Document ID: EPA-HQ-OPPT-2009-0150-0027
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2015-06-10T04:00Z

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

	

                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       

                                       
                                        
                                        
                                     NOTICE
                                        
  	This guideline is one of a series of test guidelines established by the Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a), referred to hereinafter as the harmonized test guidelines. Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides, and Toxic Substances (OPPTS).  To distinguish these guidelines from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS as part of the guideline's number. Any test guideline developed after April 22, 2010 will use the new acronym (OCSPP) in their title.  
  
  	The OCSPP test guidelines serve as a compendium of accepted standardized methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the tests, and are also used by EPA, the public, and the companies that are subject to data submission requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the EPA may depart from these guidelines where circumstances warrant and without prior notice. At places in this guidance, the Agency uses the word "should."  In this guidance, use of "should" with regard to an action means that the action is recommended rather than mandatory. The procedures contained in this guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. You may propose alternatives to the recommendations described in these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis.  
  
  	For additional information about these test guidelines and to access these guidelines electronically, please go to http://www.epa.gov/ocspp and select the topic entitled, "Test Methods and Guidelines." You may also access the guidelines in http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
  
  DRAFT DOCUMENT DISCLAIMER:  This draft document is distributed solely for the purpose of external review. It has not been formally disseminated by the EPA and should not be construed to represent any Agency determination or policy. The information correction process under the Agency's Information Quality Guidelines does not apply until the document is formally disseminated by the EPA in its final form. This draft document should only be cited or quoted in the context of providing comments.  

OCSPP 810.2000: General considerations for testing antimicrobial agents.

(a) Scope.

      (1) Applicability. OCSPP Test Guideline Series 810.2100 through 810.2700 describes general information regarding product performance testing of antimicrobial pesticides to meet requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)(7U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).
            
      (2) Background. Microbial pests can be categorized into two basic types: 1) those that present potential public health hazards because of their infectious nature to humans (public health claims) and 2) those that cause economic or aesthetic problems such as spoilage, fouling, or production of offensive odors in the substrate in which they grow (non-public health claims). The OCSPP Test Guideline Series 810.2000 addresses antimicrobial pesticide products with public health uses for which efficacy test data are required to be submitted to the Agency to support registration. The source material used in developing the OCSPP Test Guideline is OPP guideline 91-1: General Require - ments 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 of product use, compatibility of common practices associated with the sites, and ingredient status of chemicals in the formulation.
                  
            (i) Laboratory and/or simulated-use testing is conducted to determine the effectiveness of a substance, or mixture of substances, to control or kill specific pest organisms, and in some cases to determine whether the substance has sufficient pesticide potential to warrant larger scale testing (e.g., swimming pool disinfectants).
                  
            (ii) In some cases, effectiveness and usefulness of the proposed product is further determined through advanced large-scale laboratory tests, field tests, in-use tests, or simulated-use tests by procedures which closely approximate actual use and which employ typically used application equipment (e.g. fumigant sterilants).
         
      (2) Series organization. Table 1 outlines the organization of the OCSPP Test Guideline Series 810.2000.
      
      Table 1.  Organization of the OCSPP Test Guideline Series 810.2000.
                                Guideline Name
                               Guideline Number
                 Previous Subdivision  - G Guideline Number(s)
Sterilants  -  Efficacy Data Recommendations
810.2100 
91-2(a)

Disinfectants for Use on Hard Surfaces  -  Efficacy Data Recommendations
810.2200 
91-2(b)(c)(d)(e)(f)(g)(i)
91-7(a)(1)
91-3
Sanitizers for Use on Hard Surfaces  -  Efficacy Data Recommendations
810.2300 
91-2(j)(k)(l)(m)
91-3
Disinfectants and Sanitizers for Use on Textiles  -  Efficacy Data Recommendations
810.2400
91-4(a)(1)(2)(3)(4)
91-4(b)(c)(d)
Air Sanitizers  -  Efficacy Data Recommendations 
810.2500
91-5
Disinfectants for Use in Water  -  Efficacy Data Recommendations
810.2600
91-8
Prions  -  Efficacy Data Recommendations
810.2700
Not Applicable
      
      (3) Good Laboratory Practice Standards. Good Laboratory Practice Standards (GLP) as defined in 40 CFR Part 160 apply to studies to support the registration of all antimicrobial products.  According to 40 CFR §160.17: "EPA may refuse to consider reliable for purposes of supporting an application for a research or marketing permit any data from a study which was not conducted in accordance with this part." 40 CFR §160.12 (b) requires with any submitted research data "[a] A statement that the study was conducted in accordance with this part; [b] A statement describing in detail all differences between the practices used in the study and those required by this part; or [c] A statement that the person was not a sponsor of the study, did not conduct the study, and does not know whether the study was conducted in accordance with this part."
            
      (4) Data for modifications of recommended methods. When recommended methods are modified by the applicant to support specific claims and/or use patterns for a product, the applicant should submit the complete testing protocol, identifying and describing each modification to the Agency for review and evaluation prior to initiation of the test. The study report should identify and describe each modification.
            
      (5) Data for other methods. When recommended methods, or modifications thereto, are not employed to develop efficacy data, complete testing protocols should be submitted to the Agency for review and evaluation prior to initiation of the test. All pertinent information, including protocols, should be included with the final test reports. All materials and procedures employed in testing should be fully described. Where references to published reports or papers are made, copies or reprints of such references should be provided with the test reports.
            
      (6) Confirmatory data. In certain situations, an applicant may rely on previously submitted product efficacy data to support a new application or amendment for registration of a product and submit only confirmatory efficacy data on the subject product to verify the product's antimicrobial efficacy. These situations are as listed below:

            (i) Duplicated Product Formulations. In this situation, the applicant manufactures a formulation which duplicates a product that is already registered and has complete supporting efficacy data. The chemical composition, manufacturing procedure, label claims, and directions for use are identical in substance to those of the original registration, and specific references (including Master Record ID [MRID] Numbers) to the supporting data developed for the duplicated product are cited by the applicant.

            (ii) Minor Formulation Change in a Registered Product. In this situation, the change in the formulation is relatively minor. The label claims and directions for use are unchanged from those accepted for the registered formulation, and specific references (including MRID numbers) to the supporting data developed for the original formulation are cited by the applicant. If the only change in the formulation is the addition of a fragrance or dye, confirmatory data do not need to be submitted. However, when the product is an aerosol formulation, confirmatory data should be submitted for all formulation changes, including the addition of fragrances and dyes.
      
            (iii) The confirmatory data are to be developed from testing the applicant's own finished product. If the test methodology utilized in the supporting (cited) efficacy data was modified to include additional elements not specified in the recommended method (e.g., organic soil, hard water, longer or shorter contact time), the confirmatory data should be produced under similarly modified conditions.
                  
      (7) Verification of efficacy. The Agency reserves the option to perform its own testing, on a case-by-case basis, following the product specific test conditions used to register or amend the registration of the product (contact time, hard water etc.). The current version of the standard test method will be used in the evaluation of efficacy.  
      
      (8) Test failure. Failure of a product formulation to meet the specified testing or evaluation of success (i.e. the performance standard), constitutes evidence that the product is unlikely to be effective as claimed in actual use and is reportable under FIFRA section 6(a)(2).

      (9) Guideline revisions. The Agency recognizes that novel technologies associated with antimicrobial products may evolve over time and would potentially involve test methods that are not referenced in the guideline series. In addition, the Agency is considering adopting the use of quantitative test methods as an alternative or replacement to several current qualitative methods [e.g., Association of Official Analytical Chemists (AOAC International) Use-Dilution Methods]. The Agency expects to update the guidelines periodically to address such changes; however, the use of new methods may occur prior to guideline updates. In these instances, new methods will be published on the EPA Antimicrobials Division website until such time that they can be added to the guidelines. 
      
(c) Public health and non-public health uses of antimicrobial products.
      
      (1) Antimicrobial products with public health claims. An antimicrobial pesticide is considered to make a public health claim if the pesticide product bears a claim to control one or more pest microorganisms that pose a threat to human health, and whose presence cannot readily be observed by the user, including but not limited to, microorganisms infectious to man in any area of the environment. Efficacy data are required to be submitted to support registration of antimicrobial products with public health uses. A product makes a public health claim if one or more of the following apply:
      
            (i) A claim is made for control of one or more specific microorganisms that are directly or indirectly infectious or pathogenic to man (or both man and animals). Examples of specific microorganisms include, but are not limited to, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Escherichia coli (E. coli), human immunodeficiency virus (HIV), Streptococcus sp. and Staphylococcus aureus. Claims for control of microorganisms infectious or pathogenic only to animals (such as canine distemper virus or hog cholera virus) are not considered public health claims.
                  
            (ii) A claim is made for the pesticide product as a sterilant, disinfectant, virucide, sanitizer, fungicide or tuberculocide against one or more microorganisms that are infectious or pathogenic to man. Surrogate microorganisms accepted by the Agency may also be used in testing. 
                  
            (iii) A claim is made for the pesticide product as a microbiological water purifier or microbial purification system or swimming pool disinfectant/sanitizer.
                  
            (iv) A non-specific claim is made that the pesticide product will beneficially impact or affect public health at the site of use on/or in the environments in which it is applied.	
      
      (2) Antimicrobial products with non-public health uses. Applicants who propose non-health related claims for their product (e.g., control of odor-causing bacteria) should be aware that generally the Agency does not require submission of efficacy data to support such claims. However, the applicant is still responsible for ensuring that these products perform as intended by developing efficacy data and keeping the data on file. The Agency still has the responsibility of making sure that the use directions proposed for non-public health related claims are appropriate and adequate. Therefore, the Agency retains the option of requiring the submission of efficacy data for non-public health related claims on a case-by-case basis.
            
            (i) Non-public health claim. An antimicrobial pesticide is considered to make a non-public health claim if the pesticide product bears a claim to control microorganisms of economic or aesthetic significance, where the presence of the microorganism would not normally lead to infection or disease in humans. Examples of non-public health claims would include, but are not limited to:
                  
                  (A) Slimicides and odor control agents: Slimicides and odor control agents, preservatives, algicides, and other products expressly claiming control of microorganisms of economic or aesthetic significance are not considered to be human health related, but nevertheless must bear accurate labeling claims, adequate dosage recommendations, and complete directions for use.
                        
                  (B) Bacteriostatic and Fungistatic products: Since elimination or significant reduction in the number of microorganisms (e.g., sterilization, disinfection, sanitization) should be demonstrated before a product is considered acceptable for use against microorganisms infectious to humans or for use in medical environments, products bearing lower level labeling claims for effectiveness at the bacteriostatic/fungistatic (inhibition of growth) level would generally not be appropriate for such uses. Bacteriostatic/fungistatic claims are generally acceptable only for products expressly recommended for control of microorganisms of aesthetic significance (e.g., spoilage bacteria, odor-causing bacteria).

                  (C) Treated articles: The Agency has clarified its policy on applicability of the treated articles exemption to antimicrobial pesticides and provided guidance on appropriate language or label claims in Pesticide Registration Notice 2000-1. The treated articles exemption (40 CFR Part 152.25(a)) covers qualifying articles and substances bearing claims to merely protect the article or substance itself, if, among other things, the treating pesticide is registered for such use. This exemption does not include articles or substances bearing implied or explicit public health claims against human pathogens. Applicants who intend to market products with claims (such as public health claims) that go beyond the scope of the treated articles exemption should contact the Antimicrobials Division prior to conducting testing to support such a use.

                  (D) Animal disease pathogens and zoonotic microorganisms: For products labeled for public health and/or non-public health uses, submission of studies to EPA on certain animal disease pathogens and zoonotic microorganisms may be appropriate prior to approval of the label claim. For example, although label claims against foot and mouth disease virus, Newcastle disease virus, and avian influenza A virus are not considered to be human health related, the Agency requires, on a case-by-case basis, the submission of efficacy data to support these claims because these pathogens have animal health significance and/or the potential to infect humans. Applicants should consult the Agency for a current listing of organisms which meet these criteria. These organisms are also listed on the World Organization for Animal Health's website, see http://www.oie.int/animal -health-in-the-world/oie-listed-diseases-2011/.
      
(d) Definitions. Because of the variety of microorganisms to be con - trolled 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 per - formance. Even though the OCSPP Test Guideline Series 810.2000 guidelines cover only public health uses, terms covering non-public health use patterns and/or organisms are included in order to support consistency and clarity. The terms in the OCSPP Test Guideline Series 810.2000, are generally used with the meanings set forth in this paragraph.
      
      Algicide means any substance, or mixture of substances, which kills or effectively reduces the number of living algae in water.
      
      Algistat means any substance, or mixture of substances, that inhibits the growth of algae in water.
      
      Antibacterial means any substance, or mixture of substances, that destroys or eliminates bacteria in the inanimate environment. 
      
      Antibiotic resistant means the ability of a bacterial cell to resist the effects of antibiotics.
      
      Antifoulant means any substance, or mixture of substances, that is used to prevent the biological fouling of underwater structures or objects.
      
	Bacteriostat means a substance, or mixture of substances, that inhibits the growth of bacteria in the inanimate environment.
      
      Biocide/Microbicide means any substance, or mixture of substances, that kills living microorganisms (e.g., virucide--virus, mycobactericide--mycobacteria, algicide--algae; bactericide--bacteria; fungicide--fungi; slimicide--slime-forming microorganisms).  
      
      Biofilm means a dynamic, self-organized accumulation of a microorganism or microorganisms and environmental by-products immobilized on a substrate and embedded in an organic polymer mix. This organic polymer mix is also known by the term "glycocalyx."  
      
      Confirmatory data is a reduced set of data which may be used to support an application or amendment for registration of a product, for an additional microbe, or a minor formulation change of a registered product. Each guideline further addresses confirmatory data
      
      Deodorizers means a substance, or mixture of substances, that are of two basic types: (1) Those that prevent or delay the formation of bacterial odors by killing 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 bacterial spores, in the inanimate environment.
      
      Fungicide means a substance, or mixture of substances, that destroys fungi (including yeasts) and fungal spores pathogenic to man or other animals in the inanimate environment. 
      
      Fungistat means a substance, or mixture of substances, that inhibits the growth of fungi in the inanimate environment.
      
      Microbiological water purifier means any unit, water treatment product or system that removes, kills or inactivates disease-causing microorganisms from the water, including bacteria, viruses and protozoan cysts, so as to render the treated water safe for drinking.
      
      Microbiostat means a substance, or mixture of substances, that inhibits the growth of microorganisms (e.g., bacteriostat, fungistat, algistat).
      
      Mycobactericide means a substance, or mixture of substances, that destroys or irreversibly inactivates mycobacteria in the inanimate environment.
      
      Non-volatile means a substance that does not evaporate readily.
      
      One-Step Disinfectant means a substance, or mixture of substances, that has been tested and found to be effective in the presence of light to moderate soil, and, therefore, may be  used without a pre-cleaning step in the use directions.
      
      Preservative means a substance, or mixture of substances that inhibits the growth of microorganisms capable of causing biological deterioration of a material(s).
      
      Prion means proteinaceous infectious particle.
       
      Production batch refers to the antimicrobial test chemical generated using standard manufacturing processes and considered representative of the final product. 
       
      Product performance refers to all pesticidal aspects of a product's effectiveness and usefulness.
      
      Sanitizer means a substance, or mixture of substances, that reduces the bacterial population in the inanimate environment by significant numbers, (e.g., 3 log10 reduction or more), but does not destroy or eliminate all bacteria. Sanitizers meeting Public Health Ordinances are used on food contact surfaces and are termed sanitizing rinses.
      
      Slimicide means a substance, or mixture of substances, that reduces the number of slime-forming microorganisms in industrial water systems (e.g. paper mills). 
      
      Sterilant means a substance, or mixture of substances, that destroys or eliminates all forms of microbial life in the inanimate environment, including all forms of vegetative bacteria, bacterial spores, fungi, fungal spores, and viruses.
      
      Sporicide means a substance, or mixture of substances, that irreversibly inactivates bacterial spores in the inanimate environment.
      
      Tuberculocide means a substance, or mixture of substances, that destroys or irreversibly inactivates tubercle bacilli in the inanimate environment.
      
      Two-Step Sanitizer or Two-Step Disinfectant means a substance or mixture of substances that has not been registered for effectiveness against microorganisms in the presence of light to moderate soil, and, therefore, needs a pre-cleaning step for surfaces prior to sanitizing or disinfecting. 
       
      Virucide means a substance, or mixture of substances, that destroys or irreversibly inactivates viruses in the inanimate environment.
      Volatile means a substance that evaporates readily.
      
      Zoonotic microorganism means an infectious agent that can be transmitted between animals and humans.
      
(e) General testing considerations.
      
      (1) Test substance.
            
            (i) Antimicrobial pes - ticides should be tested on the formulation with active ingredient(s) concentrations that comply with Table 2 below. In some cases (e.g., pressurized sprays, towelettes) products should also be tested in the same packaging in which they are intended to be marketed. For all efficacy testing the following information should be provided:
                  
                  (A) Identification of the test substance and quantitative description of its chemical composition.
                  
                  (B) Manufacturer and production batch numbers of the test substance. If a product is diluted, the report should specify the quantities and identification of each diluent.
                  
                  (C) Data to show that the manufacturer can consistently reproduce the formulation (batch replication), as well as to show that the product will retain its effectiveness for a minimal period of storage under aver - age conditions to which it is likely to be exposed (shelf-life stability).
                  
                  (D) In situations where it may not be technically feasible to test within the Lower Certified Limits (LCL) range specified in this section, a detailed justification should be provided to the Agency for review prior to testing. Efforts should be made to test as close to the LCL of each individual active ingredient as possible.

                  (E) Efficacy testing at the Lowest Certified Limit (LCL) is necessary to demonstrate an antimicrobial product's ability to consistently perform as labeled. However, it is the Agency's view that not all efficacy data submissions necessitate this testing approach. Table 2 specifies the types or classes of efficacy data which should employ testing at the LCL for new and amended registrations, as well as circumstances where testing at or below the nominal concentration is considered appropriate. As described below, testing at the lower certified limit(s) should be performed for the required microbes (per the 810 series guidelines) needed to support a specific efficacy class. LCL testing of additional microbes should also be done for the tuberculocidal, sterilant/sporicidal, and food-contact sanitizer efficacy classes. LCL testing for fungicidal disinfection will be addressed under a separate guidance document.

Table 2.  Lower Certified Limit (LCL) for Product Efficacy Testing
                                Efficacy Class 
               (see appropriate 810 guideline for test microbe) 
                                 Testing Type
                                       
                          New Registration Submission
                       Amendments to Registered Product

Disinfection (Hospital, Broad or Limited-Spectrum):
   Required Microbes: 
                                 Test at LCL 
                                 Test at LCL 
   Additional Microbes
                           Test at or below nominal
                           Test at or below nominal
Tuberculocidal Disinfection:
   Required  Microbe and Additional Microbes
                                 Test at LCL 
                                 Test at LCL 
Fungicidal Disinfection:
   Required Microbe
                                   Reserved
                                   Reserved
Virucidal Disinfection:
   Hardest to Kill strain on label [See revised viral hierarchy section in Agency Guidance 810.2200]
                                 Test at LCL 
                                 Test at LCL 
   Additional Viruses: Easier to Kill strains on label [See Agency Guidance]
                           Test at or below nominal
                           Test at or below nominal
                                       
Non-Food Contact Sanitizer:
   Required Microbes
                                 Test at LCL 
                                 Test at LCL 
   Additional Microbes: (e.g. MRSA, E. coli)
                           Test at or below nominal
                           Test at or below nominal
                                       
Food Contact Sanitizer:
    Required Microbes: and Additional Microbes
                                 Test at LCL 
                                 Test at LCL 
                                       
Sterilant/General Sporicidal:
    Required Microbes and Additional Microbes
                                 Test at LCL 
                                 Test at LCL 
                                       
Clostridium difficile -sporicidal:
   Required Microbe
                                 Test at LCL 
                                 Test at LCL 

      Note that the guidance concerning LCL testing applies to all batches tested in a study. Consultation with the Agency prior to the initiation of testing is recommended for efficacy data types that do not fall into the efficacy classes identified in Table 2. This listing may be revised to address LCL testing for other organisms as the Agency's concerns change regarding emerging pathogens. In addition, the Agency is aware of the potential difficulties associated with generating test samples exactly at the lower certified limit. To address this issue, the Agency has specified an active ingredient concentration range above the lower certified limit which may be used for efficacy testing. Individual active ingredient concentrations within this range (above the LCL) are considered representative of the actual lower certified limit for efficacy testing purposes. The test concentration range above the LCL is determined as follows:

                        *                         For products with a nominal concentration less than or equal to 1.0%, the tested value for that active ingredient may be up to 2.0% above the lower certified limit stated on the CSF.
                              
                        *                         For products with a nominal concentration above 1.0% and less than or equal to 20.0%, the tested value for that active ingredient may be up to 1.0% above the lower certified limit stated on the CSF.

                        *                         For products with a nominal concentration above 20.0% and less than or equal to 100.0%, the tested value for that active ingredient may be up to 0.6% above the lower certified limit stated on the CSF.
                        
      Using this approach, a product that has an active ingredient with a nominal concentration of 7.00% and a lower certified limit of 6.65% (based on 40 CFR 158.350), would have a testing range of 6.65% to 6.71%. In this example the nominal concentration is greater than 1.0% and less than 20%, therefore the appropriate testing range would be up to 1.0% above the LCL of 6.65% (or 6.65% to 6.71%). Products with multiple active ingredients should use this approach to determine the usable testing range for each active in the product.
      
      Registrants/applicants are expected to develop formulated product samples for efficacy testing within this range. In cases where efforts to formulate product within this range have failed, product samples may be diluted from a higher active ingredient concentration to achieve the target range. If product dilution is performed, a diluent that is not expected to increase product efficacy should be employed. Water is the preferred diluent in these situations. Products likely to be reactive or less stable in the presence of water may be diluted with a primary solvent already present in the product formulation. The use of emulsifiers or surfactants as diluents should be avoided even if they are already present in the subject formulation, as these may alter product efficacy. Registrants/applicants should consult with the Agency prior to testing if an appropriate diluent cannot be found. Efficacy studies developed using test samples diluted with material considered likely to increase sample efficacy may be rejected by the Agency. When dilution or any other alteration is performed to achieve the acceptable LCL range, the efficacy report should specify exactly how the product (test material) was modified prior to testing.  
      (2) Test methods. 
            
            (i) Test method selection. The method used for efficacy testing should be relevant to the pest(s), the use sites and the label claims, as well as the application process and product formulation type. Since the identification of appropriate methods for all public health uses is not feasible, the applicant is responsible for the applicability of the test method selected to substantiate product efficacy. The applicant should also ensure that selected methods are current and valid for the purpose of pesticide registration.
                  
            (ii) Use conditions. Efficacy testing should address factors or conditions that would normally be encountered with the use pattern(s) intended for the product. This could include the nature of the surface to be treated, the presence or absence of organic burden (soil) or other interfering materials, water hardness, and the number of times or duration of time that the product could be used. Representative surfaces for testing are identified in each 810 Guideline.

            (iii) New or modified methods. If there is no existing standard method, registrants may, in consultation with the Agency, develop and submit protocols for claims where no standard test methods have been developed. If the standard method must be modified, the alternate protocols for demonstrating the efficacy of a product should be developed and submitted to the Agency for review prior to testing. All new or modified protocols should be submitted to the Agency for review and approval prior to data generation and collection. An assessment of method variability (e.g. within and between lab standard deviation) is critical to the Agency's approval process. Thus, an assessment of variability and a means to mitigate variability should be contained in the submission. 

      (3) Test organisms. Test microorganisms are specified in the guidelines for basic efficacy claims. The detailed final report should include information and descriptions regarding: preparation of the inoculum; application of the inoculum to the carrier; the time/temperature and relative humidity conditions for drying the microorganisms on the carrier; the technique for removal of the microorganisms from the carrier; and the specific assay procedure indicating such details as replication, subculture media, diluents, and the incubation time/temperature conditions for the enumeration procedure employed.
            
            (i) Surrogate microorganisms. Agency-approved surrogate microbes may be employed when applicable. A surrogate is considered representative of a pathogenic strain and is usually a non-pathogen or exhibits limited pathogenicity to humans. Use of surrogates may be necessary due to biosafety concerns, ease of culturing, and availability. Applicants should consult with the Agency for guidance on additional surrogates. Examples of surrogate organisms are as follows.
                  
                  (A) For tuberculocidal claims, Mycobacterium bovis (BCG) has been adopted as a surrogate for human Mycobacterium tuberculosis. See Guideline 810.2200.
                        
                  (B) The duck hepatitis B virus (DHBV) has been adopted as a surrogate for the chimpanzee test used in testing efficacy of disinfectants against human hepatitis B virus. The data consistency control (method validation) stated in the referenced protocol utilizing two dilutions of BTC 835 has been eliminated. 
                        
                  (C) The bovine viral diarrhea virus (BVDV) has been adopted as a surrogate for the hepatitis C virus. The data consistency control (method validation) stated in the referenced protocol utilizing two dilutions of BARDAC 2280 has been eliminated.
                        
                  (D) The feline calicivirus has been adopted as a surrogate for the Noroviruses. The data consistency control (method validation) stated in the referenced protocol utilizing two dilutions of bleach has been eliminated.
                        
            (ii) Use of Antibiotic Resistant Test Organisms. Organisms to be labeled as antibiotic resistant should be accompanied by scientific data that substantiates the antibiotic resistance. Demonstration of antibiotic resistance should be conducted and documented using the organism(s) listed on the label, and, if possible, should be performed at the same time as the efficacy testing. The confirmation may also be conducted within the usual transfer cycle or other appropriate transfer depending upon organism's growth requirements. The following information should be submitted with the Antibiotic Resistance Confirmation testing or included in the final report.
                  
                  (A) The source and identity (e.g. ATCC, private source, other).
                        
                  (B) The method of preparation prior to testing (e.g. transfer history).
                        
                  (C) The method used to confirm the identity (e.g. biochemical test, Gram stain, morphology).
                        
                  (D) The method of preservation/storage (e.g. refrigerated agar slants, cryogenic beads, other).
                        
                  (E) Results of the testing including the numerical values of all antibiotics tested. An example of relevant values would be Minimum Inhibitory Concentrations (MIC) for automated tests, zone sizes for manual tests, and comparison to a standard Clinical and National Standards Institute interpretation of such tests.
                        
                  (F) The scientific method used to obtain the results (Kirby-Bauer, disc agar diffusion, or gradient agar diffusion; automated MIC procedures or equivalent). If automated procedures are used, the manufacturer of such automation should be specified.
                        
                  (G) Quality control procedures used to verify results.
                  
                  (H) GLP statement.
                         
            (iii) Additional microorganisms. Label claims for additional microbes other than the designated test microorganism(s) are permitted, provided that the target microbe is likely to be present in or on the recommended use areas and surfaces. 
                        
            (iv) Control carrier counts. 
                  
                  (A) For carrier-based assays, quantitative determinations of the microbial counts on the untreated control carriers after drying should be conducted in order to determine the validity of the test results obtained with the treated carriers as specified in the method. These quantitative determinations should be performed for all carrier-based assays, whether or not modifications are made to the method being used. The test results should include the individual dried carrier counts obtained by the method.
                        
                  (B) For suspension tests. Quantitative determination of the microbial count of the inoculum in a parallel untreated diluent should be conducted in order to determine the level of microbial challenge in the test (Numbers Control). These quantitative determinations should be performed for all suspension assays, whether or not modifications are made to the method being used.
                        
      (4) Porous test materials. When an antimicrobial product is intended to be effective in treating a specific hard, porous surface, a method modification may be necessary which specifies the use of the porous material (as a carrier) in the test protocol. In addition, control data (e.g., quantitation of dried carrier, neutralization confirmation, sterility controls) should be developed to assure the validity of the test results when this modification of the method is employed. 
                  
      (5) Hard water testing. Any product that bears a label claim for effec - tiveness using hard water as the product diluent should be tested by the appropriate efficacy method using synthetic hard water (hardness expressed as ppm CaCO3) at the level claimed. A minimum of hardness of 200 ppm should be used for a hard water claim. The hard water tolerance level may differ with the level of antimicrobial activity claimed (e.g., sterilization, disinfection, or sanitization). To support an efficacy claim in hard water, all microorganisms (bacteria, viruses, and fungi) claimed to be controlled by the product should be tested by the appropriate recommended method at the same level of hard water. Refer to the most recent version of the AOAC Germicidal and Detergent Sanitizing Action of Disinfectants test (Official Method 960.09) for guidance on the preparation of synthetic hard water. The use of deionized water as a diluent for concentrates is not recommended unless the label specifies deionized water. If a product requires dilution but does not bear a claim of effectiveness in hard water, the product should be tested in sterile tap water. The ppm hard water level of the tap water should be measured and reported with the submitted study. The registrant should be aware that tap water cannot be standardized and subsequent testing (e.g. post registration testing) may use a source of tap water exhibiting a different level of hardness. 
            
      (6) Organic burden.

            (i) The effectiveness of antimicrobial agents should be demonstrated in the presence of a specific organic soil at an appropriate concentration level when specifically claimed and/or indicated by the product's pattern of use. 
                  A sug - gested procedure to simulate in-use conditions where the antimicrobial agent is intended to treat dry inanimate surfaces contaminated with an or - ganic soil load involves contamination of the appropriate carrier surface with each test microorganisms culture containing 5% v/v blood serum (e.g., 19 mL test microorganism culture + 1 mL blood serum) prior to the carrier inoculation and specified carrier-drying step in the method. Control data (e.g., quantitation of dried carrier counts, neutralization confirmation, sterility controls) should also be developed to assure the validity of the test results when an organic burden is used. The 5% organic soil level suggested is considered appropriate for simulating lightly or mod - erately soiled surface conditions. When the surface to be treated has heavy soil deposits, a cleaning step should be included on the label prior to the application of the antimicrobial agent.
                  
            (ii) An antimicrobial substance identified as a one-step cleaner-disinfectant or cleaner-sanitizer, or intended to be effective in the presence of light to moderate amounts of organic burden, should be tested for efficacy with a minimum of a 5% organic soil added to the inoculum. Registrants should check with the Agency to determine the acceptability of an organic burden other than blood serum.
                   
                  For products with a pre-cleaning step prior to application, the addition of an organic burden to the inoculum may not be necessary. 
                  
            (iii) A suggested procedure for incorporating a light to moderate organic soil load where the antimicrobial agent is not tested against a dry inanimate surface, such as the AOAC International Fungicidal Activity of Disinfectants test and the Quantitative Tuberculocidal Test, involves adding a minimum of 5% (v/v) blood serum directly to the test organism (e.g., 4.75 mL test organism + 0.25 mL blood serum) before inoculating the test solution.
                  
            (iv) When a product is recommended for certain patterns of use where the organic soil claimed is of a specific type, such as soap film residue, the product should be tested in the presence of that specific organic soil. Registrants should provide specific information in the data report regarding the way in which the organic soil, such as soap film residue was prepared (e.g., percentages of ingredients) and employed in the testing.
                   
      (7) Contact time (exposure period). The contact time used in efficacy testing should be the same or shorter than the contact time identified on the product label. If a contact time different from the range identified in the test method or guideline is preferred, consultation with the Agency prior to testing is recommended and a modification to the standard approach may be needed. In most cases a modification to provide a longer exposure period is limited by the practical considerations of the use patterns (e.g., an exposure period of >10 min for a product that will likely evaporate from the treated surface within 10 min). All method modifications need to be clearly identified and justified in the test protocol. For liquid or spray products containing volatile active ingredients where the product is applied to a hard non- porous surface, the minimum contact time may be determined by visually inspecting evaporation over the proposed contact period. As identified in the AOAC International Germicidal Spray Products as Disinfectants test method, this approach should involve product application (using the proposed application method) to glass slide carriers. Use of methods that immerse contaminated carriers in the disinfectant fluid do not accurately simulate the way in which volatile disinfectants perform on environmental surfaces.
            
      (8) Neutralization. Neutralization is the process of inactivating a test material's antimicrobial activity at the end of the contact time. This may be achieved through physical (e.g., filtration, dilution, secondary subculture) and/or chemical (e.g., addition of sodium thiosulfate to the diluent) means. For each efficacy test, neutralization should be employed at the completion of the contact time in order to preclude residual effects of the active ingredient(s) in the subculture medium. If neutralization is not properly performed, the results of efficacy testing may not be deemed valid. The method of neutralization, whether physical or chemical, should be confirmed in the laboratory in advance or concurrently with testing. Neutralization confirmation data should be submitted in the final report to demonstrate that the neutralization process sufficiently inactivated the active ingredient(s) and that the neutralizer media used did not possess antimicrobial activity. All subculture techniques employed to preclude residual carryover of active ingredient should be described in detail in the study report. To demonstrate the absence of residual effects of the active ingredient in the subculture media, the examples of neutralization techniques in the paragraphs below should be followed.
            
            (i) In qualitative test methods, after treatment the inoculated carrier is deposited in a tube of growth media (i.e., primary subculture). If the primary subculture is not sufficient to achieve neutralization by the use of a chemical agent and/or through dilution of test substance, the carrier may then be transferred to a second tube of growth media (or secondary subculture) to achieve neutralization. The neutralization procedure employed should be confirmed and reported by running a parallel test with uninoculated carriers being added to growth media containing a low level (i.e., 10  -  100 CFU/mL) of bacteria. Both the primary cultures and secondary subcultures should be incubated and checked for growth in the test and the neutralization confirmation. Dried inoculated test carriers should not be used to test the ability of a subculture medium to support organism growth, as this would provide too large a bioburden and may lead to an inaccurate evaluation of the presence of bacteriostasis from the carry-over of the antimicrobial substance on the carrier to the subculture medium. Growth results for both primary and secondary subcultures should be reported for the test and neutralization confirmation in the final report.
                  
            (ii) A neutralization confirmation for suspension based test methods should be conducted for all neutralization/recovery methods employed in testing. Neutralization confirmation may be conducted by neutralizing the test substance, without the organism, as in the test. After neutralization, inoculation of a low level of organism (10-100 CFU/mL) and subsequent plating should follow. Plate counts should be within 50% of a parallel population control or as specified in the relevant method.
                   
                  (A) For virucidal tests, scientifically accepted controls including proper neutralization controls should be performed (e.g., ASTM E1482).
                        
      (9) Batch replication for modified testing. For certain use modifications to a registered product, the Agency allows a reduced number of product batches (lots) to be tested in support of these modifications. In order to be eligible to use this approach however, the registered product should have acceptable efficacy data supporting the product that were performed without modification and included the number of batches specified in the relevant method/guideline. Under these conditions, additional data performed at the same use concentration previously tested may be conducted with reduced batch replications to support a single modified use condition such as organic soil load, hard water concentration or use of porous surface carriers. The batch number reductions are as follows:
            
            (i) For basic efficacy claims (e.g., sterilants, disinfectants, sanitizers), two samples, representing two different batches may be tested, instead of three.
                  
            (ii) For supplemental efficacy claims (e.g., fungicides and virucides), one sample instead of two may be tested.

      (10) Performance Standards. Performance standards are a set of test criteria used to establish product effectiveness, such as the number of positive carriers allowed for a passing product or a minimum log reduction of bacteria following treatment. The testing criteria may be different for each standard method and test microbe, thus the applicant should consult the appropriate guideline for this information. 

      (11) Repeat Testing Policy  -  Refer to the methods cited in "Disinfectants for Use on Hard Surfaces  -  Efficacy Data Recommendations" OCSPP 810.2200, to determine allowable levels of contamination for a particular test method.  If the  product performance test data is deemed invalid due to an unacceptable level of contamination, the following re-testing scenarios apply:

            (i) When an unacceptable level of contamination is present, retesting may be conducted using the same test conditions if the efficacy test meets the required performance standard. Retesting of the product lot(s) associated with the invalid test(s) should be conducted.   
                  
            (ii) When an unacceptable level of contamination is present and the product does not meet the required performance standard, the product is considered a failure.     
                  
(f) Data collection and reporting.

      (1) General. To assist in the proper review and evaluation of product performance, complete descriptions of the test employed and the results obtained should be submitted to the Agency. All test reports should include, at the least, the following information:
            
            (i) Study title;
                  
            (ii) Complete product identity (Certificate of Analysis);
                  
            (iii) Guideline number/Data Requirement;
                   
            (iv) Identification of the testing laboratory or organization;
                  
            (v) Location where the test was performed;
                  
            (vi) Name(s) of the person(s) responsible for the test;
                  
            (vii) Statement of Confidentiality Claims;
                  
            (viii) Statement of 40 CFR Part 160 Good Laboratory Practice compliance and Quality Assurance Statement; all protocol deviations should be identified.
                  
            (ix) Purpose of the study;
                  
            (x) Date and time of the start and end of the test;

            (xi) Name of the test method and appropriate references;
                  
            (xii) Test employed (include the version or edition) and any modifications (e.g., organic soil, hard water, etc.), when using standard tests (e.g., AOAC, ASTM, etc.) all modifications to the test methods should be reported and justified;
                  
            (xiii) Test microorganisms employed, including identification of the specific strain (ATCC or other);
                  
            (xiv) Description of the test substance, including the percent of active ingredient;
                  
            (xv) Concentration or dilution of the product tested and how prepared;
                  
            (xvi) Number of samples, batches and replicates tested;
                  
            (xvii) Manufacture date of each product batch
                  
            (xviii) Identification of all material or procedural options employed, where such choice is provided for or recommended in the test method selected (e.g., growth media, drying time for inoculated carriers, neutralization confirmation and/or subculture media, secondary subculturing);
                  
            (xix) Test exposure conditions (e.g., contact time, temperature, and relative humidity);
                  
            (xx) Complete reports of results obtained for each replication;
                  
            (xxi) Any control data, including neutralization confirmation results, essential to establish the validity of the test.
                  
            (xxii) Control carrier counts (i.e., microbial load);
                  
            (xxiii) Formula used to calculate control counts and log reduction values (if applicable) as well as any statistical treatment of the data;
                  
            (xxiv) Conclusions;
                  
            (xxv) Any pictures or figures explaining the outcome or observations;
                  
            (xxvi) References;
                  
      (2) Appendices, including study protocol and all raw data reports associated with the conduct of the study. The applicant is encouraged to use the EPA's standard efficacy report format, which may be found at
            http://www.epa.gov/oppad001/efficacystudystandards.htm.

(g) References. The references in this paragraph may be consulted for additional background information:
      
      (1) US EPA; Regulating Antimicrobial Pesticides. See http://www.epa.gov/oppad001.
      (2) Official Methods of Analysis of AOAC International. Chapter 6, Disinfectants, Current edition.  AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.
            
      (3) Annual Book of ASTM Standards, Standard Test Methods. American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition.