United States Environmental Protection Agency Office of Prevention, Pesticides And Toxic Substances ( 7510P ) EPA 712
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- 07
- 005 Draft November 5 ,

2009 

# Product Performance Test Guidelines OPPTS 810.2000 : General Considerat ions for Public Health Uses of Antimicrobial Agents

Public Review Draft1 NOTICE This guideline is one of a series of test guidelines established by the Office of Prevention, Pesticides and Toxic Substances (OPPTS), United States Environm ental 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 (FIF RA) (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

recommende d 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 DO CUMENT 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 pro cess 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 In secticide, 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 A gents (Pesticide Assessment Guidelines, Subdivi sion 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 o r 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, benef its 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) La boratory and/or simulated

- use testing is conducted to determine the eff ectiveness 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) Ins ome cases, effectiveness and usefulness of the proposed product is further proven through advanced large

- scale laboratory t ests, field tests, in

- use tests, or simulated

- use tests by procedures which closely a pproximate actual use and which employ typ ically u sed application equipment (e.g. fumigant sterilants).

(2) [Reserved] (3 ) W aiver policy. As outlined in 40 CFR Part 158, the Agency h as waived all requirements to submit efficacy data unless the pesticide product b ears a claim to control pest microorganisms that pose a threat to human health and whose presence cannot readily b e 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 o f efficacy i s based. The agency

reserves the right to require, on a case -by

-3

case basis (e.g., zoonotic microorganisms ) submission of efficacy data for any

p esticide 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 8 10.2100 91
- 2(a) Antimicrobials for use as Disinfectants, Fungicides, Virucides, & Tuberculocides 810.2 2 00 91
- 2(b)(c)(d)(e)(f)(g)(i) 91
- 7(a)(1) 91
- 3 Antimicrobials for use as Sanitizers

– Food & Non
Food Contact Surfaces , Residual 810.2 3 00 91
- 2(j)( k)( l)(m) 91
- 3
Antimicrobials for use on Textiles 810.2 4 00 91
- 4(a)(1)(2)(3)(4) 91
- 4(b)(c)(d) Antimicrobials for use in the Air 810.2 5 00 91
- 5 Antimicrobials for use in Wa ter (Swimming pool, Drinking water) 810.2 6 00 91
- 8

( 5 ) F uture guidelines. The A gency 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 i s considering adopting the use of quantitative test methods as a possible replacement for current qualitative methods [ e.g., Association of Official Analy tical Chemist s (AOAC ) Use

- Dilution Methods ] in the future. The Agency

intends to update these guidelines periodically

. H owever, 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 product s (1) A ntimicrobial products with public health uses. (i) Health

- related considerations. Microbial pests can be categorized into two basic ty pes: Those that present potential public health hazards because of their infectious nature and those that cause economic or aes thetic 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

t est data are required to be submitted to support re gistration. These include all antimicrobial products intended to control micro organisms infectious to man in any area of the4

inanimate environment where these microorganisms may p resent a hazard to human health. The label claims for an antimicrobial product determine whether it is cons idered 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 e ffectiveness 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 c onsidered as human health

- related

. S uch 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 ty pes 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 microorga nisms that are infectious for man on/in environmental surfaces or the environment, such as Staphylococcus aureus, Myco bacterium 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

- contac t surface sanitizers are considered to be human health

- related, whether control of infectious microorganisms is specifically claimed. (2) Antimicrobial products with nonpublic health uses. Registrants who propose non

health related claims for their pro duct (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. H owever, 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 ty pes of products in paragraphs (c)(2)(A) through (c)(2)(D) in this guideline are considered to be non

-public health related products. (A) S lime, odor control and other non -public health agents. S lime and odor control agents, preservatives, algicides, and other products expressly c laiming control of microorganisms of economic or aesthetic significance are not considered to be human health

- relat ed, but nevertheless must bear accurate labeling claims and adequate dosage recommendatio ns, and complete directions for use. (B) B acteriostatic products. S ince elimination or significant reduction in the number of microorganisms (sterilization, disinfe ction, sanitization) should be demonstrated before a product is5

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. B acteriostatic 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 h as clarified its policy

o n applicability

o f the treated articles exemption to antimicrobial pesticides and provided guidance on appropriate language or label claims in Pesticide Registration Notice 2000

- 1 (see ref erence (i) (1) of this guideline). The exemption (40 CFR 152.25 (a)) covers qualifying articles and substances bearing claims to merely protect th e 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. F or products labeled for public health and/or non -public health uses, submission of studies to EPA on certain animal di sease 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 i s 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 o f microorganisms to be controlled and the different claims and many

u se patterns of antimicrobial products, uniform product terminology a nd a common understanding of a few key wo rds are important to a program for evaluating product performance. Even though the OPPTS Test Guideline Series 810.2000 guideline s 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 o f man or other animals. Antiseptic products are not identified as pesticides and are regulated by t he 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 microo rganisms). Note: The terms bactericide and fungicide, as used in conjunction with the term microbiocide , are only related to industrial uses

. Biofilm means a community o f 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 ty pes: (1) Those that prevent or delay the formation of bacterial odors by killing micro organisms which produce them, and (2) those that mask, chemically de stroy, 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 o r public health microorganism, but not necessarily b acterial spores, in the inanimate environment. Fungicide means a substance, or mixture of substances that destroys fungi (including ye asts) 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.7

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 tes ted 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 microorganis ms 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 s ignificant numbers, but does not destroy o r eliminate all bacteria or other microorganisms. Slimicide means a substance, or mixture of substances that reduces the number of slime

forming microorganisms in industrial water systems (e.g., paper mills). For the purposes of these guidelines, slimicide claims are reserved for non

-public health industrial label claims. Sterilant means a substance, or mixture of substances that destroys or eliminates all forms of microbial life in the inanimate environment, including all forms of vegetative bacteria, bacterial spores, fungi, fungal spores, and viruses. Sporicide means a substance, or mixture of substances, that irreversibly inactivates bacterial spores in the inanimate environm ent. 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 be en registered for effectiveness against microorganisms in the presence of a bioburden

. The sanitizer or disinfectant use directions should state the need to pre - cle an surfaces prior to sanitizing or disinfecting. Virucide means a substance, or mixture of substances that destroys or irreversibly inactivates viruses in the inanimate environment. Zoonotic microorganism means an infectious agent that can be transmitted between animals and humans.8

( e) General testing consideration s (1) T est substance. ( i ) Unless otherwise specified, antimicrobial pes ticides 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 c an consistently

reproduce the formulation (batch replication), as well as to s how 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 consideration s (i) Good Laboratory Practice Standards. Antimi crobial products should be tested in ac cordance 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 ty pe of antimicrobial agent, target microorgani sms, and the site to be treated, all tests should address those factors that would normally b e 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 tre ated, 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) A dditional 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 o f 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 o f a product, such protocols should be submitted to the Agency f or review. Inaddition 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) U se of Antibiotic Resistant Test Organisms . Organisms to be labeled as antibiotic resistant should be accompanied by scientific data that substantiates the antibioti c 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 t esting. The confirmation may also be conducted within the usual transfer cycle or other appropriate transfer depending upon organism’s growth requirements. The information in paragra phs ( e)(3)(i) through (e )(3)(iv) in this guideline should be submitted from the Antibiotic Resistance Confirmation testing

. (i) Test org anis ms 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, cry ogenic beads, other). (ii) Results of the testing including the numerical values of all antibiotics tested. An example of values would be Minimum Inhibitory Concentration (MI C ) s for automated test, zone sizes for manual tests, and a standard National Clinical Laboratory S tandards (NCCL S) Interpretation of such tests. ( iii ) The scientific method used to obtain the results (Kirby

- Bauer, disc agar diffusion, or gradient agar diffusion; automated MI C procedures or equivalent). I f automated procedures are used, the manufacturer of such automation should be specified. ( iv) Quality

control proce dures used to verify results. ( f ) Special considerations ( 1 ) H ard Surface Carrier Test vs. Use -Dilution Methods

. The AOAC International Hard Surface Carrier Test Method (Ref. 3 ) has only b een validated for use with distilled water. F or other conditi ons (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 t ests. The phenol resistance assay i s a component of AOAC Use

- Dilution Test methods, as well as the Tuberculocidal Activity of Disinfectants method. (3 ) Surrogate microorganisms . The Agency h as approved the use of several surrogate10

organisms to be used as replacements for microorganisms that cannot be tested because of biohazards or unavailability o f scientifically

accepted methods

. Applicants shoul d 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

. (i i ) 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 ). (ii i ) 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 ) A ntimicrobial rinses for fruits and vegetables. To support label claims for consumeruse produc ts 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. C urrently

there is no standard method for assessing the effic acy o f antimicrobial rinses for pathogen reduction on fruits and vegetables. Applicants should consult with the Agency p rior 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) S pecial 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 devel oped 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) T ype of surface. When an antimicrobial product is intended to be effective in treating a hard, porous surface, some of the recommended methods may b e 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 confirmatio n, sterility

controls) should be developed to assure the validity of the test results when this modification of the method is employed. S ince 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 o f the product on hard, non -porous surfaces as well.11

(2) Hard water claim . Any p roduct that bears label claims for effec tiveness in hard water should be tested by

the appropriate method which has been modified to demonstrate effectiveness of the product in sy

nthetic hard water at the level claimed. The hard water tolerance level may differ with the level of antimicrobial activity c laimed (e.g., sterilization, disinf ection, or sanitization). T o 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 l evel. Refer to the AOAC International Germicidal and Detergent Sanitizing Action of Disinfectants test (Official Method 960.09) for guidance on the preparation of sy nthetic hard water (Ref. 10 ). (3) Organic burden . (i) An antimicrobial substance identified as a one - step cleaner

- disinfectant or cleaner

sanitizer, or intended to be effective in the presence of light to moderate amounts of organic burden should be tested for efficacy b y the appropriate methods which have been modified to in clude a minimum of a 5% representative organic soil such as blood serum or scientifically accepted equivalent as serum m ay b e 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) Asug gested 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 mLt est microorganism culture +1 mLb lood 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. C ontrol data (e.g., quantitation of dried carrier counts , neutralization confirmation, sterility

c ontrols) should also be developed to assure the validity o f the test results when this modification is incorporated into the method. The organic soil level suggested is considered appropriate for simulating lightly o r moderately

s oiled 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) Asuggested 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 Intern ational Fungicidal Activity

o f 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 mLt est solution + 0.25 mLblood serum) before adding 0.5 mLof the test organism

. (iv) When a product is recommended for certain patterns of use where the organic soil claimed is of a specific ty pe, 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 report12

regarding the way

in which the organic soil, such as soap film residue was prepared ( e.g., percentages of ingredients). (4) E xposure period. The exposure period required for an antimicrobial product to be effective may

b e 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. Am odification to provide a longer exposure period is restricted by t he 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 produc t 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. F or liquid products containing vo latile active ingredients where the product is applied to an environmental surface, the exposure period should be determined by the AOAC International Germicidal Spray P roducts As Disinfectants test (Ref. 14 ). U se of methods that immerse contaminated carriers in the disinfectant fluid would not closely s imulate the way

in which the volatile disinfectants perform on environmental surfaces. ( h ) Microbiological technique considerations ( 1) Microorganism survival after drying on a hard surface. Quantitativ e determinations of the microbial counts on the untreated control carrier after drying should be conducted in order to determine the validity

o f 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 c onditions for drying the microorganisms on the carrier; the technique for removal of the microorganisms from the carrier; and the specific assay p rocedure indicating such details as replication, subculture media , diluents, and the incubation time/temperature conditions for the enumeration procedure employed. (2) Microorganism s urvival for suspension t ests. 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 m ethod. The detailed final report for suspension testing should include information and des criptions 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 proc edure employed. (3 ) N eutralization. Neutralization is a process for inactivating or quenching antimicrobial activity d uring efficacy

t esting. This may

b e 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

t est , 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. I f neutralization is not properly employed, the results of efficacy t esting may

b e exaggerated. A specific medium capable of neutralizing the antimicrobial effects of a product should be employed prior to the microbiological assay . Inaddition , data shoul d be submitted to demonstrate that the neutralizer employed inactivates the active ingredients and does not possess any antimicrobial activity itself. S ome of the recommended methods described in this section rely s olely u pon the selection of an appropriate subculture medium to neutralize the antimicrobial effects of certain general ty pes 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

f inal reports should describe the neutralization techniques employed during the study

. Inaddition, evidence should be submitted to demonstrate that the neutralizer identified inactivates the antimicrobial ingredient and that the neutralization process itself does not possess any antimicrobial activity

. These controls are termed Neutralization Confirmation

. This confirmation is conducted by

demonstrating the growth of an inoculum of 5

- 100 CFU test organism/mLgrowth media into a parallel test (including the neutralization process) conducted without the test organism or following incubation of the actual test. Inaddition, 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) Inc arrier

-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 b e helpful in achieving neutralization either through dilution or inc orporation 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 neutraliz ation 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 carrier s should not be used to test the ability o f a subculture medium to support organism growth, as this would provide too large a bioburden and may l ead to an inaccurate evaluation of the presence of any b acteriostasis that may

result from the carry- over of th e 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 suspen sion based test methods should be conducted for all neutralization/recovery m ethods employed in testing. N eutralization confirmation may b e conducted by

n eutralizing the test substance, without the organism, as in the test. F ollow by inoculation of a low level of organism (5

- 100 CFU/mL) and subsequent plating. P late counts should14

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 b een 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, po rous surface carriers, etc.) may b e conducted with reduced batch replications as in paragraphs (h)(4)(i) and (h)(4)(ii) in this guideline

. (i) For basic efficacy

c laims (e.g., sterilants, disinfectants, sanitizers) , two samples, representing two different batches, instead of three. (ii) For supplemental efficacy c laims (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 ) T est failure

. F ailure of a product to meet the specified testing or evaluation of success , c onstitutes 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 b e consulted for additional background information : (1) Environmental Protection Agency, Pesticide Registration Notice PR 2000

- 1, Applicability

o f the Treated Articles Exemption to Antimicrobial Pesticides, March 6, 2000. O ffice of Pesticide Programs, Antimicrobials Division

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

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

- 2, Accuracy o f Stated Percentages for Ingredients Statement, May 2 , 1991. Office of Pesticide Programs, Antimicrobials Division

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

. ( 3 ) Official Me thods of Analysis of AOAC International

. C hapter 6, Disinfectants, Hard Surface Carrier Test Methods, Eighteenth edition. A OAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417. (4 ) Official Methods of Analysis of AOAC I nternational. Chapter 6, Disinfectants, Use

Dilution 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, Elimina tion of Phenol Resistance Testing for Antimicrobial Disinfectant and Sanitizer Pesticides. O ffice of Pesticide Programs, Antimicrobials Division

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

. ( 6 ) Protocols for Testing the Efficacy o f 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 Pes ticide 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 o f Virucidal Agents Intended for In animate Environmental Surfaces, Designation E1053

- 9 7. American Society f or Testing and Materials, 100 Barr Harbor Drive, West Conshohocken , PA 19428, current edition.

. (1 2 ) Official Methods of

Analysis ofAOAC International

. Chapter 6, Disinfectants, Official Method 955.17 Fungicidal Activity o f Disin fectants. Eight eenth 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 Pro grams, 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 961.0 2

- Germicidal Spray P roducts 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 f or 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 Viruc idal Efficacy Evaluations, Designation E1482

- 04. American Society f or Testing and16

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