United States

Environmental Protection

Agency

Office of Chemical Safety and 

Pollution Prevention

(7510P)

EPA 712-C-07-005

January (insert 

date), 2012

Product Performance 

Test Guidelines 

OCSPP 810.2000:

General Considerations 

for Public Health Uses of 

Antimicrobial Agents
2

NOTICE

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

Chemical Safety and Pollution Prevention (OCSPP) (formerly the Office of Prevention, 

Pesticides and Toxic Substances (OPPTS) prior to April 22, 2010), United States

Environmental Protection Agency for use in testing pesticides and chemical substances to 

develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) 

(15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) 

(7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug, and Cosmetic (FFDCA) 

(21 U.S.C. 346a), referred to hereinafter as the harmonized test guidelines.

The harmonized test guidelines serve as a compendium of accepted scientific

methodologies and protocols that are intended to provide data to inform regulatory decisions 

under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting 

appropriate tests, and is also used by EPA, the public, and the companies that are subject to 

data submission requirements under TSCA, FIFRA and/or the FFDCA. At places in this 

guidance, the Agency uses the word “should.” In this guidance, use of “should” with regard 

to an action means that the action is recommended rather than mandatory. As a guidance 

document, these guidelines are not binding on either EPA or any outside parties, and the EPA 

may depart from the guidelines where circumstances warrant and without prior notice. The 

methods contained in this guideline are strongly recommended for generating the data that 

are the subject of the guideline, but EPA recognizes that departures may be appropriate in 

specific situations. You may propose alternatives to the methods recommended in these 

guidelines, with your supporting rationale. The Agency will assess such proposals on a caseby-case basis. 

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

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

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

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

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OCSPP 810.2000: General considerations for testing public health antimicrobial 

agents.

(a) Scope.

 (1) Applicability. This guideline describes test methods that EPA believes will 

generally satisfy testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act 

(FIFRA)(7U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 

I/S/C. 346a).

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

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

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

(b) Overview—Product performance.

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

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

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

situations, nature and level of pest control, duration of pest control, compatibility with other 

chemicals, benefits and/or adverse effects of product use, compatibility of common practices 

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

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

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

cases to determine whether the substance has sufficient pesticide potential to warrant larger scale 

testing (e.g., swimming pool disinfectants).

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

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

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

equipment (e.g. fumigant sterilants).

 (2) [Reserved]

 (3) Waiver policy. As outlined in 40 CFR Part 161, the Agency has waived all 

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

more pest microorganisms that pose a threat to human health and whose presence cannot readily 

be observed by the user, including but not limited to, microorganisms infectious to man in any 

area of the inanimate environment. However, pursuant to FIFRA, each registrant must ensure 

through testing that its products are efficacious when used in accordance with label directions 

and commonly accepted pest control practices. The registrant must develop and maintain the 

relevant data upon which the determination of efficacy is based. The agency reserves the right to 

require, on a case-by-case basis submission of efficacy data for any pesticide product, registered 
4

or proposed for registration.

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

Guideline Series 810.2000.

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

Guideline Name 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

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

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

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

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

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

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

methods may occur, on a case-by-case basis, prior to guideline updates and these new methods 

will be published on the EPA Antimicrobial Policy and Guidance Documents website until such 

time that they can be added to the guidelines.

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

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

considerations. Microbial pests can be categorized into two basic types: Those that present 

potential public health hazards because of their infectious nature to humans and those that cause 

economic or aesthetic problems such as spoilage, fouling, or production of offensive odors in the 

substrate in which they grow. The OCSPP Test Guideline Series 810.2000 addresses

antimicrobial pesticide products with public health uses for which efficacy test data are required 
5

to be submitted to support registration. These include all antimicrobial products intended to 

control microorganisms infectious to man in any area of the inanimate environment where these 

microorganisms may present a hazard to human health. The label claims for an antimicrobial 

product determine whether it is considered to be related to human health.

(ii) Public health claim. An antimicrobial pesticide is considered to make a public health 

claim if the pesticide product bears a claim to control one or more pest microorganisms that pose 

a threat to human health, and whose presence cannot readily be observed by the user, including 

but not limited to, microorganisms infectious to man in any area of the environment. A product 

makes a public health claim if one or more of the following apply:

(A) A claim is made for control of one or more specific microorganisms that are directly 

or indirectly infectious or pathogenic to man (or both man and animals). Examples of specific 

microorganisms include, but are not limited to, Mycobacterium tuberculosis, Pseudomonas 

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

Staphylococcus aureus. Claims for control of microorganisms infectious or pathogenic only to 

animals (such as canine distemper virus or hog cholera virus) are not considered public health 

claims.

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

sanitizer or tuberculocide against one or more microorganisms that are infectious or pathogenic 

to man.

(C) A claim is made for the pesticide product as a fungicide against one or more fungi 

infectious or pathogenic to man, or the product does not clearly state that it is intended for use 

only against non-public health fungi.

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

microbial purification system or swimming pool disinfectant/sanitizer.

(E) A non-specific claim is made that the pesticide product will beneficially impact or 

affect public health at the site of use on in the environments in which it is applied.

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

(i) Non-public health claim. An antimicrobial pesticide is considered to make a nonpublic health claim if the pesticide product bears a claim to control 

microorganisms of economic or aesthetic significance, where the presence of the 
6

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, odor control and other non-public health agents. Slimicides and odor 

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

microorganisms of economic or aesthetic significance are not considered to be human healthrelated, but nevertheless must bear accurate labeling claims and adequate dosage 

recommendations, and complete directions for use.

(B) Bacteriostatic/Fungistatic products. Since elimination or significant reduction in the 

number of microorganisms (sterilization, disinfection, sanitization) should be demonstrated 

before a product is considered acceptable for use against microorganisms infectious for humans, 

or for use in medical or sickroom environments, products bearing lower level labeling claims for 

effectiveness at the bacteriostatic/fungistatic (inhibition of growth) level would generally not be

appropriate for such uses. Bacteriostatic/fungistatic claims are generally acceptable only for 

products expressly recommended for control of microorganisms of aesthetic significance (e.g., 

spoilage bacteria, odor-causing bacteria).

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

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

label claims in Pesticide Registration Notice 2000-1 (Ref. 1). The treated articles exemption (40 

CFR 152.25(a)) covers qualifying articles and substances bearing claims to merely protect the 

article or substance itself, if, among other things, the pesticide is registered for such use. This

exemption does not include articles or substances bearing implied or explicit public health claims 

against human pathogens. Applicants who intend to market products with claims (such as public 

health claims) that go beyond the scope of the treated articles exemption should contact the 

Antimicrobials Division prior to conducting testing to support such a use.

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

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

disease pathogens and zoonotic microorganisms may be appropriate prior to approval of the label 

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

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

Agency requires, on a case-by-case basis, the submission of efficacy data to support these claims 

because these pathogens have animal health significance or the potential to infect humans.

Applicants should consult the Agency for a current listing of organisms which meet these

criteria. These organisms are also listed on the World Organization for Animal Health’s website, 

see, http://www.oie.int/animal -health-in-the-world/oie-listed-diseases-2011/.

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

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

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

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

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

included in order to support consistency and clarity. The terms in the OCSPP Test Guideline 
7

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 

bacteria in the inanimate environment.

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

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

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

Biofilm means a dynamic, self-organized accumulation of microorganisms and 

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

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

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

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

product. Each guideline further addresses confirmatory data

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

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

produce them, and (2) those that mask, chemically destroy or neutralize odors (refer to 40 CFR 

152.10). Products that claim deodorization by antimicrobial means are subject to registration as 

pesticides under FIFRA.

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

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

environment.

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

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

Fungistat means a substance, or mixture of substances, that inhibits the growth of fungi 
8

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.

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

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

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

usefulness.

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

population in the inanimate environment by significant numbers, (e.g., 3 log 10 reduction) or 

more, but does not destroy or eliminate all bacteria. Sanitizers meeting Public Health Ordinances 

are used on food contact surfaces and are termed sanitizing rinses.

Slime means a dynamic, self-organized accumulation of microorganisms and 

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

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

Slimicide means a substance, or mixture of substances, that reduces the number of 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.
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Two-Step Sanitizer or Two-Step Disinfectant means a substance or mixture of substances 

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

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

disinfecting.

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

inactivates viruses in the inanimate environment.

Volatile means a substance that evaporates readily.

Zoonotic microorganism means an infectious agent that can be transmitted between

animals and humans.

(e) General testing considerations

(1) Test substance. 

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

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

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

The following information should be provided: 

(ii) Identification of the test substance and quantitative description of its chemical 

composition should be reported.

(iii) Manufacturer and production batch numbers of the test substance. If a product is 

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

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

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

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

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

(v) In situations where it may not be technically feasible to test at the exact Lower 

Certified Limits (LCL) (e.g., reactive chemistries with a degradation profile, multiple active 

ingredients, formulations with inversely related active ingredients), a rationale should be 

provided to the Agency for approval of the testing if testing cannot be performed at the LCL 

Efforts should be made to test as close to the LCL as possible.

(2) General considerations

(i) Good Laboratory Practice Standards. Good Laboratory Practice Standards (GLP) as 

defined in 40 CFR Part 160 apply to studies to support all antimicrobial products used on hard 

surfaces. According to 40 CFR §160.17: “EPA may refuse to consider reliable for purposes of 
10

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.” 

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

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

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

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

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

that the use solution can be used. Representative surfaces for testing are identified in each 810 

Guideline.

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

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

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

applicant should be responsible for the validity of the test method selected to substantiate a 

product's efficacy. The applicants should ensure themselves that the selected method is current 

and applicable to the product and uses proposed for registration.

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

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

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

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

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

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

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

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

confirmation may also be conducted within the usual transfer cycle or other appropriate transfer 

depending upon organism’s growth requirements. The following information identified in 

paragraphs (e)(3)(i) through (e)(3)(iv) in this guideline should be submitted with the Antibiotic 

Resistance Confirmation testing or included in the final report.

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

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

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

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

(C) The method used to confirm the identity (e.g. biochemical test, Gram stain, 
11

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 comparison to a standard Clinical and National Standards Institute 

interpretation of such tests.

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

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

used, the manufacturer of such automation should be specified.

(iv) Quality control procedures used to verify results.

(f) Special considerations

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

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

For other conditions (hard water, organic soil, 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 was a 

component of the AOAC Use-Dilution Test methods, as well as the Tuberculocidal Activity of 

Disinfectants method.

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

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

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

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

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

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

tuberculosis. See Guideline 810.2200.

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

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

The data consistency control (method validation) stated in the referenced protocol utilizing two 

dilutions of BTC 835 has been eliminated.

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

hepatitis C virus (Ref. 7). The data consistency control (method validation) stated in the 
12

referenced protocol utilizing two dilutions of BARDAC 2280 has been eliminated.

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

The data consistency control (method validation) stated in the referenced protocol utilizing two 

dilutions of bleach has been eliminated.

(4) Antimicrobial rinses for fruits and vegetables. To support label claims for 

consumer-use products as antimicrobial rinses for fruits and vegetables, products should meet a 

two log reduction of five outbreak strains of Salmonella spp., Listeria monocytogenes, and 

Escherichia coli O157:H7. Currently there is no standard method for assessing the efficacy of 

antimicrobial rinses for pathogen reduction on the surfaces of fruits and vegetables (raw 

agricultural commodities). Applicants should consult with the Agency prior to conducting testing 

to support this use.

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

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

products, as a method modification to the AOAC Sporicidal Activity Test. Consult the Agency 

for other chemistries. (Ref. 9).

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

manner that is not reflected by the test conditions specified in the recommended AOAC methods 

(e.g., inclusion of organic soil or hard water), one or more test conditions specified in the method 

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

relative to the conditions of use of the product. The information in paragraphs (g)(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 suffice to support an analogous claim for efficacy of the product on 

hard, non-porous surfaces as well. 

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

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

effectiveness of the product in synthetic hard water at the level claimed. The hard water tolerance 

level may differ with the level of antimicrobial activity claimed (e.g., sterilization, disinfection, 

or sanitization). To establish efficacy in hard water, all microorganisms (bacteria, viruses, and 

fungi) claimed to be controlled by the product should be tested by the appropriate recommended 

method at the same hard water tolerance level. Refer to the AOAC International Germicidal and 
13

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 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
14

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 this deviation in the method should be included in the test protocol 

submitted with the data. For liquid products containing volatile active ingredients where the 

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

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

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

in which the volatile disinfectants perform on environmental surfaces.

(h) Microbiological technique considerations

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

determinations of the microbial counts on the untreated control carriers after drying should be 

conducted in order to determine the validity of the test results obtained with the treated carriers. 

The control carrier count evaluation should be performed at the completion of the test. These 

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

modifications are made to the method being used. The test results should include the individual 

dried carrier counts obtained by the method. The detailed final report for this testing should 

include information and descriptions regarding: preparation of the inoculum; application of the 

inoculum to the carrier; the time/temperature and relative humidity conditions for drying the 

microorganisms on the carrier; the technique for removal of the microorganisms from the carrier; 

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

diluents, and the incubation time/temperature conditions for the enumeration procedure 

employed.

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

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

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

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

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

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

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

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

and the incubation time/temperature conditions for the enumeration procedure employed.

(3) Neutralization. Neutralization is the process for inactivating or quenching 

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

filtration, dilution, secondary subculture) and/or chemical (e.g., addition of sodium thiosulfate to 

the diluent) means. For each efficacy test, neutralization procedures should be employed, at the 

completion of the contact time, in order to preclude residual effects of the active ingredients in 

the subculture medium. If neutralization is not properly employed, the results of efficacy testing 

may be exaggerated. The method of neutralization, whether that be physical or chemical, should 

be confirmed. Data should be submitted (Neutralization Confirmation) in the final report to 

demonstrate that the neutralization method sufficiently inactivated the active ingredient(s) and 
15

that any neutralizer medium used did not possess any antimicrobial activity. 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 examples of neutralization 

techniques procedures in paragraphs (h)(3)(i) through (h)(3)(ii) in this guideline should be 

followed.

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

in a tube of growth media (i.e., primary subculture). If the primary subculture tube is not 

sufficient to achieve neutralization by the use of a chemical agent and/or through dilution of the 

test substance, the carrier may then be transferred to a second tube of growth media (i.e., 

secondary subculture). Secondary subculture may be helpful in achieving neutralization. The 

neutralization procedure employed should be confirmed and reported by running a parallel test 

with uninoculated carriers being added to growth media containing a low level (i.e., 10 – 100 

CFU/mL) of bacteria. Both the primary cultures and secondary subcultures should be incubated 

and checked for growth in the test and the neutralization confirmation. Dried test carriers should 

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

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

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

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

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

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

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

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

neutralization, inoculation of a low level of organism (5-100 CFU/mL) and subsequent plating

should follow. Plate counts should be within 1.0 log of a parallel population control: 

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

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

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

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

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

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

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

guideline.

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

representing two different batches, instead of three.

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

sample instead of two.

(5) Validation of efficacy. The Agency reserves the option to perform its own tests, on a 
16

case-by-case basis, following the protocol submitted with the registration if that protocol meets 

current agency standards. In all other cases, the Agency will conduct the validation studies using the 

current agency protocol.”

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

evaluation of success, constitutes evidence that the product is unlikely to be effective as claimed 

in actual use and is reportable under FIFRA section 6(a)(2).

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

background information:

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

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

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

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

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

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

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

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

Frederick Avenue, Gaithersburg, MD 20877-2417.

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

Gaithersburg, MD 20877-2417.

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

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

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

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

Office of Pesticide Programs, Antimicrobials Division. See 

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

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

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

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

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

Office of Pesticide Programs, Antimicrobials Division. See 

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

(9) McDonnell, G. (2003) J. AOAC Int. 86,407-411. 
17

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

Official Method 960.09 - Germicidal and Detergent Sanitizing Action of Disinfectants, Current

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

2417.

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

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

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

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

Official Method 955.17 Fungicidal Activity of Disinfectants. Current edition. AOAC 

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

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

Effectiveness Data for All Antimicrobial Pesticides with Tuberculocidal Claims (Registration 

Division, Office of Pesticide Programs, June 13, 1986). See 

http://www.epa.gov/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, Current edition. AOAC 

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

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

of Antimicrobial Agents, Designation E1054. American Society for Testing and Materials, 100 

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

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

Virucidal Agents in Virucidal Efficacy Evaluations, Designation E1482. American Society for 

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