*** Deliberative Internal Document

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1 United States Environmental Protection Agency Office of Chemical Safety and Pollution Prevention (7503P) EPA XXX

-X

-XX

-XXX April 2015 

# Product Performance Test **Guidelines** OCSPP 810.2000 : General Considerations for Testing Antimicrobial Agents*** Deliberative Internal Document

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2 **NOTICE** This guideline is one of a series of test guidelines established by the Office of Chemical Safety

and Pollution Prevention (OCSPP) for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a), referred to hereinafter as the harmonized test guidelines. Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides, and Toxic Substances (OPPTS). To distinguish these guidelines from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS as part of the guideline’s number. Any test guideline developed after April 22, 2010 will use the new acronym(OCSPP) in their title. The OCSPP test guidelines serve as a compendium of accepted standardized methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the tests , and are also used by EPA, the public, and the companies that are subject to data submission requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the EPA may

depart from these guidelines where circumstances warrant and without prior notice. At places in this guidanc e, the Agency uses the word “should.” In this guidance, use of “should” with regard to an action means that the action is recommended rather than mandatory

. The procedures contained in this guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA recognizes that departures may b e appropriate in specific situations. You may propose alternatives to the recommendations described in these guidelines, and the Agency will assess them for appropriateness on a case -by

- case basis. For additional information about these test guidelines and to access the se guidelines electronically, please go to http://www.epa.gov/ocspp and select the topic entitled, “Test Methods and Guidelines

. ” You may also access the guidelines in http://www.regulations.gov grouped by S eries 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 f or the purpose of external review. It has not been formally disseminated by

the EPA and should not be construed to represent any Agency determination or policy

. The information correction process under the Agency ’ s Information Quality Guidelines does not apply until the document is formally disseminated by the EPA in its final form. This draft document should only

be cited or quoted in the context of providing comments.*** Deliberative Internal Document

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3 **OCSPP 810.2000**  **: General considerations fo**  **r testing**  **antimicrobial agents**

. **(a) Scope**  **. (1)**  **Applicability.** OCSPP Test Guideline Series 810.2100 through 810.2700 describes general information regarding product performance testing of antimicrobial pesticides to meet requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)(7U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C . 346a). **(2) Background.**  Microbial pests can be categorized into two basic ty pes: 1) those that present potential public health hazards because of their infectious nature to humans (public health claims) and 2) those that cause economic or aesthetic problems such as spoilage, fouling, or producti on of offensive odors in the substrate in which they grow (non

-public health claims). The OCSPP Test Guideline Series 810.2000 addresses antimicrobial pesticide products with public health uses for which efficacy test data are required to be submitted to the Agency to support registration. The source material used in developing the OCSPP T est Guideline is OPP guideline 91

- 1: General Requirements for Antimicrobial Agents (Pesticide Assessment Guidelines, Subdivi sion G, Product Performance, EPA report 540/9

- 82

- 026, October 1982). **(b) Purpose and**  **Overview**

**— Product performance**  **. (1)**  **General concepts.**  Evaluation of product performance is conducted in consideration of expressed and implied labeling claims or recommendations which may

include pests, sites, methods of application, application equipment, dosage rates , timing and number of applications, use situations, nature and level of pest control, duration of pest control, compatibility with other chemicals, benefits and/or adverse effects o f product use, compatibility of common practices associated with the sites, and ingredient status of chemicals in the formulation

. (i) Laboratory

and/or simulated

-use testing is conducted to determine the effectiveness of a substance, or mixture of substances , to control or kill specific pest organisms, and in some cases to determine whether the substance has sufficient pesticide potential to warrant larger scale testing (e.g., swimming pool disinfectants)

. (ii) Ins ome cases, effectiveness and usefulness of the proposed product is further determined through advanced large

- scale laboratory tests, field tests, in

-use tests, or simulated

-use tests by procedures which closely approximate actual use and which employ typically used application equipment (e.g. fumigant sterilants).*** Deliberative Internal Document

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4 **(2) Series**  **organization.** Table 1 outlines the organization of the OCSPP Test Guideline Series 810.2000. **Table 1. Organization of the OCSPP Test Guideline Series 810.2000.** Guideline Name Guideline Number Previous Subdivision

–G Guideline Number(s) Sterilants

– Efficacy

Data Recommendations 810.2100 91

-2(a) Disinfectants for Use on Hard Surfaces

– Efficacy

Data Recommendations 810.2200 91

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

- 7(a)(1) 91

- 3 Sanitizers for Use on Hard Surfaces

– Efficacy

Data Recommendations 810.2300 91

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

- 3 Disinfectants and Sanitizers for Use on Textiles

– Efficacy Data Recommendations 810.2400 91

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

-4(b)(c)(d) Air Sanitizers

– Efficacy

Data Recommendations 810.2500 91

- 5 Disinfectants for Use in Water

– Efficacy Data Recommendations 810.2600 91

- 8 Prions

– Efficacy

Data Recommendations 810

. 2700 Not Applicable
**(3) Good**  **Laboratory Practice**  **Standards.** Good Laboratory Practice Standards (GLP) as defined in 40 CFR Part 160 apply to studies to supp ort the registration of all antimicrobial products

. According to 40 CFR §160.17: “EPA may refuse to consider reliable for purposes of supporting an application for a research or marketing permit any data from a study which was not conducted in accordance with this part.” 40 CFR §160.12 (b) requires with any submitted research data “[a] A statement that the study was conducted in accordance with this part; [b] A statement describing in detail all differences between the practices used in the study and those required by

this part; or [c] A statement that the person was not a sponsor of the study, did not conduct the study, and does not know whether the study was conducted in accordance with this part.” **(4) Data**  **for modifications**  **of recommended**  **methods.** When recommended methods are modified by the applicant to support specific claims and/or use patterns for a product, the applicant should submit the complete testing protocol, identifying and describing each modification to the Agency f or review and evaluation prior to initiation of the test. The study report should identify and describe each modification.*** Deliberative Internal Document

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5 **(5) Data**  **for other**  **methods.** When recommended methods, or modifications thereto, are not employed to develop efficacy data, complete testing protocols should be submitted to the Agency for review and evaluation prior to initiation of the test
. All pertinent information, including protocols, should be included with the final test reports. All materials and procedures employed in testing should be fully describ ed

. Where references to published reports or papers are made, copies or reprints of such references should be provided with the test reports. **(6) Confirmatory**  **data**

. In certain situations , an applicant may rely on previously submitted product efficacy data to support a new application or amendment for registration of a product and submit only confirmatory

efficacy data on the subject product to verify

the product’s antimicrobial efficacy

. These situations are as listed below : (i) **Duplicated Produc** **t Formulations.**  In this situation, the applicant manufactures a formulation which duplicates a product that is already registered and has complete supporting efficacy data. The chemical composition, manufacturing procedure, label claims, and directions for use are identical in substance to those of the original registration, and specific references (including Master Record ID [MRID] Numbers) to the supporting data developed for the duplicated product are cited by the applicant. (ii) **Minor Fo**  **rmulation Change**  **in a**  **Registered Product.**  In this situation, the change in the formulation is relatively minor. The label claims and directions for use are unchanged from those accepted for the registered formulation, and specific references (including MRID numbers) to the su pporting data developed for the original formulation are cited by the applicant. If the only

change in the formulation is the addition of a fragrance or dye, confirmatory data do not need to be submitted. However, when the product is an aerosol formulation, confirmatory data should be submitted for all formulation changes, including the addition of fragrances and dyes. (iii) The confirmatory data are to be developed from testing the applicant's own finished product. If the test methodology utilized in the supporting (cited) efficacy

data was modified to include additional elements not specified in the recommended method (e.g., organic soil, hard water, longer or shorter contact time) , the confirmatory data should be produced under similarly

modified conditions. **(7) Verification**  **of efficacy** 

. The Agency reserves the option to perform its own testing , on a case -by

- case basis, following the product specific test conditions*** Deliberative Internal Document

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6 used to register or amend the registration of the product (contact time, hard water etc.)

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

. **(8) Test**  **failure**

. Failure of a product formulation to meet the specified testing or evaluation of success (i.e. the performance standard), constitutes evidence that the prod uct is unlikely

to be effective as claimed in actual use and is reportable under FIFRA section 6(a)(2). **(9) G** **uideline revisions**  **.** The Agency recognizes that novel technologies associated with antimicrobial products may evolve over time and would potentially involve test methods that are not referenced in the guideline series

. Inaddition, the Agency i s considering adopting the use of quantitative test methods as an alternative or replacement to several current qualitative methods [ e.g., Association of Official Analytical Chemists (AOAC International) Use

-Dilution Methods ]

. The Agency expects to update the guidelines periodically to address such changes ; however, the use of new methods may occur prior to guideline updates

. Inthese instances , new methods will b e published on the EPA Antimicrobial s Division website until such time that they can be added to the guidelines. **(c) Public health and non** 
**- public health uses of antimicrobial product** **s .**  **(1) Antimicrobial products with public health**  **claims .**  An antimicrobial pesticide is considered to make a public health claim if the pesticide product bears a claim to control one or more pest microorganisms that pose a threat to human health, and whose presence cannot readily b e observed by

the user, including but not limited to, microorganisms infectious to man in any area of the environment. Efficacy data are required to be submitted to support registration of antimicrobial products with public health uses. A product makes a public health claim if one or more of the following apply: (i) A claim is made for control of one or more specific microorganisms that are directly or indirectly

infectious or pathogenic to man (or both man and animals). Examples of specific microorganisms include, but are not limited to, Mycobacterium tuberculosis , Pseudomonas aeruginosa , Escherichia coli (

E. coli), human immunodeficiency virus (HIV), Streptococcus sp. and Staphylococcus aureus . Claims for c ontrol of microorganisms infectious or pathogenic only to animals (such as canine distemper virus or hog cholera virus) are not considered public health claims. (ii) A claim is made for the pesticide product as a sterilant, disinfectant, virucide, sanitizer, fungicide or tuberculocide against one or more microorganisms that are infectious or pathogenic to man . Surrogate*** Deliberative Internal Document

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7 microorganisms accepted by the Agency

may also be used in testing. (iii) A claim is made for the pesticide product as a microbiological water purifier or microbial purification system or swimming pool disinfectant/sanitizer. (iv) A non- specific claim is made that the pesticide product will beneficially impact or affect public health at the site of use on/or in the environments in which it is applied. **(2) Antimicrobial**  **products with**  **non -** **public health**  **uses.** Applicants who propose non-health related claims for their product (e.g., control of odor

- causing bacteria) should be aware that generally the Agency does not require submission of efficacy data to support such claims. However, the applicant is still responsible for ensuring that these products perform as intended by developing efficacy

data and keeping the data on file

. The Agency still has the responsibility o f making sure that the use directions proposed for non-public health related claims are appropriate and adequate. Therefore, the Agency retains the option of requiring the submission of efficacy data for non-public health related claims on a case -by

case basis. (i) **Non -** **public health**  **claim.** An antimicrobial pesticide is considered to make a non-public health claim if the pesticide product bears a claim to control microorganisms of economic or aesthetic significance, where the presence of the microorganism would not normally l ead to infection or disease in humans. Examples of non-public health claims would include, but are not limited to: (A) Slimicides and odor control agents : Slimicides and odor control agents, preservatives, algicides, and other products expressly claiming control of microorganisms of economic or aesthetic significance are not considered to be human health related, but nevertheless must bear accurate labeling claims , adequate dosage recommendations, and complete directions for use. (B) Bacteriostatic and Fungistatic products : Since elimination or significant reduction in the number of microorganisms (e.g., sterilization, disinfection, sanitization) should be demonstrated be fore a product is considered acceptable for use against microorganisms infectious to humans or for use in medical environments, products bearing lower level labeling claims for effectiveness at the bacteriostatic/fungistatic (inhibition of growth) level would generally not be appropriate for such uses. Bacteriostatic/fungistatic claims are generally acceptable only for*** Deliberative Internal Document

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8 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

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

- 1

. The treated articles exemption (40 CFR Part 152.25(a)) covers qualifyingarticles and substances bearing claims to merely protect the article or substance itself, if , among other things, the treating pesticide is registered for such use . This exemption does not include articl es 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 c onsidered to be human health related, the Agency requires, on a case -by

- case basis, the submission of efficacy data to support these claims because these pathogens have animal health significance and/or the potential to infect humans. Applicants should consult the Agency for a current listing of organisms which meet these criteria. These organisms are also listed on the World Organization for Animal Health’s website, s ee 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 guideline s cover only

public health uses, terms covering non-public health use patterns and/or organisms are included in order to support cons istency and clarity . The terms in the OCSPP Test Guideline Series 810 .2000 , are generally used with the meanings set forth in this paragraph

. Algicide means any substance, or mixture of substances, which kills or effectively*** Deliberative Internal Document

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9 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 destro ysor 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 living microorganisms (e.g., virucide

- -virus, mycobactericide

- -mycobacteria, algicide

- - algae; bactericide

- -bacteria; fungicide

- - fungi; slimicide

- - slime

- forming microorganisms). Biofilm means a dy

namic, self

- organized accumulation of a microorganism or microorganisms and environmental by

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

. This organic poly mer mix is also known by the term “glyc ocalyx .” Confirmatory data is a reduced set of data which may b e used to support an application or amendment for registration of a product, for an additional microbe, or a minor formulation change of a registered product. Each guideline further addresses confirmatory data Deodorizers means a substance, or mixture of substances , that are of two basic 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 irreversibly inactivates bacteria, fungi and viruses, but not necessarily b acterial spores, in the inanimate environment. Fungicide means a substance, or mixture of substances, that destro ysf ungi (including ye asts) and fungal spores pathogenic to man or other animals in the inanimate environment. Fungistat means a substance, or mixture of substances , that inhibits the growth of fungi in the inanimate environment.*** Deliberative Internal Document

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10 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 b e used without a pre

- cleaning step in the use directions. Preservative means a substance, or mixture of substances that inhibits the growth of microorganisms capabl e of causing biological deterioration of a material(s). Prion means proteinaceous infectious particle. Production batch refers to the antimicrobial test chemical generated using standard manufacturing processes and considered representative of the final product. Product performance refers to all pesticidal aspects of a product’s effectiveness and usefulness. Sanitizer means a substance, or mixture of substances , that reduces the bacterial population in the inanimate environment by s ignificant 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. Slimicide means a substance, or mixture of substances, that reduces the number of slime

forming microorganisms in industrial water s ystems (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 destro ysor irreversibly inactivates tubercle bacilli in the inanimate environment.*** Deliberative Internal Document

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11 Two

-Step Sanitizer or Two

-Step Disinfectant means a substance or mixture of substances that has not been registered for effectiveness against microorganisms in the presence of light to moderate soil , and, therefore , needs a pre - cleaning step for surfaces prior to sanitizing or disinfecting. Virucide means a substance, or mixture of substances , that destro ysor irreversibly inactivates viruses in the inanimate environment. Volatile means a substance that evaporate s readily. Zoonotic microorganism means an infectious agent that can be transmitted between animals and humans. **(e) General testing**  **consideration s**  **. (1)**  **T est substance.**  (i) Antimicrobial pesticides should be tested on the formulation with active ingredient(s) concentrations that comply with Table 2 below. In some cases (e.g., pressurized sprays, towelettes) products should also be tested in the same packaging in which they are intended to be marketed. For all efficacy testing the following information should be provided: (A) Identification of the test substance and quantitative description of its chemical composition. (B) Manufacturer and production batch numbers of the test substance

. If a product is diluted, the report should specify the quantities and identification of each diluent. (C) D ata to show that the manufacturer can consistently reproduce the formulation (batch replication), as well as to show that the product will retain its effectiveness for a minimal period of storage under average conditions to which it is likely to be exposed (shelf

- life stability). (D) In situations where it may not be technically f easible to test within the Lower C ertified Limits (LCL) range specified in this section, a detailed justification should be provided to the Agency f or review prior to testing

. Efforts should be made to test as close to the LC L of each individual active ingredient as possible. (E) E fficacy testing at the Lowest Certified Limit (LCL) is necessary to demonstrate an antimicrobial product’s ability to consistently*** Deliberative Internal Document

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12 perform as labeled. However, it is the Agency ’s view that not all efficacy data submissions necessitate this testing approach . Table 2 specifies the ty

pes or classes of efficacy data which should employ testing at the LC L for new and amended registrations , as well as circumstances where testing at or below the nominal concentration is considered appropriate

. As described below, testing at the lower certified limit(s) should be performed for the required microbes (per the 810 series guidelines) needed to support a specific efficacy class. LC L testing of additional microbes should also be done for the tuberculocidal, sterilant/sporicidal, and food

- contact sanitizer efficacy c lasses. LC L testing for fungicidal disinfection will be addressed under a separate guidance document

.*** Deliberative Internal Document

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

**Efficacy Class (see appropriate 810 guideline for**  **test microbe)**  **Testing Type**  **New Registration**  **Submission Am** **endments**

**to Registered Product**  **Disinfection (Hospital, Broad or Limited**
**- Spectrum):**  Required Microbes: Test at LC L Test at LC L Additional Microbes Test at or below nominal Test at or

below nominal **Tuberculocidal Disinfection:** Required Microbe and Additional Microbes Test at LC L Test at LC L **Fungicidal Disinfection:** Required Microbe Reserved Reserved **Virucidal Disinfection:** Hardest to Kill strain on label [See revised viral hierarchy section in Agency

Guidance 810.2200 ] Test at LC L Test at LC L Additional Viruses: Easier to Kill strains on label [See Agency Guidance ] Test at or below nominal Test at or

below nominal
**Non -** **Food Contact Sanitizer:** Required Microbes Test at LC L Test at LC L Additional Microbes: (e.g. MRSA, E. coli) Test at or below nominal Te st at or

below nominal
**Food Contact Sanitizer:** Required Microbes: and Additional Microbes Test at LC L Test at LC L **Sterilant/General Sporicidal:** Required Microbes and Additional Microbes Test at LC L Test at LC L
**Clostridium d** 

**ifficile**
**- s** **poricidal:** Required Microbe Test at LC L Test at LC L

Note that the guidance concerning LC L testing applies to all batches tested in a study. Consultation with the Agency prior to the initiation of testing is recommended for efficacy data ty pes that do not fall into the efficacy classes identified in Table 2

. This listing may

be revised to address LC L testing for other organisms as the Agency ’s*** Deliberative Internal Document

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14 concerns change regarding emerging pathogens. Inaddition, the Agency is aware of the potential difficulties associated with generating test samples exactly at the lower certified limit. To address this issue, the Agency has specified an active ingredient concentration range above the lower certified limit which may be used for efficacy

testing. Individual active ingredient concentrations within this range (above the LCL) are considered representative of the actual lower certified limit for efficacy testing purposes. The test concentration range above the LCL is determined as follows:

 For products with a nominal concentration less than or equal to 1.0%, the tested value for that active ingredient may b e up to 2.0% above the lower certified limit stated on the CSF.

 For products with a nominal concentration above 1.0% and less than or equal to 20.0%, the tested value for that active ingredient may b e up to 1.0% above the lower certified limit stated on the CSF.

 For products with a nominal concentration above 20.0% and less than or equal to 100.0%, the tested value for that active ingredient may b e up to 0.6% above the lower certified limit stated on the CSF. Using this approach, a product that has an active ingredient with a nominal concentration of 7. 0 0% and a lower certified limit of 6.65% (based on 40 CFR 158.3 5 0), would have a testing range of 6.65% to 6.71%. Inthis example the nominal concentration is greater than 1.0% and less than 20%, therefore the a ppropriate testing range would be up to 1.0% above the LCLo f 6.65% (or 6.65% to 6.71%). Products with multiple active ingredients should use this approach to determine the usable testing range for each active in the product. Registrants/applicants are expected to develop formulated product samples for efficacy testing within this range. In cases where efforts to formulate product within this range have failed, product samples may b e diluted from a higher active ingredient concentration to achieve the target range. If product dilution is performed, a diluent that is not expected to increase product efficacy should be employed. Water is the preferred diluent in these situations. Products likely to be reactive or less stable in the presence of water may be diluted with a primary s olvent already

present in the product formulation. The use of emulsifiers or surfactants as diluents should be avoided even if they are already present in the subject formulation, as these may alter product efficacy

. Registrants/applicants should consult with the Agency prior to testing if an appropriate diluent cannot be found. Efficacy studies developed using test samples diluted with material considered likely to increase sample efficacy may b e rejected by

the Agency

. When dilution or any

other alteration is performed to achieve the acceptable LC L range,*** Deliberative Internal Document

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15 the efficacy

report should specify

exactly how the product (test material) was modified prior to testing. **(2) Test**  **m ethods.**  (i) **Test method**  **selection .**  The method used for efficacy testing should be relevant to the pest(s), the use sites and the label claims, as well as the application process and product formulation ty pe . Since the identification of appropriate methods for all public health uses is not feasible, the applicant is re sponsible for the applicability of the test method selected to substantiate product efficacy

. The applicant should also ensure that selected methods are current and valid for the purpose of pesticide registration. (ii) **Use conditions**  **.** Efficacy testing should address factors or conditions that would normally

b e encountered with the use pattern(s) intended for the product

. This could include the nature of the surface to be treated, the presence or absence of organic burden ( soil) or other interfering materials , water hardness , and the number of times or duration of time that the product could be used. Representative surfaces for testing are identified in each 810 Guideline. (iii) **New or**  **modified methods.**  If there is no existing standard method, registrants may, in consultation with the Agency, develop and submit protocols for claims where no standard test methods have been developed. If the standard method must be modified, the alternate protocols for demonstrating the efficacy o f a product should be developed and submitted to the Agency for review prior to testing

. All new or modified protocols should be submitted to the Agency for review and approval prior to data generation and collection. An assessment of method variability (e.g. within and between lab standard deviation) is critical to the Agency ’s approval process. Thus, an assessment of variability and a means to mitigate variability

should be contained in the submission. **(3) Test**  **organisms.** Test microorganisms are specified in the guidelines for basic effic acy c laims. The detailed final report should include information and descriptions regarding: preparation of the inoculum; application of the inoculum to the carrier; the time/temperature and relative humidity conditions for dryingthe 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 em ployed. (i) **Surrogate microorganisms.**  Agency

- approved surrogate microbes may be employed when applicable. A surrogate is considered representative of*** Deliberative Internal Document

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

to humans

. Use of surrogates may b e necessary due to bios afety concerns , ease of culturing, and availability

. Applicants should consult with the Agency for guidance on additional surrogates. Examples of surrogate organisms are as follows. (A) For tuberculocidal claims, Mycobacterium bovis (BCG) has been adopted as a surrogate for human Mycobacterium tuberculosis . See Guideline 810.2200. (B) The duck hepatitis B virus (DHBV) has been adopted as a surrogate for the chimpanzee test used in testing efficacy o f disinfectants against human hepatitis B virus. The data consistency control (method validation) stated in the referenced protocol utilizing two dilutions of BTC 835 has been eliminated. (C) The bovine viral diarrhea virus (BVDV) has been adopted as a surrogate for the hepatitis C virus. The data consistency c ontrol (method validation) stated in the referenced protocol utilizing two dilutions of BARDAC 2280 has been eliminated. (D) The feline calicivirus has been adopted as a surrogate for the Noroviruses. The data consistency c ontrol (method validation) stated in the referenced protocol utilizing two dilutions of bleach has been eliminated. (ii) **Use of**  **Antibiotic Resistant**  **Test Organisms.**  Organisms to be labeled as antibiotic resistant should be accompanied by s cientific data that substantiates the antibiotic resistance

. Demonstration of antibiotic resistance should be conducted and documented using the organism(s) listed on the label, and, if possible, should be performed at the same time as the efficacy

testing. The confirmation may also be conducted within the usual transfer cycle or other appropriate transfer depending upon organism’s growth requirements. The following information should be submitted with the Antibiotic Resistance Confirmation testing or included in the final rep ort. (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).*** Deliberative Internal Document

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17 (D) The method of preservation/storage (e.g. refrigerated agar slants, cryo genic beads, other). (E) Results of the testing including the numerical values of all antibiotics tested. An example of relevant values would be Minimum Inhibitory

C oncentrations (MIC) for automated tests , zone sizes for manual tests, and comparison to a standard Clinical and National Standards Institute interpretation of such tests. (F) The scientific method used to obtain the results (Kirby

- Bauer, disc agar diffusion, or gradient agar diffusion; automated MIC procedures or equivalent). If automated procedures are used, the manufacturer of such automation should be specified. (G) Quality

control procedures used to verify results. (H) GLP statement. (iii) **Additional microorganisms.**  Label claims for additional microbes other than the designated test microorganism(s) are permitted, provided that the target microbe is likely

to be present in or on the recommended use areas and surfaces. (iv) **Control carrier**  **counts .**  (A) **For carrier** 
**- based**  **assays** , quantitative determinations of the microbial counts on the untreated control carriers after drying should be conducted in order to determine the validity o f the test results obtained with the treated carriers as specified in the method. These quantitative determinations should be performed for all carrier

-based assays, whether or not modifications are made to the method being used. The test results should include the individual dried carrier counts obtained by the method. (B) **For suspension**  **tests.** Quantitative determination of the microbial count of the inoculum in a parallel untreated diluent should be conducted in order to determine the level of microbial challenge in the test (Numbers Control). These quantitative determinations should be performed for all suspension assays, whether or not modifications are made to the method being used. **(4) Porous**  **test materials**  **.** When an antimicrobial product is intended to be effective in treating a specific hard, porous surface, a method modification may b e necessary which specifies the use of the porous material (as a carrier) in the test*** Deliberative Internal Document

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18 protocol. In addition, control data (e.g. , quantitation of dried carrier, neutralization confirmation, sterility

c ontrols) should be developed to assure the validity o f the test results when this modification of the method is employed. **(5) Hard**  **water testing**  **.** Any product that bears a label claim for effectiveness using hard water as the product diluent should be tested by the appropriate efficacy method using sy

nthetic hard water (hardness expressed as ppm CaCO 3 ) at the level claimed. A minimum of hardness of 2 00 ppm should be used for a hard water claim. The hard water tolerance level may differ with the level of antimicrobial activity

c laimed (e.g., sterilization, disinfection, or sanitization). To support an efficacy claim in hard water, all microorganisms (bacteria, viruses, and fungi) claimed to be controlled by the product should be tested by the appropriate recommended method at the same level of hard water
. Refer to the most recent version of the AOAC Germicidal and Detergent Sanitizing Action of Disinfectants test (Official Method 960.09) for guidance on the preparation of sy nthetic hard water. The use of deionized water as a diluent for concentrates is not recommended unless the label specifies deionized water. If a product requires dilution but does not bear a claim of effectiveness in hard water, the product should be tested in sterile tap water. The ppm hard water level of the tap water should be measured and reported with the submitted study

. The registrant should be aware that tap water cannot be standardized and subsequent testing (e.g. post registration testing) may use a source of tap water exhibiting a different level of hardness. **(6) Organic burden.**  (i) The effectiveness of antimicrobial agents should be demonstrated in the presence of a specific organic soil at an appropriate concentration level when specifically claimed and/or indicated b y the product’s pattern of use. A suggested procedure to simulate in

-use conditions where the antimicrobial agent is intended to treat dry inanimate surfaces contaminated with an organic soil load involves contamination of the appropriate carrier surface with each test microorganisms culture containing 5% v/v blood serum (e.g., 19 mLtest microorganism culture + 1 mLb lood serum) prior to the carrier inoculation and specified carrier

drying step in the method. Control data (e.g., quantitation of dried carrier counts, neutralization confirmation, sterility c ontrols) should also be developed to assure the validity o f the test results when an organic burden is used. The 5% organic soil level suggested is considered appropriate for simulating lightly

or moderately soiled surface conditions. When the surface to be treated has heavy so il deposits, a cleaning step should be included on the label prior to the application of the antimicrobial agent. (ii) An antimicrobial substance identified as a one - step cleaner

- disinfectant or*** Deliberative Internal Document

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19 cleaner

- sanitizer, or intended to be effective in the presence of light to moderate amounts of organic burden, should be tested for efficacy with a minimum of a 5% organic soil added to the inoculum. Registrants should check with the Agency

to determine the acceptability of an organic burden other than blood serum. For products with a pre - cleaning step prior to application, the addition of an organic burden to the inoculum may not be necessary. (iii) A suggested procedure for incorporating a light to moderate organic soil load where the antimicrobial agent is not tested against a dry inanimate surface, such as the AOAC International Fungicidal Activity o f Disinfectants test and the Quantitative Tuberculocidal Test, involves adding a minimum of 5% (v/v) blood serum directly to the test organism (e.g., 4.75 mLtest organism + 0.25 mLb lood serum) before inoculating the test solution. (iv) When a product is recommended for certain patterns of use where the organic soil claimed is of a specific 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 report regarding the way

in which the organic soil, such as soap film residue was prep ared (e.g., percentages of ingredients) and employed in the testing

. **(7) Contact time**  **( ex** **posure period )**  **.** The contact time used in efficacy testing should be the same or shorter than the contact time identified on the product label. If a contact time different from the range identified in the test method or guideline is preferred, consultation with the Agency prior to testing is recommended and a modification to the standard approach may be needed. In most cases a modification to provide a longer exposure period is limited by the practical considerati **o**ns of the use patterns (e.g., an exposure period of >10 min for a product that will likely evaporate from the treated surface within 10 min). All method modifications need to be clearly identified and justified in the test protocol. For liquid or spray products containing volatile active ingredients where the product is applied to a hard non- porous surface, the minimum contact time may b e determined by visually inspecting evaporation over the proposed contact period

. As identified in the AOAC Internati onal Germicidal Spray Products as Disinfectants test method, this approach should involve product application (using the proposed application method) to glass slide carriers

. Use of methods that immerse contaminated carriers in the disinfectant fluid do not accurately simulate the way in which volatile disinfectants perform on environmental surfaces. **(8) Neutralization.**  Neutralization is the process of inactivating a test material’s antimicrobial activity at the end of the contact time. This may b e achieved*** Deliberative Internal Document

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20 through physical (e.g. , filtration, dilution, secondary subculture) and/or chemical (e.g., addition of sodium thiosulfate to the diluent) means. For each efficacy test, neutralization should be employed at the completion of the contact time in order to preclude residual effects of the active ingredient( s) in the subculture medium. If neutralization is not properly performed, the results of efficacy testing may not be deemed valid. The method of neutralization, whether physical or chemical, should be confirmed in the laboratory in advance or concurrently with testing

. Neutralization confirmation data should be submitted in the final report to demonstrate that the neutralization process sufficiently inactivated the active ingredient(s) and that the neutralizer medi a used did not possess antimicrobial activity

. All subculture techniques employed to preclude residual carryover of active ingredient should be described in detail in the study report
. To demonstrate the absence of residual effects of the active ingredient in the subculture medi a, the examples of neutralization techniques in the paragraphs below should be followed

. (i) In qualitative test methods, after treatment the inoculated carrier is deposited in a tube of growth media (i.e., primary subculture). If the primary subculture is not sufficient to achieve neutralization by the use of a chemical agent and/or through dilution of test substance, the carrier may then be transferred to a second tube of growth media (or secondary subculture) to achieve neutralization. The neutralization procedure employed should be confirmed and reported by r unning a parallel test with uninoculated carriers being added to growth media containing a low level (i.e., 10

– 100 CFU/mL) of bacteria. Both the primary cultures and secondary subcultures should be incubated and checked for growth in the test and the neutralization confirmation. Dried inoculated test carriers should not be used to test the ability o f a subculture medium to support organism growth, as this would provide too large a bioburden and may lead to an inaccurate evaluation of the presence of bacteriostasis from the carry- over of the antimicrobial substance on the carrier to the subculture medium. Growth results for b oth 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 b e conducted by

neutralizing the test substance, without the organism, as in the test. After neutralization, inoculation of a low level of organism ( 10

- 100 CFU/mL) and subsequent plating should follow

. Plate counts should be within 50% of a parallel population control or as specified in the relevant method

. (A) For virucidal tests, scientifically accepted controls including proper*** Deliberative Internal Document

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21 neutralization controls should be performed (e.g., ASTM E1482). **(9) Batch**  **replication for**  **modified test** **ing .**  For certain use modifications to a registered product, the Agency allows a reduced number of product batches (lots) to be tested in support of these modifications. Inorder to be eligible to use this approach however, the registered product should have acceptable efficacy data supporting the product that were performed without modification and included the number of batches specified in the relevant method /guideline. Under these conditions, additional data performed at the same use concentration previously tested may

b e conducted with reduced batch replications to support a single modified use condition such as organic soil load, hard water concentration or use of porous surface carriers. The batch number reductions are as follows: (i) For basic efficacy c laims (e.g., sterilants, disinfectants, sanitizers) , two samples, representing two different batches may b e tested, instead of three. (ii) For supplemental efficacy c laims (e.g., fungicides and virucides) , one sample instead of two may be tested

. **(10) Performance**  **Standards**

. Performance standards are a set of test criteria used to establish product effectiveness, such as the number of positive carriers allowed for a passing product or a minimum log reduction of bacteria following treatment. The testing criteria may

b e different for each standard method and test microbe, thus the applicant should consult the appropriate guideline for this information. **(11) Repeat**  **Testing Policy** 

– Refer to the methods cited in “Disinfectants for Use on Hard Surfaces

– Efficacy D ata Recommendations ” OCSPP 810.2200, to determine allowable levels of contamination for a particular test method. If the product p erformance test data is deemed invalid due to an unacceptable l evel of c ontamination, the following re

-testing scenarios apply : (i) When an unacceptable level of contamination is present, retesting may b e conducted using the same test conditions if the efficacy test meets the required performance standard. Retesting of the product lot(s) associated with the invalid test(s) should be conducted. (ii) When an unacceptable level of contamination is present and the product does not meet the required performance standard, the product is considered a failure. **(f) Data collection and r** **eporting. (1)**  **General.** To assist in the proper review and evaluation of product performance,*** Deliberative Internal Document

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22 complete descriptions of the test employed and the results obtained should be submitted to the Agency. All test reports should include, at the least, the following information: (i) Study

title; (ii) Complete product i dentity (Certificate of Analys is) ; (iii) Guideline number/Data Requirement; (iv) Identification of the testing laboratory or organization; (v) Location where the test was performed; (vi) Name(s) of the person(s) responsible for the test; (vii) Statement of Confidentiality Claims; (viii) Statement of 40 CFR Part 160 Good Laboratory Practice compliance and Quality Assurance Statement; all protocol deviations should be identified. (ix) Purpose of the study; (x) Date and time of the start and end of the test; (xi) Name of the test method and appropriate references ; (xii) Test emplo yed (include the version or edition) and any modifications (e.g., organic soil, hard water, etc.), when using standard tests (e.g., AOAC, ASTM, etc.) all modifications to the test methods should be reported and justified ; (xiii) Test microorganisms employed, including identification of the specific strain (ATCC or other); (xiv) Description of the test substance, including the percent of active ingredient; (xv) Concentration or dilution of the product tested and how prepared; (xvi) Number of samples, batches and replicates tested; (xvii) Manufacture date of each product batch*** Deliberative Internal Document

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23 (xviii) Identification of all material or procedural options employed, where such choice is provided for or recommended in the test method selected (e.g., growth media, drying time for inoculated carriers, neutralization confirmation and/or subculture media, secondary subculturing); (xix) Test exposure conditions (e.g., contact time, temperature, and relative humidity ); (xx) Complete reports of results obtained for each replication; (xxi) Any

control data, including neutralization confirmation results, essential to establish the validity

of the test. (xxii) Control c arrier counts (i.e., microbial load) ; (xxiii) Formula used to calculate control counts and log reduction values (if appli cable) as well as any statistical treatment of the data; (xxiv) Conclusions; (xxv) Any

pictures or figures explaining the outcome or observations ; (xxvi) References; **(2)** Appendices, including study protocol and all raw data reports associated with the conduct of the study

. The applicant is encouraged to use the EPA’s standard efficacy report format, which may be found at http://www.epa.gov/oppad001/efficacystudystandards.htm

. **(g) References.**  The references in this paragraph may be consulted for additional background information : **(1)** US EPA; Regulating Antimicrobial Pesticides. See http://www.epa.gov/oppad001

. **(2)** Official Methods of Analysis of AOAC International

. Chapter 6, Disinfectants, Current edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

-2417. **(3)** Annual Book of ASTM Standards , Standard Test Methods . American Society f or Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition.