United States

Environmental Protection

Agency

Office of Prevention, Pesticides

And Toxic Substances

(7510P)

EPA 712-C-07-005

Draft November 5,

2009

Product Performance 

Test Guidelines 

OPPTS 810.2000:

General Considerations 

for Public Health Uses of 

Antimicrobial Agents

Public Review Draft
1

NOTICE

 This guideline is one of a series of test guidelines established by the Office of 

Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental 

Protection Agency for use in testing pesticides and chemical substances to develop data for 

submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, 

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

seq.), and section 408 of the Federal Food, Drug, and Cosmetic (FFDCA) (21 U.S.C. 346a).

The OPPTS test guidelines serve as a compendium of accepted scientific methodologies 

and protocols that are intended to provide data to inform regulatory decisions under TSCA, 

FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also 

used by EPA, the public, and the companies that are subject to data submission requirements 

under TSCA, FIFRA and/or the FFDCA. As a guidance document, these guidelines are not 

binding on either EPA or any outside parties, and the EPA may depart from the guidelines 

where circumstances warrant and without prior notice. The procedures contained in this 

guideline are strongly recommended for generating the data that are the subject of the 

guideline, but EPA recognizes that departures may be appropriate in specific situations. You 

may propose alternatives to the recommendations described in these guidelines, and the 

Agency will assess them for appropriateness on a case-by-case basis. 

For additional information about OPPTS harmonized test guidelines and to access the 

guidelines electronically, please go to http://www.epa.gov/oppts and select “Test Methods & 

Guidelines” on the left side navigation menu. You may also access the guidelines in 

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

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

DRAFT DOCUMENT DISCLAIMER: This draft document is distributed solely for the 

purpose of external review. It has not been formally disseminated by the EPA and should not 

be construed to represent any Agency determination or policy. The information correction 

process under the Agency’s Information Quality Guidelines does not apply until this 

document is formally disseminated by the EPA in its final form. This draft document should 

only be cited or quoted in the context of providing comments. 
2

OPPTS 810.2000: General considerations for public health uses of antimicrobial 

agents.

(a) Scope.

 (1) Applicability. This guideline is intended to meet testing requirements of the Federal 

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

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

 (2) Background. The source material used in developing this OPPTS test guideline is 

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

Guidelines, Subdivision G, Product Performance, EPA report 540/9-82-026, October 1982).

(b) Overview—Product performance.

 (1) General concepts. Any evaluation of product performance is conducted in light of 

expressed and implied labeling claims or recommendations concerning pests, sites, methods of 

application, application equipment, dosage rates, timing and number of applications, use situations, 

nature and level of pest control, duration of pest control, compatibility with other chemicals, benefits 

and/or adverse effects of product use, compatibility of common practices associated with the sites, 

active ingredient status of chemicals in the formulation, and equipment.

(i) Laboratory and/or simulated-use testing is conducted to determine the effectiveness of a 

substance, or mixture of substances, to control or kill specific pest organisms, and in some cases to 

determine whether the substance has sufficient pesticide potential to warrant larger scale testing (e.g., 

swimming pool disinfectants).

(ii) In some cases, effectiveness and usefulness of the proposed product is further proven 

through advanced large-scale laboratory tests, field tests, in-use tests, or simulated-use tests by

procedures which closely approximate actual use and which employ typically used application 

equipment (e.g. fumigant sterilants).

 (2) [Reserved]

 (3) Waiver policy. As outlined in 40 CFR Part 158, the Agency has waived all 

requirements to submit efficacy data unless the pesticide product bears a claim to control pest 

microorganisms that pose a threat to human health and whose presence cannot readily be observed 

by the user, including but not limited to, microorganisms infectious to man in any area of the 

inanimate environment. However, pursuant to FIFRA, each registrant must ensure through testing 

that his products are efficacious when used in accordance with label directions and commonly

accepted pest control practices. The registrant must develop and maintain the relevant data upon 

which the determination of efficacy is based. The agency reserves the right to require, on a case-by-
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case basis (e.g., zoonotic microorganisms) submission of efficacy data for any pesticide product, 

registered or proposed for registration.

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

Series 810.2000.

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

Pesticide Type Guideline Number Previous Subdivision –G 

Guideline Number(s)

Antimicrobials for use as 

Sterilants

810.2100 91-2(a)

Antimicrobials for use as 

Disinfectants, Fungicides, 

Virucides, & Tuberculocides

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

91-7(a)(1)

91-3

Antimicrobials for use as 

Sanitizers – Food & NonFood Contact Surfaces, 

Residual

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

91-3

Antimicrobials for use on 

Textiles

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

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

Antimicrobials for use in the 

Air 

810.2500 91-5

Antimicrobials for use in 

Water (Swimming pool, 

Drinking water)

810.2600 91-8

 (5) Future guidelines. The Agency recognizes the fact that novel technologies 

associated with antimicrobial products may evolve over time and would potentially involve test 

methods that are not referenced in this current guideline. In addition, the Agency is considering 

adopting the use of quantitative test methods as a possible replacement for current qualitative 

methods [e.g., Association of Official Analytical Chemists (AOAC ) Use-Dilution Methods] in the 

future. The Agency intends to update these guidelines periodically. However, the use of new 

methods may be approved, on a case-by-case basis, prior to guideline updates.

(c) Public health and nonpublic health uses of antimicrobial products

 (1) Antimicrobial products with public health uses. (i) Health-related considerations. 

Microbial pests can be categorized into two basic types: Those that present potential public health 

hazards because of their infectious nature and those that cause economic or aesthetic problems such 

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

OPPTS Test Guideline Series 810.2000 address antimicrobial pesticide products with public health 

uses for which efficacy test data are required to be submitted to support registration. These include 

all antimicrobial products intended to control microorganisms infectious to man in any area of the 
4

inanimate environment where these microorganisms may present a hazard to human health. The 

label claims for an antimicrobial product determine whether it is considered to be related to human 

health.

(ii) Products bearing claims to control organisms that may pose a threat to human health, 

either directly or through transmission of disease-causing organisms on environmental surfaces or 

the environment, are considered public health related antimicrobials, and require specific efficacy

data to support labeling claims and patterns of use. Unqualified and non-specific claims for products 

as sterilants, disinfectants, or sanitizers are considered to include or imply effectiveness against 

microorganisms infectious to man. Antimicrobial products recommended for use in hospital or 

medical environments, including but not limited to; sickrooms in public or private dwellings, are 

similarly considered as human health-related. Such claims or recommendations need to be expressly

qualified or deleted in order to remove implications of human health significance. 

(iii) Products of human health significance. The types of products in paragraphs (c)(1)(iii)(A) 

and (c)(1)(iii)(B) in this guideline are considered to be of human health significance.

(A) Products bearing labeling claims to control specific microorganisms that are infectious 

for man on/in environmental surfaces or the environment, such as Staphylococcus aureus, 

Mycobacterium tuberculosis, and Pseudomonas aeruginosa, are considered to be directly related to 

human health.

(B) All sterilants, disinfectants, swimming pool water disinfectants/sanitizers, human 

drinking water disinfectants and purifiers, and food-contact surface sanitizers are considered to be 

human health-related, whether control of infectious microorganisms is specifically claimed.

(2) Antimicrobial products with nonpublic health uses. Registrants who propose nonhealth related claims for their product (e.g., control of odor-causing bacteria) should be aware that 

generally the Agency does not require submission of efficacy data to support such claims. However, 

the registrant is still responsible for ensuring that these products perform as intended by developing 

efficacy data which should be kept on file. The Agency still has the responsibility of making sure 

that the use directions proposed for non-public health related claims are appropriate and adequate. 

Therefore, the Agency retains the option of requiring the submission of efficacy data for non-public 

health related claims on a case-by-case basis. The types of products in paragraphs (c)(2)(A) through 

(c)(2)(D) in this guideline are considered to be non-public health related products.

(A) Slime, odor control and other non-public health agents. Slime and odor control agents, 

preservatives, algicides, and other products expressly claiming control of microorganisms of 

economic or aesthetic significance are not considered to be human health-related, but nevertheless 

must bear accurate labeling claims and adequate dosage recommendations, and complete directions 

for use.

(B) Bacteriostatic products. Since elimination or significant reduction in the number of 

microorganisms (sterilization, disinfection, sanitization) should be demonstrated before a product is 
5

considered acceptable for use against microorganisms infectious for humans, or for use in medical or 

sickroom environments, products bearing labeling claims for effectiveness at the bacteriostatic 

(inhibition of growth) level are not appropriate for such uses. Bacteriostatic claims are generally

only acceptable for products expressly recommended for control of microorganisms of aesthetic 

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

(C) Treated articles. The Agency has clarified its policy on applicability of the treated 

articles exemption to antimicrobial pesticides and provided guidance on appropriate language or 

label claims in Pesticide Registration Notice 2000-1 (see reference (i)(1) of this guideline). The 

exemption (40 CFR 152.25 (a)) covers qualifying articles and substances bearing claims to merely

protect the article or substance itself, if the pesticide is registered for such use. This exemption does 

not include articles or substances bearing implied or explicit public health claims against human 

pathogens. Applicants who intend to market products with claims (such as public health claims) that 

go beyond the scope of the treated articles exemption should contact the Antimicrobials Division 

prior to conducting testing to support this use.

(D) Animal disease pathogens and zoonotic microorganisms. For products labeled for public 

health and/or non-public health uses, submission of studies to EPA on certain animal disease 

pathogens and zoonotic microorganisms may be required prior to approval of the label claim. For 

example, although label claims against foot and mouth disease virus, Newcastle disease virus, and 

avian influenza A virus are not considered to be human health related, the Agency is requesting the 

submission of efficacy data to support these claims because these pathogens have animal health 

significance or the potential to infect humans. Applicants should consult the Agency for a current 

listing of organisms which meet these criteria.

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

claims and many use patterns of antimicrobial products, uniform product terminology and a common 

understanding of a few key words are important to a program for evaluating product performance. 

Even though the OPPTS Test Guideline Series 810.2000 guidelines cover only public health uses, 

terms covering non-public health use patterns and/or organisms are included in order to support 

consistency and clarity in the regulations of antimicrobial pesticides. The terms in the OPPTS Test 

Guideline Series 810.2000 are generally used with the meanings set forth in this paragraph.

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

number of living algae in water.

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

water.

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

bacteria in the inanimate environment. 

Antibiotic resistant means the ability of a bacterial cell to resist the effects of antibiotics.
6

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

of underwater structures or objects.

Antiseptic means a drug product applied topically to the skin to help prevent infection or to 

help prevent cross contamination. Antiseptic products are applied on or in the living body of man or 

other animals. Antiseptic products are not identified as pesticides and are regulated by the Food and 

Drug Administration.

Aseptic means free of microbial contamination.

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

in the inanimate environment.

Biocide/ Microbiocide mean any substance, or mixture of substances, that kills a number of 

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

bactericide-bacteria; fungicide-fungi; slimicide-slime-forming microorganisms). Note: The terms 

bactericide and fungicide, as used in conjunction with the term microbiocide, are only related to 

industrial uses.

Biofilm means a community of bacteria or other microorganisms encased in an extracellular 

polysaccharide substance that attach to a variety of substrates (such as hard surfaces and liquids). 

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

amendment for registration of a product, or a minor formulation change of a registered product.

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

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

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

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

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

specific species of infectious or public health microorganism, but not necessarily bacterial spores, in 

the inanimate environment.

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

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

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

inanimate environment.

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

removes, kills, or inactivates microorganisms from the water, including bacteria, viruses and 

protozoan cysts so as to render the treated water safe for drinking.
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Microbiostat means a substance, or mixture of substances, that inhibit the growth of 

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

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

inactivates mycobacteria in the inanimate environment.

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

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

without a pre-cleaning step in the use directions.

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

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

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

usefulness.

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

the inanimate environment by significant numbers, but does not destroy or eliminate all bacteria or 

other microorganisms.

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

guidelines, slimicide claims are reserved for non-public health industrial label claims.

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

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

spores, fungi, fungal spores, and viruses.

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

spores in the inanimate environment.

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

inactivates tubercle bacilli in the inanimate environment.

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

has not been registered for effectiveness against microorganisms in the presence of a bioburden. The 

sanitizer or disinfectant use directions should state the need to pre-clean surfaces prior to sanitizing 

or disinfecting.

Virucide means a substance, or mixture ofsubstances that destroys or irreversibly inactivates 

viruses in the inanimate environment.

Zoonotic microorganism means an infectious agent that can be transmitted between animals 

and humans.
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(e) General testing considerations

(1) Test substance. 

(i) Unless otherwise specified, antimicrobial pesticides should be tested on the formulation 

with the lowest certified limit(s) of the active ingredient(s) and, in some cases (e.g., pressurized 

sprays, towelettes) with the product in the same packaging intended to be marketed. 

(ii) Identification should be made of the test substance and quantitative description of its 

chemical composition should be reported.

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

a product is diluted, the report should specify the quantities and identification of each diluent.

(iv) The manufacturer should also submit effectiveness data to show that they can 

consistently reproduce the formulation (batch replication), as well as to show that the product will 

retain its effectiveness for a minimal period of storage under average conditions to which it is likely

to be exposed (shelf-life stability)(Ref. 2). 

(2) General considerations

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

the proposed directions for use. 

(ii) Use pattern. Depending upon the type of antimicrobial agent, target microorganisms, 

and the site to be treated, all tests should address those factors that would normally be expected to be 

encountered in the use pattern intended for the product, such as the method of application, the nature 

of the surface, item or substrate to be treated, the presence or absence of soil or other interfering 

conditions, temperature, exposure period, and the number of times or duration of time that the use 

solution can be used or reused.

(iii) Additional factors. The actual test procedure to be employed will vary according to the 

characteristics of the product, the target pests and the pattern of use intended. A specification of 

methods in these guidelines for all conceivable public health uses is not feasible, and the applicant 

should be responsible for the validity of the test method selected to substantiate a product's efficacy. 

The applicants should ensure themselves that the selected method is current and applicable to the 

product and uses proposed for registration.

(iv) New methods. If applicants believe they have alternative protocols for demonstrating 

the efficacy of a product, such protocols should be submitted to the Agency for review. In addition 

to modifying the standard methods, registrants may, in consultation with the Agency, develop and 

submit protocols for claims where no standard test methods have been developed.
9

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

resistant should be accompanied by scientific data that substantiates the antibiotic resistance. The 

Antibiotic Resistance confirmation should be conducted using the organism(s) listed on the label, 

and, if possible, should be performed at the same time as the efficacy testing. The confirmation may

also be conducted within the usual transfer cycle or other appropriate transfer depending upon 

organism’s growth requirements. The information in paragraphs (e)(3)(i) through (e)(3)(iv) in this 

guideline should be submitted from the Antibiotic Resistance Confirmation testing.

(i) Test organisms should be characterized according to paragraphs (e)(3)(i)(A) through 

(e)(3)(i)(D) of this guideline:

(A) the source and identity (e.g. ATCC, private source, other).

(B) the method of preparation prior to testing (e.g. transfer history).

(C) the method used to confirm the identity (e.g. biochemical test, Gram stain, morphology).

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

(ii) Results of the testing including the numerical values of all antibiotics tested. An 

example of values would be Minimum Inhibitory Concentration (MIC) s for automated test, zone 

sizes for manual tests, and a standard National Clinical Laboratory Standards (NCCLS) 

Interpretation of such tests.

(iii) The scientific method used to obtain the results (Kirby-Bauer, disc agar diffusion, or 

gradient agar diffusion; automated MIC procedures or equivalent). If automated procedures are 

used, the manufacturer of such automation should be specified.

(iv) Quality control procedures used to verify results.

(f) Special considerations

(1) Hard Surface Carrier Test vs. Use-Dilution Methods. The AOAC International Hard 

Surface Carrier Test Method (Ref. 3) has only been validated for use with distilled water. For other 

conditions (hard water, organic soil, and/or distilled water), the AOAC International Use-Dilution 

Method (Ref. 4) is the recommended method. 

(2) Elimination of Phenol Resistance Testing. As described in Pesticide Registration 

Notice 2001-4 (Ref. 5) the Agency is no longer recommending the use of the phenol resistance assay

when conducting carrier-based efficacy tests. The phenol resistance assay is a component of AOAC 

Use-Dilution Test methods, as well as the Tuberculocidal Activity of Disinfectants method.

(3) Surrogate microorganisms. The Agency has approved the use of several surrogate 
10

organisms to be used as replacements for microorganisms that cannot be tested because of 

biohazards or unavailability of scientifically accepted methods. Applicants should consult with the 

Agency for guidance on additional surrogates. Examples of surrogate organisms are as follows in 

paragraphs (f)(3)(i) through (f)(3)(iv) of this guideline.

(i) Mycobacterium bovis BCG has been adopted as a surrogate for human Mycobacterium 

tuberculosis.

(ii) The duck hepatitis B virus test (DHBV) has been adopted as a surrogate for the 

chimpanzee test used in testing efficacy of disinfectants against human hepatitis B virus (Ref. 6). 

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

C virus (Ref. 7).

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

(4) Antimicrobial rinses for fruits and vegetables. To support label claims for consumeruse products as antimicrobial rinses for fruits and vegetables, products should meet a two log 

reduction of five outbreak strains of Salmonella spp., Listeria monocytogenes, and Escherichia coli

O157:H7. Currently there is no standard method for assessing the efficacy of antimicrobial rinses for 

pathogen reduction on fruits and vegetables. Applicants should consult with the Agency prior to 

conducting testing to support this use.

(5) Use of Dacron Loops. The AOAC International has accepted the use of Dacron loops

(also termed braided polyester), instead of silk suture loops, for peracetic acid containing products, as

a method modification to the AOAC Sporicidal Activity Test. (Ref. 9).

(g) Special situations. When it is intended that an antimicrobial product be used in a manner 

that is not reflected by the test conditions specified in the recommended AOAC methods (e.g., 

inclusion of organic soil or hard water), one or more test conditions specified in the method should 

be modified and/or supplementary data developed in order to provide meaningful results relative to 

the conditions of use of the product. The information in paragraphs (g)(1) through (g)(4) in this 

guideline is critical to the development and submission of the appropriate supportive efficacy data.

(1) Type of surface. When an antimicrobial product is intended to be effective in treating a 

hard, porous surface, some of the recommended methods may be modified to simulate this more 

stringent condition by substitution of a hard, porous surface carrier (e.g., porcelain penicylinder or 

unglazed ceramic tile) for the hard, nonporous surface carrier (stainless steel cylinder or glass slide) 

specified in the method. In addition, control data (e.g., quantitation of dried carrier, neutralization 

confirmation, sterility controls) should be developed to assure the validity of the test results when 

this modification of the method is employed. Since the use of a hard, porous surface would simulate 

the more stringent test condition, demonstrated efficacy on hard, porous surfaces would generally

suffice to support an analogous claim for efficacy of the product on hard, non-porous surfaces as 

well. 
11

(2) Hard water claim. Any product that bears label claims for effectiveness in hard water 

should be tested by the appropriate method which has been modified to demonstrate effectiveness of 

the product in synthetic hard water at the level claimed. The hard water tolerance level may differ 

with the level of antimicrobial activity claimed (e.g., sterilization, disinfection, or sanitization). To 

establish disinfectant efficacy in hard water, all microorganisms (bacteria, viruses, and fungi) 

claimed to be controlled by the product should be tested by the appropriate recommended method at 

the same hard water tolerance level. Refer to the AOAC International Germicidal and Detergent 

Sanitizing Action of Disinfectants test (Official Method 960.09) for guidance on the preparation of 

synthetic hard water (Ref. 10). 

(3) Organic burden.

(i) An antimicrobial substance identified as a one-step cleaner-disinfectant or cleanersanitizer, or intended to be effective in the presence of light to moderate amounts of organic burden

should be tested for efficacy by the appropriate methods which have been modified to include a 

minimum of a 5% representative organic soil such as blood serum or scientifically accepted 

equivalent as serum may be inhibitory to some viruses (Ref. 11). Registrants should check with the 

Agency to determine the acceptability of an organic burden other than blood serum. 

(ii) A suggested procedure to simulate in-use conditions where the antimicrobial agent is 

intended to treat dry inanimate surfaces contaminated with an organic soil load involves 

contamination of the appropriate carrier surface with each test microorganisms culture containing 5%

v/v blood serum (e.g., 19 mL test microorganism culture +1 mL blood serum) prior to the specified 

carrier-drying step in the method. Additional organic material need not be incorporated into those 

procedures where at least 5 percent blood serum is already present in the microbial inoculum to be 

dried on the surface. Control data (e.g., quantitation of dried carrier counts, neutralization 

confirmation, sterility controls) should also be developed to assure the validity of the test results 

when this modification is incorporated into the method. The organic soil level suggested is 

considered appropriate for simulating lightly or moderately soiled surface conditions. When the 

surface to be treated has heavy soil deposits, a cleaning step should be recommended on the label 

prior to the application of the antimicrobial agent. The effectiveness of antimicrobial agents should 

be demonstrated in the presence of a specific organic soil at an appropriate concentration level when 

specifically claimed and/or indicated by the pattern of use. 

(iii) A suggested procedure for incorporating a light to moderate organic soil load where the 

antimicrobial agent is not tested against a dry inanimate surface, such as the AOAC International 

Fungicidal Activity of Disinfectants test (Ref. 12) and the Quantitative Tuberculocidal Test (Ref. 13) 

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

solution + 0.25 mL blood serum) before adding 0.5 mL of the test organism.

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

claimed is of a specific type, such as soap film residue, the product should be tested in the presence 

of that specific organic soil. Registrants should provide specific information in the data report 
12

regarding the way in which the organic soil, such as soap film residue was prepared (e.g., 

percentages of ingredients). 

(4) Exposure period. The exposure period required for an antimicrobial product to be 

effective may be shorter than the exposure period specified in the recommended method. A

modification to provide a shorter exposure period is restricted by the manipulative limitations 

inherent in the test procedure. A modification to provide a longer exposure period is restricted by the 

practical considerations of the use patterns (e.g., an exposure period of >10 min cannot be 

recommended for a product that will effectively evaporate from the treated surface in 10 min). If 

the product is to be represented in labeling for use at exposure periods shorter than those specified in 

the method, the method should be modified in a manner acceptable to the Agency, to reflect the 

deviation in exposure intended. For liquid products containing volatile active ingredients where the 

product is applied to an environmental surface, the exposure period should be determined by the 

AOAC International Germicidal Spray Products As Disinfectants test (Ref. 14). Use of methods that 

immerse contaminated carriers in the disinfectant fluid would not closely simulate the way in which 

the volatile disinfectants perform on environmental surfaces.

(h) Microbiological technique considerations

(1) Microorganism survival after drying on a hard surface. Quantitative determinations 

of the microbial counts on the untreated control carrier after drying should be conducted in order to 

determine the validity of the test results obtained with the treated carriers. These quantitative 

determinations should be performed for all carrier-based assays, whether or not modifications are 

made to the method being used. The test results should include the individual dried carrier counts 

obtained by the method. The detailed final report for this testing should include information and 

descriptions regarding: preparation of the inoculum; application of the inoculum to the carrier; the 

time/temperature and relative humidity conditions for drying the microorganisms on the carrier; the 

technique for removal of the microorganisms from the carrier; and the specific assay procedure 

indicating such details as replication, subculture media, diluents, and the incubation time/temperature 

conditions for the enumeration procedure employed.

(2) Microorganism survival for suspension tests. Quantitative determination of the 

microbial count of the inoculum in a parallel untreated diluent should be conducted in order to 

determine the level of microbial challenge in the test (Numbers Control). These quantitative 

determinations should be performed for all suspension assays, whether or not modifications are made 

to the method being used. The test results should include the individual counts obtained by the 

recovery method. The detailed final report for suspension testing should include information and 

descriptions regarding: preparation of the inoculum, the volumes used for inoculation, and the 

specific assay procedure indicating such details as replication, subculture media, diluents, and the 

incubation time/temperature conditions for the enumeration procedure employed.

(3) Neutralization. Neutralization is a process for inactivating or quenching antimicrobial 

activity during efficacy testing. This may be achieved through physical (e.g. filtration, dilution, 

secondary subculture) and/or chemical (e.g., addition of sodium thiosulfate to the diluent) means. 
13

For each efficacy test, neutralization procedures should be employed, at the completion of the contact

time, in order to preclude residual effects of the active ingredients in the subculture medium. If 

neutralization is not properly employed, the results of efficacy testing may be exaggerated. A 

specific medium capable of neutralizing the antimicrobial effects of a product should be employed 

prior to the microbiological assay. In addition, data should be submitted to demonstrate that the 

neutralizer employed inactivates the active ingredients and does not possess any antimicrobial 

activity itself. Some of the recommended methods described in this section rely solely upon the 

selection of an appropriate subculture medium to neutralize the antimicrobial effects of certain 

general types of chemical compounds (active ingredients). In lieu of chemical neutralization, it 

should be documented that appropriate subculture techniques have been employed that preclude 

residual carryover of active ingredients. To document the absence of residual effects of the active 

ingredients in the subculture medium, the procedures in paragraphs (h)(3)(i) through (h)(3)(ii)(C) in 

this guideline should be followed.

(i) Efficacy final reports should describe the neutralization techniques employed during the 

study. In addition, evidence should be submitted to demonstrate that the neutralizer identified 

inactivates the antimicrobial ingredient and that the neutralization processitself does not possess any

antimicrobial activity. These controls are termed Neutralization Confirmation. This confirmation is 

conducted by demonstrating the growth of an inoculum of 5-100 CFU test organism/mL growth 

media into a parallel test (including the neutralization process) conducted without the test organism 

or following incubation of the actual test. In addition, the Neutralization Confirmation inoculum is 

inoculated into the parallel test without the neutralization process to confirm lack of antimicrobial 

activity of the process (Ref. 15).

(ii) Examples of neutralization techniques: (A) In carrier-based test methods, the carrier is 

initially deposited in a tube of growth media (i.e., primary subculture). The carrier may then be 

transferred to a second tube of growth media (i.e., secondary subculture). The primary and/or the 

secondary subculture may include a chemical agent to achieve neutralization. Secondary subcultures 

may be helpful in achieving neutralization either through dilution or incorporation of chemical 

agents in the growth media. A neutralization confirmation for carrier-based test methods may be 

conducted by demonstrating the growth an inoculum of 5-100 CFU of test organism/ml growth 

media, into a parallel test with the neutralization process conducted without the test organism or 

following incubation of the actual test. Both the primary cultures and secondary subcultures should 

be incubated and checked for growth in the test and the neutralization confirmation. Dried test 

carriers should not be used to test the ability of a subculture medium to support organism growth, as 

this would provide too large a bioburden and may lead to an inaccurate evaluation of the presence of 

any bacteriostasis that may result from the carry-over of the antimicrobial substance on the carrier to 

the subculture medium. Growth results for both primary and secondary subcultures should be 

reported for the test and neutralization confirmation in the final report.

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

all neutralization/recovery methods employed in testing. Neutralization confirmation may be 

conducted by neutralizing the test substance, without the organism, as in the test. Follow by

inoculation of a low level of organism (5-100 CFU/mL) and subsequent plating. Plate counts should 
14

be within 1.0 log of a parallel population control. 

(C) For virucidal tests, scientifically accepted controls, including proper neutralization 

controls should be performed (e.g., ASTM E1482) (Ref. 16).

(4) Batch replication for modified tests. Where batch replication has already been 

performed and accepted for a product registration with unmodified tests by the recommended 

methods, additional testing at the same use concentration under modified conditions (e.g., different 

exposure period, presence of organic soil or hard water, porous surface carriers, etc.) may be 

conducted with reduced batch replications as in paragraphs (h)(4)(i) and (h)(4)(ii) in this guideline.

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

representing two different batches, instead of three.

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

sample instead of two.

(5) Validation of efficacy. The Agency reserves the option to perform its own tests for 

validation of efficacy of products selected on a case-by-case basis.

(6) Test failure. Failure of a product to meet the specified testing or evaluation of success,

constitutes evidence that the product is unlikely to be effective as claimed in actual use and is 

reportable under FIFRA section 6(a)(2).

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

information:

(1) Environmental Protection Agency, Pesticide Registration Notice PR 2000-1, 

Applicability of the Treated Articles Exemption to Antimicrobial Pesticides, March 6, 2000. Office 

of Pesticide Programs, Antimicrobials Division. See http://www.epa.gov/PR_Notices/.

(2) Environmental Protection Agency, Pesticide Registration Notice PR 91-2, Accuracy of 

Stated Percentages for Ingredients Statement, May 2, 1991. Office of Pesticide Programs, 

Antimicrobials Division. See http://www.epa.gov/PR_Notices/.

(3) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants, Hard 

Surface Carrier Test Methods, Eighteenth edition. AOAC International, Suite 500, 481 North 

Frederick Avenue, Gaithersburg, MD 20877-2417.

(4) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants, UseDilution Methods, Eighteenth edition. AOAC International, Suite 500, 481 North Frederick Avenue, 

Gaithersburg, MD 20877-2417.
15

(5) Environmental Protection Agency, Pesticide Registration Notice 2001-4, Elimination of 

Phenol Resistance Testing for Antimicrobial Disinfectant and Sanitizer Pesticides. Office of 

Pesticide Programs, Antimicrobials Division. See http://www.epa.gov/PR_Notices/.

(6) Protocols for Testing the Efficacy of Disinfectants Against Hepatitis B Virus (HBV). 

Office of Pesticide Programs, Antimicrobials Division. See 

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

(7) Virucidal Effectiveness Test Using Bovine Viral Diarrhea Virus (BVDV) as a Surrogate 

for Human Hepatitis C Virus. Office of Pesticide Programs, Antimicrobials Division. See 

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

(8) Virucidal Effectiveness Test Using Feline Calicivirus as a Surrogate for Norovirus. 

Office of Pesticide Programs, Antimicrobials Division. See 

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

(9) McDonnell, G. (2003) J. AOAC Int. 86,407-411. 

(10) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants, Official 

Method 960.09 - Germicidal and Detergent Sanitizing Action of Disinfectants, Eighteenth edition. 

AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.

(11) Annual Book of ASTM Standards, Test Method for Efficacy of Virucidal Agents 

Intended for Inanimate Environmental Surfaces, Designation E1053-97. American Society for 

Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition. .

(12) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants, Official 

Method 955.17 Fungicidal Activity of Disinfectants. Eighteenth edition. AOAC International, Suite 

500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.

(13) Environmental Protection Agency, Data Call-in Notice for Tuberculocidal Effectiveness 

Data for All Antimicrobial Pesticides with Tuberculocidal Claims (Registration Division, Office of 

Pesticide Programs, June 13, 1986). See http://www.epa.gov/oppad001/dis_tss_docs/dis-06.htm.

(14) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants, Official 

Method 961.02 - Germicidal Spray Products as Disinfectants, Eighteenth edition. AOAC 

International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.

(15) Annual Book of ASTM Standards, Standard Test Methods, Evaluation of Inactivators of 

Antimicrobial Agents, Designation E1054-02. American Society for Testing and Materials, 100 Barr 

Harbor Drive, West Conshohocken, PA 19428, current edition.

(16) Annual Book of ASTM Standards, Standard Test Method for Neutralization of Virucidal 

Agents in Virucidal Efficacy Evaluations, Designation E1482-04. American Society for Testing and 
16

Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition.