual Book of ASTM Standards, Standard Test Methods. American Society for Testing and
Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition.

(4) Environmental Protection Agency, Biological and Economical Analysis Division, Office of
Pesticide Programs. Antimicrobial Testing Methods & Procedures Developed by EPA’s
Microbiology Laboratory.

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