United States

Environmental Protection

Agency

Office of Chemical Safety 

and Pollution Prevention

(7503P)

EPA XXX-X-XX-XXX

April 2015

Product Performance Test 

Guidelines 

OCSPP 810.2000:

General Considerations for 

Testing Antimicrobial Agents

1
NOTICE

This guideline is one of a series of test guidelines established by the 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 their 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 the 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, please go to http://www.epa.gov/ocspp and select the topic entitled, “Test 

Methods and Guidelines.” You may also access the guidelines in 

http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

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 

the 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. 

OCSPP 810.2000: General considerations for testing antimicrobial agents.

(a) Scope.

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(1) Applicability. OCSPP Test Guideline Series 810.2100 through 810.2700 describes 

general information regarding product performance testing of antimicrobial 

pesticides to meet requirements of the Federal Insecticide, Fungicide, and 

Rodenticide Act (FIFRA)(7U.S.C. 136, et seq.) and the Federal Food, Drug, and 

Cosmetic Act (FFDCA) (21 U.S.C. 346a).

(2) Background. Microbial pests can be categorized into two basic types: 1) those that 

present potential public health hazards because of their infectious nature to humans 

(public health claims) and 2) those that cause economic or aesthetic problems such 

as spoilage, fouling, or production of offensive odors in the substrate in which they 

grow (non-public health claims). The OCSPP Test Guideline Series 810.2000 

addresses antimicrobial pesticide products with public health uses for which 

efficacy test data are required to be submitted to the Agency to support registration. 

The source material used in developing the OCSPP Test Guideline is OPP guideline 

91-1: General Requirements for Antimicrobial Agents (Pesticide Assessment 

Guidelines, Subdivision G, Product Performance, EPA report 540/9-82-026, 

October 1982).

(b) Purpose and Overview—Product performance.

(1) General concepts. Evaluation of product performance is conducted in 

consideration of 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 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 determined 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) Series organization. Table 1 outlines the organization of the OCSPP Test 

Guideline Series 810.2000.

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

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Guideline Name Guideline Number Previous Subdivision –G 

Guideline Number(s)

Sterilants – Efficacy Data 

Recommendations

810.2100 91-2(a)

Disinfectants for Use on Hard 

Surfaces – Efficacy Data 

Recommendations

810.2200 91-2(b)(c)(d)(e)(f)(g)(i)

91-7(a)(1)

91-3

Sanitizers for Use on Hard 

Surfaces – Efficacy Data 

Recommendations

810.2300 91-2(j)(k)(l)(m)

91-3

Disinfectants and Sanitizers for 

Use on Textiles – Efficacy Data 

Recommendations

810.2400 91-4(a)(1)(2)(3)(4)

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

Air Sanitizers – Efficacy Data 

Recommendations 

810.2500 91-5

Disinfectants for Use in Water –

Efficacy Data 

Recommendations

810.2600 91-8

Prions – Efficacy Data 

Recommendations

810.2700 Not Applicable

(3) Good Laboratory Practice Standards. Good Laboratory Practice Standards 

(GLP) as defined in 40 CFR Part 160 apply to studies to support the registration of 

all antimicrobial products. According to 40 CFR §160.17: “EPA may refuse to 

consider reliable for purposes of supporting an application for a research or 

marketing permit any data from a study which was not conducted in accordance 

with this part.” 40 CFR §160.12 (b) requires with any submitted research data “[a] 

A statement that the study was conducted in accordance with this part; [b] A 

statement describing in detail all differences between the practices used in the study 

and those required by this part; or [c] A statement that the person was not a sponsor 

of the study, did not conduct the study, and does not know whether the study was 

conducted in accordance with this part.”

(4) Data for modifications of recommended methods. When recommended methods 

are modified by the applicant to support specific claims and/or use patterns for a 

product, the applicant should submit the complete testing protocol, identifying and 

describing each modification to the Agency for review and evaluation prior to 

initiation of the test. The study report should identify and describe each 

modification.

(5) Data for other methods. When recommended methods, or modifications thereto, 

are not employed to develop 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 test 

reports. All materials and procedures employed in testing should be fully described.

4
Where references to published reports or papers are made, copies or reprints of such 

references should be provided with the test reports.

(6) Confirmatory data. In certain situations, an applicant may rely on previously 

submitted product efficacy data to support a new application or amendment for 

registration of a product and submit only confirmatory efficacy data on the subject 

product to verify the product’s antimicrobial efficacy. These situations are as listed 

below:

(i) Duplicated Product Formulations. In this situation, the applicant 

manufactures a formulation which duplicates a product that is already 

registered and has complete supporting efficacy data. The chemical 

composition, manufacturing procedure, 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 duplicated product are cited by the 

applicant.

(ii) Minor Formulation Change in a Registered Product. In this situation, 

the change in the formulation is relatively minor. 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. If the only change in the formulation is the addition of a fragrance 

or dye, confirmatory data do not need to be submitted. However, when the 

product is an aerosol formulation, confirmatory data should be submitted 

for all formulation changes, including the addition of fragrances and dyes.

(iii) The confirmatory data are to be developed from testing the applicant's own 

finished product. 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, longer or shorter 

contact time), the confirmatory data should be produced under similarly 

modified conditions.

(7) 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 (contact time, hard water etc.). 

The current version of the standard test method will be used in the evaluation of 

efficacy. 

(8) Test failure. Failure of a product formulation to meet the specified testing or 

evaluation of success (i.e. the performance standard), constitutes evidence that the 

product is unlikely to be effective as claimed in actual use and is reportable under 

FIFRA section 6(a)(2).

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(9) Guideline revisions. The Agency recognizes that novel technologies 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 will be published on the EPA Antimicrobials Division website until such 

time that they can be added to the guidelines.

(c) Public health and non-public health uses of antimicrobial products.

(1) Antimicrobial products with public health claims. An antimicrobial pesticide is 

considered to make a public health claim if the pesticide product bears a claim to 

control one or more pest microorganisms that pose a threat to human health, and 

whose presence cannot readily be observed by the user, including but not limited 

to, microorganisms infectious to man in any area of the environment. Efficacy data 

are required to be submitted to support registration of antimicrobial products with 

public health uses. 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 man (or both man and 

animals). Examples of specific microorganisms include, but are not limited 

to, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Escherichia 

coli (E. coli), human immunodeficiency virus (HIV), Streptococcus sp. and 

Staphylococcus aureus. Claims for control of microorganisms infectious or 

pathogenic only to animals (such as canine distemper virus or hog cholera 

virus) are not considered public health claims.

(ii) A claim is made for the pesticide product as a sterilant, disinfectant, 

virucide, sanitizer, fungicide or tuberculocide against one or more

microorganisms that are infectious or pathogenic to man. Surrogate 

microorganisms accepted by the Agency may also be used in testing. 

(iii) A claim is made for the pesticide product as a microbiological water purifier 

or microbial purification system or swimming pool disinfectant/sanitizer.

(iv) 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 is applied.

(2) Antimicrobial products with non-public health uses. Applicants who propose 

6
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 applicant is still responsible for ensuring 

that these products perform as intended by developing efficacy data and keeping 

the data on file. The Agency still has the responsibility of making sure that the use 

directions proposed for non-public health related claims are appropriate and 

adequate. Therefore, the Agency retains the option of requiring the submission of 

efficacy data for non-public health related claims on a case-by-case basis.

(i) Non-public health claim. An antimicrobial pesticide is considered to make 

a non-public health claim if the pesticide product bears a claim to control 

microorganisms of economic or aesthetic significance, where the presence 

of the microorganism would not normally lead to infection or disease in 

humans. Examples of non-public health claims would include, but are not 

limited to:

(A) Slimicides and odor control agents: Slimicides and odor control 

agents, preservatives, algicides, and other products expressly 

claiming control of microorganisms of economic or aesthetic 

significance are not considered to be human health related, but 

nevertheless must bear accurate labeling claims, adequate dosage 

recommendations, and complete directions for use.

(B) Bacteriostatic and Fungistatic products: Since elimination or 

significant reduction in the number of microorganisms (e.g., 

sterilization, disinfection, sanitization) should be demonstrated

before a product is considered acceptable for use against 

microorganisms infectious to humans or for use in medical 

environments, products bearing lower level labeling claims for 

effectiveness at the bacteriostatic/fungistatic (inhibition of growth) 

level would generally not be appropriate for such uses. 

Bacteriostatic/fungistatic claims are generally acceptable only for 

products expressly recommended for control of microorganisms of 

aesthetic significance (e.g., spoilage bacteria, odor-causing 

bacteria).

(C) Treated articles: The Agency has clarified its policy on applicability 

of the treated articles exemption to antimicrobial pesticides and 

provided guidance on appropriate language or label claims in 

Pesticide Registration Notice 2000-1. 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. 

7
Applicants who intend to market products with claims (such as 

public health claims) that go beyond the scope of the treated articles 

exemption should contact the Antimicrobials Division prior to 

conducting testing to support such a use.

(D) Animal disease pathogens and zoonotic microorganisms: For 

products labeled for public health and/or non-public health uses, 

submission of studies to EPA on certain animal disease pathogens 

and zoonotic microorganisms may be appropriate prior to approval 

of the label claim. For example, although label claims against foot 

and mouth disease virus, Newcastle disease virus, and avian 

influenza A virus are not considered to be human health related, the 

Agency requires, on a case-by-case basis, the submission of efficacy 

data to support these claims because these pathogens have animal 

health significance and/or the potential to infect humans. Applicants 

should consult the Agency for a current listing of organisms which 

meet these criteria. These organisms are also listed on the World 

Organization for Animal Health’s website, see 

http://www.oie.int/animal -health-in-the-world/oie-listed-diseases2011/.

(d) Definitions. Because of the variety of microorganisms to be controlled and the different 

claims and many use patterns of antimicrobial products, consistent product terminology 

and a common understanding of a few key words are important to a program for evaluating 

product performance. Even though the OCSPP Test Guideline Series 810.2000 guidelines 

cover only public health uses, terms covering non-public health use patterns and/or 

organisms are included in order to support consistency and clarity. The terms in the OCSPP

Test Guideline Series 810.2000, are generally used with the meanings set forth in this

paragraph.

Algicide means any substance, or mixture of substances, which kills or effectively reduces 

the number of living algae in water.

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

water.

Antibacterial means any substance, or mixture of substances, that destroys or eliminates 

bacteria in the inanimate environment. 

Antibiotic resistant means the ability of a bacterial cell to resist the effects of antibiotics.

Antifoulant means any substance, or mixture of substances, that is used to prevent the 

biological fouling of underwater structures or objects.

Bacteriostat means a substance, or mixture of substances, that inhibits the growth of 

8
bacteria in the inanimate environment.

Biocide/Microbicide means any substance, or mixture of substances, that kills living 

microorganisms (e.g., virucide--virus, mycobactericide--mycobacteria, algicide--algae; 

bactericide--bacteria; fungicide--fungi; slimicide--slime-forming microorganisms). 

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

microorganisms and environmental by-products immobilized on a substrate and embedded in an 

organic polymer mix. This organic polymer mix is also known by the term “glycocalyx.” 

Confirmatory data is a reduced set of data which may be used to support an application or 

amendment for registration of a product, for an additional microbe, or a minor formulation change 

of a registered product. Each guideline further addresses confirmatory data

Deodorizers means a substance, or mixture of substances, that are of two basic types: (1) 

Those that prevent or delay the formation of bacterial odors by killing microorganisms which 

produce them, and (2) those that mask, chemically destroy or neutralize odors. Products that claim 

deodorization by antimicrobial means are subject to registration as pesticides under FIFRA.

Disinfectant means a substance, or mixture of substances that destroys or irreversibly 

inactivates bacteria, fungi and viruses, but not necessarily bacterial spores, in the inanimate 

environment.

Fungicide means a substance, or mixture of substances, that destroys fungi (including 

yeasts) and fungal spores pathogenic to man or other animals in the inanimate environment. 

Fungistat means a substance, or mixture of substances, that inhibits the growth of fungi in 

the inanimate environment.

Microbiological water purifier means any unit, water treatment product or system that 

removes, kills or inactivates disease-causing microorganisms from the water, including bacteria, 

viruses and protozoan cysts, so as to render the treated water safe for drinking.

Microbiostat means a substance, or mixture of substances, that inhibits the growth of 

microorganisms (e.g., bacteriostat, fungistat, algistat).

Mycobactericide means a substance, or mixture of substances, that destroys or irreversibly 

inactivates mycobacteria in the inanimate environment.

Non-volatile means a substance that does not evaporate readily.

One-Step Disinfectant means a substance, or mixture of substances, that has been tested

and found to be effective in the presence of light to moderate soil, and, therefore, may be used

without a pre-cleaning step in the use directions.

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

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

Prion means proteinaceous infectious particle.

Production batch refers to the antimicrobial test chemical generated using standard 

manufacturing processes and considered representative of the final product. 

Product performance refers to all pesticidal aspects of a product’s effectiveness and 

usefulness.

Sanitizer means a substance, or mixture of substances, that reduces the bacterial population 

in the inanimate environment by significant numbers, (e.g., 3 log10 reduction or more), but does 

not destroy or eliminate all bacteria. Sanitizers meeting Public Health Ordinances are used on food 

contact surfaces and are termed sanitizing rinses.

Slimicide means a substance, or mixture of substances, that reduces the number of slimeforming microorganisms in industrial water systems (e.g. paper mills). 

Sterilant means a substance, or mixture of substances, that destroys or eliminates all forms 

of microbial life in the inanimate environment, including all forms of vegetative bacteria, bacterial 

spores, fungi, fungal spores, and viruses.

Sporicide means a substance, or mixture of substances, that irreversibly inactivates 

bacterial spores in the inanimate environment.

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

inactivates tubercle bacilli in the inanimate environment.

Two-Step Sanitizer or Two-Step Disinfectant means a substance or mixture of substances 

that has not been registered for effectiveness against microorganisms in the presence of light to 

moderate soil, and, therefore, needs a pre-cleaning step for surfaces prior to sanitizing or 

disinfecting.

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

inactivates viruses in the inanimate environment.

Volatile means a substance that evaporates readily.

Zoonotic microorganism means an infectious agent that can be transmitted between

animals and humans.

(e) General testing considerations.

(1) Test substance.

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(i) Antimicrobial pesticides should be tested on the formulation with active 

ingredient(s) concentrations that comply with Table 2 below. In some cases 

(e.g., pressurized sprays, towelettes) products should also be tested in the 

same packaging in which they are intended to be marketed. For all efficacy 

testing the following information should be provided:

(A) Identification of the test substance and quantitative description of its 

chemical composition.

(B) Manufacturer and production batch numbers of the test substance. If 

a product is diluted, the report should specify the quantities and 

identification of each diluent.

(C) Data to show that the manufacturer 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).

(D) In situations where it may not be technically feasible to test within 

the Lower Certified Limits (LCL) range specified in this section, a 

detailed justification should be provided to the Agency for review 

prior to testing. Efforts should be made to test as close to the LCL 

of each individual active ingredient as possible.

(E) Efficacy testing at the Lowest Certified Limit (LCL) is necessary to 

demonstrate an antimicrobial product’s ability to consistently 

perform as labeled. However, it is the Agency’s view that not all 

efficacy data submissions necessitate this testing approach. 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. As described below, testing at the lower 

certified limit(s) should be performed for the required microbes (per 

the 810 series guidelines) needed to support a specific efficacy class. 

LCL testing of additional microbes should also be done for the 

tuberculocidal, sterilant/sporicidal, and food-contact sanitizer 

efficacy classes. LCL testing for fungicidal disinfection will be 

addressed under a separate guidance document.

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Table 2. Lower Certified Limit (LCL) for Product Efficacy Testing

Efficacy Class 

(see appropriate 810 guideline for test microbe) 

Testing Type

New 

Registration 

Submission

Amendments 

to Registered 

Product

Disinfection (Hospital, Broad or Limited-Spectrum):

 Required Microbes: Test at LCL Test at LCL 

 Additional Microbes Test at or 

below nominal

Test at or 

below nominal

Tuberculocidal Disinfection:

 Required Microbe and Additional Microbes Test at LCL Test at LCL 

Fungicidal Disinfection:

 Required Microbe Reserved Reserved

Virucidal Disinfection:

 Hardest to Kill strain on label [See revised viral 

hierarchy section in Agency Guidance 810.2200] Test at LCL Test at LCL 

 Additional Viruses: Easier to Kill strains on label [See 

Agency Guidance]

Test at or 

below nominal

Test at or 

below nominal

Non-Food Contact Sanitizer:

 Required Microbes Test at LCL Test at LCL 

 Additional Microbes: (e.g. MRSA, E. coli) Test at or 

below nominal

Test at or 

below nominal

Food Contact Sanitizer:

 Required Microbes: and Additional Microbes Test at LCL Test at LCL 

Sterilant/General Sporicidal:

 Required Microbes and Additional Microbes Test at LCL Test at LCL 

Clostridium difficile -sporicidal:

 Required Microbe Test at LCL Test at LCL 

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 as 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 

12
limit. To address this issue, the Agency has specified an active ingredient concentration 

range above the lower certified limit which may be used for efficacy testing. Individual 

active ingredient concentrations within this range (above the LCL) are considered 

representative of the actual lower certified limit for efficacy testing purposes. The test 

concentration range above the LCL is determined as follows:

• For products 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 the lower certified limit stated on the CSF.

• For products 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 the lower certified limit stated on the CSF.

• For products 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 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 6.65% to 6.71%. 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% (or 6.65% to 6.71%). Products with multiple active 

ingredients should use this approach to determine the usable testing range for each active 

in the product.

Registrants/applicants are expected to develop formulated product samples for 

efficacy testing within this range. In cases where efforts to formulate product within this 

range have failed, product samples may be diluted from a higher active ingredient 

concentration to achieve the target range. If product dilution is performed, a diluent that is 

not expected to increase product efficacy should be employed. 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. 

Registrants/applicants should consult with the Agency prior to testing if an appropriate 

diluent cannot be found. Efficacy studies developed using test samples diluted with 

material considered likely to increase sample efficacy may be rejected by the Agency. 

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 

13
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.

(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 standard method, 

registrants may, in consultation with the Agency, develop and submit 

protocols for claims where no standard test methods have been developed. 

If the standard method must be modified, the alternate protocols 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 for review and approval prior to data 

generation and collection. An assessment of method variability (e.g. within 

and between lab standard deviation) is critical to the Agency’s approval 

process. Thus, an assessment of variability and a means to mitigate 

variability should be contained in the submission. 

(3) Test organisms. Test microorganisms are specified in the guidelines for basic 

efficacy claims. The detailed final report 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.

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

employed when applicable. A surrogate is considered representative of a 

pathogenic strain and is usually a non-pathogen or exhibits limited 

pathogenicity to humans. Use of surrogates may be necessary due to 

biosafety concerns, ease of culturing, and availability. Applicants should 

consult with the Agency for guidance on additional surrogates. Examples 

of surrogate organisms are as follows.

(A) For tuberculocidal claims, Mycobacterium bovis (BCG) has been 

adopted as a surrogate for human Mycobacterium tuberculosis. See 

14
Guideline 810.2200.

(B) The duck hepatitis B virus (DHBV) has been adopted as a surrogate 

for the chimpanzee test used in testing efficacy of disinfectants 

against human hepatitis B virus. The data consistency control 

(method validation) stated in the referenced protocol utilizing two 

dilutions of BTC 835 has been eliminated. 

(C) The bovine viral diarrhea virus (BVDV) has been adopted as a 

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) The feline calicivirus has been adopted as a surrogate for the 

Noroviruses. The data consistency control (method validation) 

stated in the referenced protocol utilizing two dilutions of bleach has 

been eliminated.

(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, if possible, should be performed at the same time as 

the efficacy testing. The confirmation may also be conducted within the 

usual transfer cycle or other appropriate transfer depending upon 

organism’s growth requirements. The following information should be 

submitted with the Antibiotic Resistance Confirmation testing or 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. biochemical test, 

Gram stain, morphology).

(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.

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(F) 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.

(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.

(iv) Control carrier counts.

(A) For carrier-based assays, quantitative determinations of the 

microbial counts on the untreated control carriers after drying 

should be conducted in order to determine the validity of the test 

results obtained with the treated carriers 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.

(B) 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.

(4) Porous test materials. When an antimicrobial product is intended to be effective 

in treating a specific hard, porous surface, a method modification may be necessary 

which specifies the use of the porous material (as a carrier) in the test protocol. 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. 

(5) Hard water testing. Any product that bears a label claim for effectiveness using

hard water as the product diluent should be tested by the appropriate efficacy 

method using synthetic hard water (hardness expressed as ppm CaCO3) at the level 

claimed. A minimum of hardness of 200 ppm should be used for a hard water claim. 

The hard water tolerance level may differ with the level of antimicrobial activity 

claimed (e.g., sterilization, disinfection, or sanitization). To support an efficacy

16
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. 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. The use of 

deionized water as a diluent for concentrates is not recommended unless the label 

specifies deionized water. If a product requires dilution but does not bear a claim 

of effectiveness in hard water, the product should be tested in sterile tap water. The 

ppm hard water level of the tap water should be measured and reported with the 

submitted study. The registrant should be aware that tap water cannot be 

standardized and subsequent testing (e.g. post registration testing) may use a source 

of tap water exhibiting a different level of hardness. 

(6) Organic burden.

(i) 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 product’s pattern of use.

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.

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. The 5% 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 included on the label prior to the 

application of the antimicrobial agent.

(ii) 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 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.

For products with a pre-cleaning step prior to application, the addition of an 

organic burden to the inoculum may not be necessary. 

(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 

17
Disinfectants test and the Quantitative Tuberculocidal Test, involves adding 

a minimum of 5% (v/v) blood serum directly to the test organism (e.g., 4.75 

mL test organism + 0.25 mL blood serum) before inoculating the test 

solution.

(iv) When a product is recommended for certain patterns of use where the 

organic soil claimed is of a specific type, such as soap film residue, the 

product should be tested in the presence of that specific organic soil. 

Registrants should provide specific information in the data report regarding 

the way in which the organic soil, such as soap film residue was prepared 

(e.g., percentages of ingredients) and employed in the testing.

(7) 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 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). All method modifications need 

to be clearly identified and justified in the test protocol. For liquid or spray products 

containing volatile active ingredients where the product is applied to a hard nonporous surface, the minimum 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. 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.

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

antimicrobial activity at the end of 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. If 

neutralization is not properly performed, the results of efficacy testing may not be 

deemed valid. The method of neutralization, whether physical or chemical, should 

be confirmed in the laboratory in advance or 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 neutralizer media used did 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 in the subculture media, the 

18
examples of neutralization techniques in the paragraphs below should be followed.

(i) In qualitative test methods, after treatment the inoculated carrier is 

deposited in a tube of growth media (i.e., primary subculture). If the primary 

subculture is not sufficient to achieve neutralization by the use of a chemical 

agent and/or through dilution of test substance, the carrier may then be 

transferred to a second tube of growth media (or secondary subculture) to 

achieve neutralization. The neutralization procedure employed should be 

confirmed and reported by running a parallel test with uninoculated carriers 

being added to growth media containing a low level (i.e., 10 – 100 CFU/mL) 

of bacteria. Both the primary cultures and secondary subcultures should be 

incubated and checked for growth in the test and the neutralization 

confirmation. Dried inoculated 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 bacteriostasis from the carry-over of the antimicrobial 

substance on the carrier to the subculture medium. Growth results for both 

primary and secondary subcultures should be reported for the test and 

neutralization confirmation in the final report.

(ii) A neutralization confirmation for suspension based test methods should be 

conducted for all neutralization/recovery methods employed in testing. 

Neutralization confirmation may be conducted by neutralizing the test 

substance, without the organism, as in the test. After neutralization,

inoculation of a low level of organism (10-100 CFU/mL) and subsequent 

plating should follow. Plate counts should be within 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).

(9) Batch replication for modified testing. For certain use modifications to a 

registered product, the Agency allows a reduced number of product batches (lots) 

to be tested in support of these modifications. In order to be eligible to use this 

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 or use 

of porous surface carriers. The batch number reductions are as follows:

(i) For basic efficacy claims (e.g., sterilants, disinfectants, sanitizers), two 

samples, representing two different batches may be tested, instead of three.

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(ii) For supplemental efficacy claims (e.g., fungicides and virucides), one 

sample instead of two may be tested.

(10) Performance Standards. Performance standards are a set of test criteria used to 

establish product effectiveness, such as the number of positive carriers allowed for 

a passing product or a minimum log reduction of bacteria following treatment. The 

testing criteria may be different for each standard method and test microbe, thus the 

applicant should consult the appropriate guideline for this information. 

(11) Repeat Testing Policy – Refer to the methods cited in “Disinfectants for Use on 

Hard Surfaces – Efficacy Data Recommendations” OCSPP 810.2200, to determine 

allowable levels of contamination for a particular test method. If the product 

performance test data is deemed invalid due to an unacceptable level of 

contamination, the following re-testing scenarios apply:

(i) When an unacceptable level of contamination is present, retesting may be 

conducted using the same test conditions if the efficacy test meets the 

required performance standard. Retesting of the product lot(s) associated 

with the invalid test(s) should be conducted. 

(ii) When an unacceptable level of contamination is present and the product

does not meet the required performance standard, the product is considered 

a failure. 

(f) Data collection and reporting.

(1) General. To assist in the proper review and evaluation of product performance, 

complete descriptions of the test employed and the results obtained should be 

submitted to the Agency. All test reports should include, at the least, the following 

information:

(i) Study title;

(ii) Complete product identity (Certificate of Analysis);

(iii) Guideline number/Data Requirement;

(iv) Identification of the testing laboratory or organization;

(v) Location where the test was performed;

(vi) Name(s) of the person(s) responsible for the test;

(vii) Statement of Confidentiality Claims;

20
(viii) Statement of 40 CFR Part 160 Good Laboratory Practice compliance and 

Quality Assurance Statement; all protocol deviations should be identified.

(ix) Purpose of the study;

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

(xi) Name of the test method and appropriate references;

(xii) Test employed (include the version or edition) 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;

(xiii) Test microorganisms employed, including identification of the specific 

strain (ATCC or other);

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

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

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

(xvii) Manufacture date of each product batch

(xviii) 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);

(xix) Test exposure conditions (e.g., contact time, temperature, and relative 

humidity);

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

(xxi) Any control data, including neutralization confirmation results, essential to 

establish the validity of the test.

(xxii) Control carrier counts (i.e., microbial load);

(xxiii) Formula used to calculate control counts and log reduction values (if 

applicable) as well as any statistical treatment of the data;

(xxiv) Conclusions;

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(xxv) Any pictures or figures explaining the outcome or observations;

(xxvi) References;

(2) Appendices, including study protocol and all raw data reports associated with the 

conduct of the study. The applicant is encouraged to use the EPA’s standard 

efficacy report format, which may be found at

http://www.epa.gov/oppad001/efficacystudystandards.htm.

(g) References. The references in this paragraph may be consulted for additional background 

information:

(1) US EPA; Regulating Antimicrobial Pesticides. See http://www.epa.gov/oppad001.

(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.

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