United States Environmental Protection Agency Office of Prevention, Pesticides And Toxic Substances ( 7510P ) EPA 712

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- 07

- 074 Draft November 5 ,

2009 

# Product Performance Test Guidelines OPPTS 810.2200 : Disinfectants for Use on Hard Surfaces

— Efficacy Data Recommendations

Public Review DraftNOTICE This guideline is one of a series of test guidelines established by the Office of Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection Agency

for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq. ), the Federal Insecticide, Fungicide , and Rodenticide Act (FIF RA) (7 U.S.C. 136, et seq. ), and section 408 of the Federal Food, Drug , and Cosmetic (FFDCA) (21 U.S.C. 346a). The OPPTS test guidelines serve as a compendium of accepted scientific methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also used by EPA, the public, and the companies that are subject to data submission requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances warrant and without prior notice. The procedures contained in this guideline are strongl y recommende d for generating the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. You may propose alternatives to the recommendations described in these guidelines, and the Agency

will assess them for appropriateness on a case

-by

- case basis. For additional information about OPPTS harmonized test guidelines and to access the guidelines electronically, please go to http://www.epa.gov/oppts and select “Te st Methods & Guidelines” on the left side navigation menu. You may also access the guidelines in http://www.regulations.gov grouped by Series under Docket ID #s: EPA

- HQ

- OPPT

- 2009

- 0150 through EPA

- HQ

- OPPT

- 2009

- 01 59, and EPA

- HQ

OPPT

- 2009

- 0576.

DRAFT DOCUMENT DISCLAIMER: This draft document is distributed solely for the purpose of external review. Ithas not been formally disseminated by

the EPA and should not be construed to represent any Agency determination or policy . The information correction process under the Agency ’s Information Quality Guidelines does not apply

until this document is formally disseminated by

the EPA in its final form. This draft document should only be cited or quoted in the context of providing comments.1

OPPTS 810.2200 : Disinfectants for use on hard surfaces - efficacy d ata recommendations (a) Scope

. (1) Applicability

. This guideline is intended to meet testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)( 7U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 I/ S/C. 346a)

. It addresses testing to demonstrate effectiveness of products which are disinfectants, fungicides, virucides, and tuberculocides. (2) Background

. The source materials used in developing this OPPTS test guideline are OPP guidelines 91

- 2 : Products for use on hard surfaces and 91

- 30 : Acceptable methods (Pesticide Assessment Guidelines, Subdivision G, Product Performance. EPA report 540/9

- 82

- 026, October 1982). (b) Purpose . This guideline addresses efficacy testing for antimicrobial pesticides intended to be used on hard surfaces, namely disinfectants, fungicides, virucides, and tuberculocides in a variety o f product ty

pes (water

- soluble powders, l iquids, sprays, towelettes, etc.). (c) General considerations ( 1) This guideline recommends tests to be conducted and data to be submitted which the Agency b elieves will generall y satisfy the requirements for pesticide registration. All studies conducted under this guideline should be completed under EPA’s Good Laboratory Practice regulations (40 CFR Part 160 ). Note: The Association of Official Analy tical Chemist s (AOAC ) recommended tests are expected to be conducted as written. For deviations (e.g ., cultures grown with shaking instead of static, dilution of culture prior to drying on carriers) proposed to be used in the conduct of these tests, obtain written approval from the Agency and document such deviations in the study

reports submitted to the Agency

. The Agency may c onsult with the AOAC prior to accepting modification s to their standardized methods. Refer to OPPTS Test Guideline 810.2000 for general testing recommendations prior to initiating tests. (2) [Reserved] (3 ) Confirmatory testing. Inc ertain situations an applicant may rel y on previously submitted efficacy

data to support an application or amendment for registration of a product and submit only

c onfirmatory

e fficacy data on his own product to demonstrate his ability to produce an effective formulation. These situations are as listed in paragraphs (C)( 3 )(i) through (C)(3 )(iii) of this guideline : (i) Duplicated Product Formulations. In this situation, the applicant manufactures a formulation which duplicates a product that is already registered with 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 (Master Record ID Numbers [MRIDs]) to the supporting data developed for the original product2

are cited b y the applicant. (ii) Minor Formulation Change in a Registered Product. Inthis situation, the change in the formulation is relatively minor, e.g., a change of an inert ingredient. The label claims and directions for use are unchanged from those accepted for the registered formulation, and specific references (MRIDs) to the supporting data developed for the original formulation are cited by the applicant. If the onl y change in the formulation is the addition of a fragrance or dye, confirmatory

data does not need to be submitted. H owever, 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 must be developed on the applicant's own finished product. When the test methodology utilized in deriving the original supporting efficacy data were modified to include additional elements not specified in the recommended method, such as organic soil, hard water, longer or shorter contact time, etc., the confirmatory data should be produced under similarly modified conditions. (4 ) Efficacy claims. Table 1 provides a quick reference guide to testing for basi c claims described in this guideline . Consult the text for detailed testing descriptions

.

Table 1. T esting for basic efficacy claims Level of Efficacy Test Methods Test Organisms No. of

Batches/Carriers Evaluation

of Success Limited disinfectant/hard n on - porous surfaces. Water soluble

powders/liquids AOAC Use

Dilution Method or AOAC Hard Surface Carrier Test (distilled water only) Staphylococcus

aureus (ATCC 6538) or Salmonella enterica (ATCC 10708) Three batches, one at least 60 days old. 60 carriers against either orga nism claimed (180 carriers ). 59/60 carriers are negative for each batch tested for all methods except AOAC Hard Surface Carrier Test, which is 58/60 carriers are negative for each batch. Spray products AOAC

Germicidal Spray Produ cts Test Towelettes Modified

Germicidal Spray Test Broad

- spectrum disinfectant/hard non - porous surfaces. Water soluble

powders/liquids AOAC Use

Dilution Method or AOAC Hard Surface Carrier Test (distilled water only) Staphylococcus

aureus an d Salmonella enterica Three batches, one at least 60 days old. 60 carriers against e ach organism (360 carriers ). 59/60 carriers are negative for each batch tested for all methods except AOAC Hard Surface Carrier Test, which is 58/60 carriers are negative for each batch. Spray products AOAC

Germicidal Spray Products Test Towelettes Modified

Germicidal Spray Test3

Level of Efficacy Test Methods Test Organisms No. of

Batches/Carriers Evaluation

of Success Hospital disinfectant/hard non - porous surfaces. Water soluble

powders/liquids AOAC Use

Dilution Method or AOAC Hard Surface Carrier Test (distilled water only) Staphylococcus

aureus and Pseudomonas aeruginosa (ATCC 15442) Three batches, one at least 60 days old. 60 carriers against each organism (360 c arriers ). 59/60 carriers are negative for each batch tested for all methods except AOAC Hard Surface Carrier Test, which is 58/60 carriers are negative against Staphylococcus aureus for each batch, and 57/60 carriers are negative against Pseudomonas aeruginosa

. Spray products AOAC

Germicidal Spray Products Test Towelettes Modifie d

Germicidal Spray Test Fungicidal disinfectant/hard non - porous surfaces. Water soluble

powders/liquids AOAC Use

Dilution Test modified for fungi or AOAC Fungicidal Test Trichophyton

mentag rophytes (ATCC 9533) Two batches, ten carriers per batch for the modified AOAC Use Dilution Test, the modified AOAC Germicidal Spray Products Test, and the EPA Towelette Test. Two batches for the AOAC Fungicidal Test. All fungal spores on all carriers should be killed.

Spray products AOAC

Germicida l Spray Products Test modified for fungi Towelettes Modified

Germicidal Spray Test Virucidal disinfectant/hard non - porous surfaces. Water soluble

powders/liquids AOAC Use

Dilution Test modified for viruses or ASTM E1053

- 97 Virus claimed on

t he label or approved surrogate. Two batches. Complete inactivation of the virus. W here cytotoxicity is present, demonstrate a 3log 10 reduction. Spray products AOAC

Germicidal Spray Products Test modified for viruses or ASTM E1053

- 97 Towelettes Modi fied

Germicidal Spray Test Tuberculocidal disinfectant/hard non - porous surfaces. Water soluble

powders/liquids AOAC

Tuberculocidal Activity of Disinfectants, Quantitative Tuberculocidal Activity Test Mycobacterium

bovis BCG Two batches, ten carri ers per batch 10/10 carriers are negative for growth and there is no growth in the additional test media. Spray products AOAC

Germicidal Spray Products Test modified for tuberculocidal activity4

Level of Efficacy Test Methods Test Organisms No. of

Batches/Carriers Evaluation

of Success Towelettes Modified

Germicidal Spray Test Additiona l bacteria/hard non

porous surfaces. Water soluble

powders/liquids AOAC Use

Dilution Test or AOAC Hard Surface Carrier Test (distilled water only) Bacteria claimed on

the label. Two batches, ten carriers for each batch. All bacteria are killed

Spray products AOAC

Germicidal Spray Products Test (d) Disinfectants ( 1) Limited spectrum products. This section addresses efficacy testing for disinfectant products with limited efficacy ( effective against Gram

-negative or Gram

-positive bacteria , but not both). (i) Water
- soluble powders and non -volatile liquid products test procedure. The Agency recommends the use of the AOAC International Use

- Dilution Methods (see reference (i )( 1) of this guideline) or the AOAC International Hard Surface Carrier Test Methods (distilled water onl y)(Ref. 2 ). Sixty carriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against Salmonella enterica (S. enterica )( formerl y designated as Salmonella choleraesuis )(ATCC 10708 ) for effectiveness against Gram

-negative bacteria, or Staphylococcus aureus (S. aureus )(ATCC 6538) for effectiveness against Gram

-positive bacteria. If the product is intended to be represented as bactericidal in the presence o f organic soil (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (ii) Germicidal spray products (aerosol or pump) and volatile liquid products test procedure. The Agency recommends us e of the AOAC International Germicidal Spray Products as Disinfectants test (Ref. 3 ). S ixty c arriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against S. enterica (ATCC 1 0708) for effectiveness against Gram

-negative bacteria, or S. aureus (ATCC 6538) for effectiveness against Gram

-positive bacteria. If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (iii) Single
- use towelettes test p rocedure. The Agency recommends the use of a modified AOAC Germicidal Spray Products as Disinfectants test. Sixty carriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against S. enterica (ATCC 10708) for effectiveness against Gram

-negative bacteria, or S. aureus (ATCC 6538) for effectiveness against Gram-positive bacteria. If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ) , an5

appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. Instead of spraying the inoculated surface of the gl ass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. The towelette should be removed from its container and subse quently

handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a maximum amount of its surface in the course of wiping a set of slides. Note: Adetailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency for review and approval prior to conducting the test. (iv) Evaluation of limited disinfectant success. For the AOAC International Use

Dilution Methods, the Germicidal Spray Products as Disinfectants test, and single

-use towelettes, the product should kill the test microorganisms on 59 out of each set of 60 carriers/slides within ten minutes or less. Inaddition, per the 2009 revisions for the Use

- Dilution Method, the mean log density for S. aureus must be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10 6 ); a mean log density < 6.0 invalidates the test. For the AOAC International Hard Surface Carrier Test Methods, the product should kill the test microorganisms on 58 out of each set of 60 carriers for S. enterica or S. aureus within ten minutes or less. (2 ) Confirmatory testing for limited spectrum products (i) Water- soluble powders a nd non -volatile liquid products test procedure. The Agency recommends the use of the AOAC International Use

- Dilution Methods (Ref.1 ) or the AOAC International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2 ). T en carri ers for each of two product samples, representing two different batches of the product, should be tested against either S. aureus or S. enterica (depending on whether the product is claimed to be effective against Gram

-negative or Gram

-positive bacteria). If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (ii) Germicidal spray products (aerosol o r pump) and volatile liquid products test p rocedure. The Agency recommends the AOAC International Germicidal Spray Products as Disinfectants test (Ref. 3 ). T en carriers for each of two product samples, representing two different batches of the product, should be tested against either S. aureus or S. enterica (depending on whether the product is claimed to be effective against Gram

-negative or Gram

positive bacteria). If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. For pressurized spray p roducts, certification should be furnished specifying that all parts and materials used in manufacturing the container for pressurized spray

disinfectants are identical to those specified b y the basic manufacturer

.6

(iii) Single
- use towelettes test p rocedure. The Agency recommends the use of a modified AOAC Germicidal Spray Products as Disinfectants test

. Ten carriers for each of two product samples, representing two different batches of the product, should be tested against either S. aureus or S. enterica (depending on whether the product is claimed to be effective against Gram

-negative or Gram

-positive bacteria). If the product is intended to be represented as bactericidal in the presence of organi c soil (one

- step ) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. In stead of spraying the inoculated surface of the glass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. The towelette should be removed from its container and subs e quently handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch . The area of the towelette used for wiping should be rotated so as to expose a maximum amount of its surface in the course of wiping a set of slides

. Note: Adetailed description of the wiping procedure, including the towelette folding and rotation pro cess should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency f or review and approval prior to conducting the test. (iv) Evaluation of confirmatory limited disinfectant success
. The product should kill all the test microorganisms on all carriers/slides within ten minutes or less. In addition, per the 2009 revisions for the Use

- Dilution Method, the mean log density for S. aureus must be at least 6.0 (corresponding to a geometric mean density o f 1.0 x 10 6 ); a mean log density <6.0 invalidates the test. (3 ) General or broad spectrum efficacy products . When a disinfectant is represented in labeling as having efficacy against both Gram

-negative and Gram

-pos itive bacteria, the product is considered a general or broad spectrum disinfectant. (i) Water
- soluble powders an d non -volatile liquid products test procedure. The Agency recommends the use of the AOAC International Use

- Dilution Methods (Ref. 1 ) or the AOAC International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2 ). S ixty carriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against both S. enterica (ATCC 10708) and S. aureus (ATCC 6538). If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (ii) G ermicidal spray products (aerosol or pump) and volatile liquid products test procedure. The Agency recommends the AOAC International Germicidal Spray Products as Disinfectants test (Ref. 3 ). S ixty c arriers for each of three samples, representing three di fferent batches, one of which should be at least 60 days old, should be tested against both S. enterica (ATCC 10708) and S. aureus (ATCC 6538). If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ) , an appropriate organic soil, such as 57

percent blood serum, should be included with the bacterial inoculum. (iii) Single
- use towelettes test p rocedure. The Agency recommends the use of a modified AOAC Germicidal Spray Products as Disinfectants test. Sixty c arriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against both S. enterica (ATCC 10708) and S. aureus (ATCC 6538). If the product is intended to be represented as bacteri cidal in the presence of organic soil (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. Instead of spraying the inoculated surface of the gl ass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. The towelette should be removed from its container and subse quentl y handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette used for wiping should be rotated s o as to expose a maximum amount of its surface in the course of wiping a set of slides. Note: Adetailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency for review and approval prior to conducting the test. (iv) Evaluation of general or broad spectrum disinfectant success. For the AOAC International Use

- Dilution Methods, the Germicidal Spray Products as Disinfectants test, and single

-use towelettes , the product should kill the test microorganisms on 59 out of each set of 60 carriers/slides within ten minutes or less. In addition, per the 2009 revisions for the Use

- Dilution Method, the mean log density for S. aureus must be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10 6 ); a mean log density < 6.0 invalidates the test. For the AOAC International Hard Surface Carrier Test Methods, the product should kill the test microorganisms on 58 out of each set of 60 carriers within ten minutes or less. (4 ) Confirmatory testing for general or broad spectrum products (i) Water
- soluble powders and non -volatile liquid products. test procedure. The Agency recommends the use of the AOAC International Use

- Dilution Methods (Ref. 1 ) or the AOAC International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2 ). T en carriers for each of two product samples, representing two different batches of the p roduct, should be tested against both S. aureus and S. enterica

. If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (ii) Germicidal spray products (aerosol or pump) and volatile liquid products test procedure. The Agency recommends the AOAC International Germicidal Spray Products as Disinfectants test (Ref. 3 ). T en carriers for each of two product samples, representing two different batches of the product, should be tested against both S. aureus and S. enterica

. If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial8

inoculum. For pressurized spray p roducts, certification should be furnished specifying that all parts and materials used in manufacturing the container for pressurized spray disinfectants are identical to those specified by the basic manufacturer. (iii) Single
- use towelettes test p rocedure. The Agency recommends the use of a modified AOAC Germicidal Spray Products as Disinfectants test. T en carriers for each of two product samples, representing two different batches of the product, should be tested against both S. aureus and S. enterica

. If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. Instead of spraying the inoculated surface of the glass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slide s after the specified holding time. The towelette should be removed from its container and subse quently handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a maximum amount of its surface in the course of wiping a set of slides. Note: A detailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agenc y for review and approval prior to conducting the test. (iv) Evaluation of confirmatory general or broad spectrum disinfectant success. The product should kill all the test microorganisms on all carriers/slides within ten minutes or less

. In addition, per the 2009 revisions for the Use

- Dilution Method, the mean log density for S. aureus must be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10 6 ); a mean log density <6.0 invalidates the test. (5 ) Hospital or healthcare disinfectants. This section addresses efficacy testing for products recommended for use in hospitals, clinics, dental offices, nursing homes, sickrooms, or any other healthcare

-related facility. (i) Water
- soluble powders an d non -volatile liq uid product test procedure. The Agency recommends the use of the AOAC International Use

- Dilution Methods (Ref. 1 ) or the AOAC International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2 ). S ixty carriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against S. aureus (ATCC 6538), and Pseudomonas aeruginosa (

P. aeruginosa )(ATCC 15442). If the product is intended to be represented as bactericidal in the presence of organic so il (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (ii) Germicidal spray products (aerosol or pump) and volatile liquid products test procedure. The Agency recommends the use of the AOAC Inter national Germicidal Spray Products as Disinfectants test (Ref. 3 ). S ixty c arriers for each of three samples, representing9

three different batches, one of which should be at least 60 days old, should be tested against: S. aureus (ATCC 6538), and P. aerugin osa (ATCC 15442). If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (iii) Sin gle
- use towelettes test p rocedure. The Agency recommends the use of a modified AOAC Germicidal Spray Products as Disinfectants test. Sixty carriers for each of three samples, representing three different batches, one of which should be at least 60 days old, should be tested against S. aureus (ATCC 6538), and P. aeruginosa (ATCC 15442). If the product is intended to be represented as bactericidal in the presence of organic soil ( one - step), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. Instead of spraying the inoculated surface of the gl ass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. The towelette should be removed from its container and subse quently

handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a maximum amount of its surface in the course of wiping a set of slides. Note: Adetailed description of the wip ing procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency for review and approval prior to conducting the test. (iv) Evaluation of hospital or healthcare disinfectant success. For the AOAC International Use

- Dilution Methods, the Germicidal Spray Products as Disinfectants test, and single

-use towelettes, the product should kill the test microorganisms on 59 out of each set of 60 carriers/slides within ten minutes or less. In addition, per the 2009 revisions for the Use

- Dilution Method, the mean log density for S. aureus and P. aeruginosa must be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10 6 ); a mean log density <6.0 invalidates the test. For the AOAC International Hard Surface Carrier Test Methods, the product should kill the test microorganisms on 58 out of each set of 60 carriers for S. aureus , and 57 out of each set of 60 carriers for P. aeruginosa within ten minutes or less. (6) Confirmatory testing for products with hospital or healthcare disinfectant claim (i) Water
- soluble powders and non -volatile liquid products test procedure. The Agency recommends the use of the AOAC International Use

- Dilution Methods (Ref. 1 ) or the AOAC International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2 ). T en carriers for each of two product samples, representing two different batches of the product, should be te sted against S. aureus (ATCC 6538) P. aeruginosa (ATCC 15442). If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (ii) Germicidal spray products (aerosol or pump) and volatile liquid products test10

procedure. The Agency recommends the AOAC International Germicidal Spray Products as Disinfectants test (Ref. 3 ). T en carriers for each of two product samples, representing two different batches of the product, should be tested against S. aureus (ATCC 6538) and P. aeruginosa (ATCC 15442). If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. For pressurized spray products, certification should be furnished specifying that all parts and materials used in manufacturing the container for pressurized spray

disinfectants are identical to those specified b y the basic manufacturer. (iii) Single
- use towelettes test p rocedure. The Agency recommends the use of a modified AOAC Germicidal Spray Products as Disinfectants test. T en carriers for each of two product samples, representing two different batches of the product, should be tested against S. aureus (ATCC 6538) and P. aeruginosa (ATCC 15442). If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. In stead of spraying the inoculated surface of the glass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. The towelette should be removed from its container and subse quently handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. Alter

natively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a maximum amount of its surface in the course of wiping a set of slides

. Note: Adetailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency f or review and approval prior to conducting the test. (iv) Evaluation of confirmatory hospital or healthcare disinfectant success. The product should kill all the test microorganisms on all carriers/slides within ten minutes or less. In addition, per the 2009 revisions for the Use

- Dilution Method, the mean log density for S. aureus and P. aeruginosa must be at least 6.0 (corresponding to a geometric mean density o f 1.0 x 10 6 ); a mean log density <6.0 invalidates the test. (7) Bridging for disinfectant towelettes. In some cases, disinfectant towelette formulations are identical to registered liquid formulations. In order to bridge efficacy data from the EPA registered bulk liquid disinfectant used to saturate a towelette or other related product form, the studies in paragraphs (d)(7)(i) and (d)(7)(ii) of this guideline should be conducted and submitted to EPA for review

. (i) Chemical Testing
- Comparison of Expressed Liquid from the Towelette(s) to the EPA Registered Liquid Disinfectant Formulation to which it is being bridged: All active ingredients in the expressed liquid should be within the certified limits of the Confidential Statement of Formula of the liquid formula being referenced/bridged. The disinf ectant towelettes package should be filled according to the manufacturing specifications. Excess liquid in the bulk11

towelette containers can not be poured off for use in the chemical testing for bridging of the efficacy data. The liquid used in the chemic al testing should only be that expressed from the towelettes. Three batches (one of which is 60 days old) should be tested. Analy tical data for the active ingredients in the expressed liquid should be submitted for review. (ii) Efficacy Testing: Efficac y testing should be conducted under the same testing conditions (e.g. soil load, contact time, temperature) as use d for the bulk liquid testing

. Note: A detailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency f or review and approval prior to conducting the test. For l imited disinfectants, broad

- spectrum disinfectants, and hospital disinfectants , to bridge bacterial disinfection claims: (A) Test Procedure. The Agency recommends the use of the AOAC Germicidal Spray Products as Disinfectants test modified for towelettes, using the test organisms specified for limited, broad

- spectrum or hospital disinfectant testing. S ixty c arriers for each organism should be tested against three different batches of the product (one of which should be 60 days old). Instead of sprayingthe inoculated surface of the gl ass slide , the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. One towel will be used to treat 10 carriers. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. (B ) E valuation of bactericidal towelette success. The product should kill the test organism on 59 out of 60 carriers. This testing is intended to support bridging of all vegetative bacteria listed on the EPA registered liquid disinfectant used to saturate the towel to the EPA registered towelette product. (8) D isinfectants for Internal Toilet and Urinal Bowl Surfaces Above and Below the Water Line. This section addresses efficacy testing for products bearing label claims as disinfectants (limited, broad

- spectrum, or hospital) for internal toilet and urinal bowl surfaces. Regarding water

- soluble powders and non-volatile liquid products test procedure , the Agency recommends the use of the AOAC International Use

- Dilution Methods (see reference (i )( 1) of this guideline) modified to include a 5% organic soil challenge. Sixty c arriers for each of three samples, representing three different batches, one of which is at least 60 days old, should be tested against Salmonella enterica (ATCC 10708) or Staphylococcus aureus (ATCC 6538), for limited disinfectant products; S. enterica and S. aureus , for broad

- spectrum disinfectant products; and S. aureus and Pseudomonas aeruginosa (ATCC 15442), for hospital disinfectant products. The contained bowl water (

- 96 fl oz) should be used to calculate the appropriate use dilution for testing. (i) Evaluation of disinfectant success for internal toilet bowl and urinal bowl surfaces. For the AOAC International Use

- Dilution Methods and the Germicidal Spray Products as Disinfectants test, the product should kill the test microorganisms on 59 out of each12

set of 60 carriers/slides within ten minutes or less. Inaddition, per the 2009 revisions for the Use

- Dilution Method, the mean log density for S. aureus and P. aeruginosa must be at least 6.0 (corresponding to a geometric mean density of 1.0 x 10 6 ); a mean log density <6.0 invalidates the test. (9) Additional mi croorganism s. This section addresses efficacy testing for broad

spectrum or hospital disinfectants which bear label claims against bacteria other than Salmonella enterica (ATCC 10708), Staphylococcus aureus (ATCC 6538) or Pseudomonas aeruginosa (ATCC 15442). (i) Water- soluble powders an d non -volatile liquid products test procedure. The Agency recommends the use of the AOAC International Use

- Dilution Methods (Ref. 1 ) or the AOAC International Hard Surface Carrier Test Methods (distilled water only)(Ref. 2 ). T en car riers should be tested against each specific bacterium for each of two samples representing two different batches

. If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (ii) Germicidal spray products (aerosol or pump) and volatile liquid products test procedure. The Agency recommends the use of the AOAC International Germicidal Spray Products as D isinfectants test (Ref. 3 ). T en carriers should be tested against each specific bacterium for each of two samples representing two different batches. If the product is intended to be represented as bactericidal in the presence of organic soil (one

- step ), an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (iii) Single
- use towelettes test p rocedure. The Agency recommends the use of a modified AOAC Germicidal Spray Products as Disinfectants test. T en carriers should be tested against each specific bacterium for each of two samples representing two different batches. If the product is intended to be represented as bactericidal in the presence of organic soil (one

step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. Instead of spraying the inoculated surface of the glass slide, the product should be tested by

wiping the surface of the gl ass slide with the saturated towelette, and then subculturing the slides after the specified holding time. The towelette should be removed from its container and subse quently handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a maximum amount of its surface in the course of wiping a set o f slides . Note: Adetailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency

for review and approval prior to conducting the test. (iv) Evaluation of disinfectant success for additional microorganisms. The product should kill all the test microorganisms on all carriers/slides within ten minutes or less.13

(e) Disinfectants with fungicidal claims. This section addresses efficacy testing for broad

- spectrum or hospital disinfectant products which bear label claims of efficacy against pathogenic fungi. (1) Water soluble powders and non
-volatile liquid products (i) Test procedures

. The Agency recommends the use of the AOAC International Fungicidal Activity

o f Disinfectants test (Ref. 4 ) Two samples representing two different batches of the product should be evaluated for efficacy against Trichophyton mentagroph ytes ( T. mentagrophytes

)( ATCC 9533). The Agenc y also recommends the use of the AOAC International Use

- Dilution Methods (Ref. 1 ). This test may b e modified to conform to appropriate elements (e.g., media, growth conditions, etc.) in the AOAC International Fungicidal Activity o f Disinfectants test. T en carriers for each of two samples representing two different batches of the product should be evaluated against T. mentagrophytes (ATCC 9533 ). The inoculum employed should provide a concentration of 1 x 10 4

– 1 x 10 5 conidia per carrier. If the product is intended to be represented as fungici dal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the fungal inoculum. (ii) Evaluation of fungicidal success. For the AOAC International Fungicidal Activity of Disinfectants test, all fungal spores in all replicates for each batch should be killed. F or the AOAC International Use

- Dilution Methods, all fungal spores on all 10 carriers should be killed within ten minutes or less. (2) Germicidal spray products (aerosol or pump) and volatile liquid products

—(i) Test procedures. The Agency recommends the use of the AOAC International Germicidal Spray Products as Disinfectants test (see referenc e (i )( 3) of this guideline). This test may b e modified to conform to appropriate elements (e.g., media, growth conditions, etc.) in the AOAC International Fungicidal Activity o f Disinfectants test. T en carriers for each of two samples representing two dif

ferent batches of the product should be evaluated against T. mentagrophytes (ATCC 9533). The inoculum employed should be modified to provide a concentration of 1 x 10 4

– 1 x 10 5 conidia per carrier. If the product is intended to be represented as fungic idal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the fungal inoculum. (ii) Evaluation of fungicidal success. All fungal spores on all 10 carriers/slides should be killed within ten minutes or less. (3) Single
-Use Towelettes (i) Test Procedure. The Agency recommends the use of a modified AOAC Germicidal Spray Products as Disinfectants test. T en carriers for each of two samples representing two different batches of the product should be evaluated against T. m entagrophytes (ATCC 9533). The inoculum employed should be modified to provide a concentration of 1 x 10 4

– 1 x 10 5 conidia per carrier. If the product is intended to be represented as fungicidal in the presence of14

organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the fungal inoculum. In stead of spraying the inoculated surface of the glass slide, the product should be tested by wiping the surface of the glas s slide with the saturated towelette, and then subculturing the slides after the specified holding time. The towelette should be removed from its container and subse quently h andled with sterile gloves. One towelette should be used to wipe a minimum of 1 0 inoculated slides. Alternativel y, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a maximum amount of its surface in the course of wiping a set of slides

. Note: Adetailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency for review and approval prior to conducting the test. (ii) Evaluation of fungicidal towelette success. All fungal spores on all 10 carriers/slides should be killed within ten minute s or less. (4) Bridging for disinfectant towelettes. In some cases, disinfectant towelette formulations are identical to registered liquid formulations. In order to bridge efficacy data from the EPA registered bulk liquid disinfectant used to saturate a towelette or other related product form, the studies in paragraphs (e)(4)(1) and (e)(4)(ii) of this guideline should be conducted and submitted to EPA for review: (i) Chemical Testing
- Comparison of Expressed Liquid from the Towelette(s) to the EPA Registered Liquid Disinfectant Formulation to which it is being bridged: All active ingredients in the expressed liquid should be within the certified limits of the Confidential Statement of Formula of the liquid formula being referenced/bridged. The disinfectant towelettes package should be filled according to the manufacturing specifications. Excess liquid in the bulk towelette containers can not be poured off for use in the chemical testing for bridging of the efficacy data. The liquid used in the chemical testing should only be that expressed from the towelettes. Three batches (one of which is 60 days old) should be tested. Analy tical data for the active ingredients in the expressed liquid should be submitted for review. (ii) Efficacy Testing: Efficacy testing should be conducted under the same testing conditions (e.g. soil load, contact time, temperature) as used for the bulk liquid testing. This testing allows bridging of data from the registered bulk liquid used to saturate the towel for each ty pe of organism in this paragraph. Note: Adetailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency

f or review and approval prior to conducting the test. For f ungicidal test procedure , the Agency

recommends the use of the AOAC International Germicidal Spray Products as Disinfectants (Ref. 3 ) modified for fungicidal towelette testing. The test should be modified to conform to appropriate elements (e.g. media, growth conditions) in the AOAC International Fungicidal Activity o f Disinfectants test. T en carriers for each of two samples, representing two batches of the product should be evaluated against T. mentagrophytes (ATCC15

9533) for the label recommended contact time

. The inoculum employed should be at a count to achieve 1 x 10 4

– 1 x 10 5 conidia per carrier. Instead of spraying the inoculated surface of the glass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. One towelette should be used to wipe a minimum of 10 inoculated slides. Alternativel y, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. (A) Evaluation of Fungicidal towelette success. The product should kill the test organism on all 10 carriers. (B ) Bridging. This testing is intended to support bridging of all fungal test organisms from the EPA registered bulk liquid disinfectant used to saturate the towel to the EPA registered towelette product. (f ) D isinfectants with virucidal claims. This section addresses efficacy testing for broad

- spectrum or hospital disinfectant products that bear label claims of effectiveness against viruses. Virucidal products are intended for use on dry inanimate surfaces; therefore, virological data are usuall y develop ed by c arrier methods. E ach specific virus listed on the label should be tested. F or label claims against Hepatitis B virus, Hepatitis C virus, and Norovirus, the Duck Hepatitis B virus, Bovine Viral Diarrhea virus, and Feline Calicivirus, respectivel y, are considered acceptable surrogates for testing. Additional guidance and protocols for surrogate virus testing can be found at http://www.epa.gov/oppad001/regpolicy .htm

. To simulate in

-use conditions, the specific virus to be treated (or surrogate as noted in this paragraph) should be inoculated onto hard surfaces (e.g., Petri dishes, glass slides, stainless steel penicylinders, or other appropriate test surface), allowed to dry, and then treated with the product according to the directions for use on the product label. (1) Water soluble powders a nd non -volatil e liquid products test procedures

. The Agency recommends the use of either the AOAC International Use

- Dilution Methods (see reference (i )( 1) of this guideline) modified for virucidal testing or the ASTM E1053

- 97 Virucidal Test Method (Ref. 5)

. One surface for each of two samples, representing two different batches of disinfectant, should be tested against a recoverable virus titer of at l east 10 4 viable viral particles from the test surface for a specified exposure period at room temperature. I f the product is intended to be represented as virucidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the viral inoculum. When viral suspensions are grown in the presence of at least 5% serum, addition of serum to the inoculum is not expected as part of a study to support a one

- step label claim. (2 ) Germicidal spray products (aerosol or pump) and volatile liquid products test procedure. The Agency recommends the use of a AOAC International Germicidal Spray Products as Disinfectants test (Ref. 3 ) modified for virucidal testing or the ASTM E1053

- 97 Virucidal Test Method (Ref. 5 ). One surface for each of two samples, representing two different batches of disinfectant, should be tested against a recoverable virus titer of at least 10 4 viable16

viral particles from the test surface for the exposure period specified on the label. I f the product is intended to be represented as virucidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum , should be included with the viral inoculum. When viral suspensions are grown in the presence of at least 5% serum, addition of serum to the inoculum is not expected as part of a study to support a one

- step label claim. (3 ) Single
- use towelettes test p rocedure. The Agency recommends the use of the modified AOAC Germicidal Spray Products as Disinfectants test. One surface for each of two samples, representing two different batches of disinfectant, should be tested against a recoverable virus titer of at least 10 4 viable viral particles from the test surface for a specified exposure period at room temperature. If the product is intended to be represented as virucidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the viral inoculum. When viral suspensions are grown in the presence of at least 5% serum, addition of serum to the inoculum is not expected as part of a study to support a one - step label claim

. In stead of spraying the inoculated surface of the glass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. The towelette should be removed from its container and subse quently handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. A lternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a maximum amount of its surface in the course of wiping a set of slides. Note: Adetailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency for review and approval prior to conducting the test. (4 ) Evaluation of virucidal success. Following treatment of the test virus with the disinfectant product, the presence of remaining viable virus should then be assayed using an appropriate virological technique (e.g., cy topathogenic effect, fluorescent antibody, plaque count, or animal response)

. The protocol for the viral assay should provide the information in paragraphs (f)(4)(i)through (f)(4)(ix) of this guideline

. (i) The virus recovery

(titer) should include a minimum of four determinations per each dilution in the assay

s ystem (e.g., tissue culture, embryonated egg, animal infection, etc.)

. (ii) Cy totox icity c ontrols. The effect of the disinfectant on the viral assay s ystem should include a minimum of four determinations per each dilution. (iii) The activity

o f the disinfectant against the test virus should include a minimum of four determinations per dilution in the assay s ystem. (iv) Neutralization controls. N eutralization controls should be performed (see reference (i )( 6)) and should include a minimum of four determinations per each dilution.17

(v) Any special methods which are used to increase the virus titer and to detoxify the residual disinfectant should be described. (vi) The ID50 values calculated for each assay should be provided. (vii) The test results should be reported as the reduction of the virus titer by the activity of the disinfectant (ID 50 of the virus control less the ID 50 of the test system) expressed as the logarithm to the base 10 and calculated by a statistical method (e.g., Reed and Munch, Most Probable Number)

. (viii) The product should demonstrate complete inactivation of the virus at all dilutions. If cy totoxicity is present, at least a 3

- log 10 reduction in viral titer should be demonstrated beyond the cy totoxic level. T able 1 provides an example of a ty pical laboratory report of a single test with one virus, assayed in a tissue culture s ystem. (ix) A ty pical laboratory report of a single test with one virus (recovered from a treated surface) involving a tissue culture assay s ystem would include the details of the methods employed and the information included in Tables 2

- 1, 2

- 2 and 2

- 3 :

Table 2 - 1: Test Results

Dilution of Virus Virus

- Disinfectant* Virus

- Control* Cytotoxic

- Control 10 - 1 T T T T + + + + T T T T 10 - 2 T T T T + + + + T T T T 10 - 3 T 0 0 0 + + + + T 0 0 0 10 - 4 0 0 0 0 + + + + 0 0 0 0 10 - 5 0 0 0 0 + + + + 0 0 0 0 10 - 6 0 0 0 0 + + + 0 0 0 0 0 10 - 7 0 0 0 0 + 0 0 0 0 0 0 0 10 - 8 0 0 0 0 0 0 0 0 0 0 0 0
No te: T = toxic; + = virus recovered; 0 = no virus recovered18

Table 2 -2: Calculation of the Tissue Culture Infective Dose 50 (TCID 50 )

Values Accumulated Values
Virus Dilution Inoculated No.

Infected / No

. Inoculated No.

Infected No. not

Infected No.

Infected No. not

Infected No.

Infected / No. Inoculated % Infected

10 - 1 4/4 4 0 24 0 24/24 100 10 - 2 4/4 4 0 20 0 20/20 100 10 - 3 4/4 4 0 16 0 16/16 100 10 - 4 4/4 4 0 12 0 12/12 100 10 - 5 4/4 4 0 8 0 8/8 100 10 - 6 3/4 3 1 4 1 4/5 80 10 - 7 1/4 1 3 1 4 1/5 20 10 - 8 0/4 0 4 0 8 0/8 0
TCID 50 = 10 6.5

Table 2 -3: Calculations of the Tissue Culture Lethal Dose 50 (TCLD 50 )

Values Accumulated Values Virus Dilution Inoculated No. Toxic /

No

. Inoculated No. Toxic No. not To xic No. Toxic No. not To xic No. Toxic /

No. Inoculated % Toxic

10 - 1 4/4 4 0 9 0 9/9 100 10 - 2 4/4 4 0 5 0 5/5 100 10 - 3 1/4 1 3 1 3 1/4 25 10 - 4 0/4 0 4 0 7 0/7 0 10 - 5 0/4 0 4 0 11 0/11 0 10 - 6 0/4 0 4 0 15 0/15 0 10 - 7 0/4 0 4 0 19 0/19 0 10 - 8 0/4 0 4 0 23 0/23 0 TCLD 50 = 10 2.7 Therefore : Virus inactivation = TCID 50 - TCLD 50 = 10 3.8 log 1019

(5) Bridging for disinfectant towelettes. In some cases, disinfectant towelette formulations are identical to registered liquid formulations. In order to bridge efficacy data from the EPA registered bulk liquid disinfectant used to saturate a towelette or other related product form, the studies in paragraphs (f)(5)(1) and (f )(5)(ii) of this guideline should be conducted and submitted to EPA for review

. (i) Chemical T esting

—Comparison of Expressed Liquid from the Towelette(s) to the EPA Registered Liquid Disinfectant Formulation to which it is being bridged: All active ingredients in the express ed liquid should be within the certified limits of the Confidential Statement of Formula of the liquid formula being referenced/bridged. The disinfectant towelettes package should be filled according to the manufacturing specifications. Excess liquid in the bulk towelette containers can not be poured off for use in the chemical testing for bridging of the efficacy data. The liquid used in the chemical testing should only be that expressed from the towelettes. Three batches (one of which is 60 days old) should be tested. Analy tical data for the active ingredients in the expressed liquid should be submitted for review. (ii) Efficacy Testing: Efficacy testing should be conducted under the same testing conditions (e.g. soil load, contact time, temperature) as used for the bulk liquid testing. This testing allows bridging of data from the registered bulk liquid used to saturate the towel for each ty pe of virus in paragraphs (f)(5)(ii)(A )(1) through (f)(5)(ii)(A) (3 ) of this guideline. Note: A detailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency f or review and approval prior to conducting the test. (A) Virucidal Test Procedure. The Agency recommends the use of either the AOAC International Germicidal Spray Products as Disinfectants (see reference (i )( 3) of this guideline) modified for virucidal towelette testing or the ASTM E1053 / current edition Virucidal Test Method (see reference (i) (5)) modified for virucidal towelette testing. (1 ) To support bridging of all viral claims, the most difficult to inactivate small

- sized non- enveloped virus, from the viral strains registered for the bulk liquid , should be selected for testing

. Examples of small

- sized non- enveloped viral families include members of the Picornaviridae famil y (e.g., poliovirus, enterovirus, hepatitis A virus, rhinovirus), and Parvoviridae family

(e.g., parvovirus). (2 ) To support bridging of viral claims for large

- sized non- enveloped and enveloped viral strains, the most difficult to inactivate large

- sized non- enveloped virus , from the viral strains registered for the bulk liquid , should b e selected for testing . Examples of large

- sized non

enveloped viral families include members of the Adenoviridae family ( e.g., adenovirus), Reoviridae family (e.g., rotavirus), and Papillomaviridae family (e.g., papillomavirus). (3 ) To support bridging of viral claims for enveloped viral strains, the most difficult to20

inactivate enveloped virus , from the viral strains registered for the bulk liquid , should be selected for testing

. Examples of enveloped viral families include members of the Coronaviridae famil y (e.g., coronavirus), Flaviviridae famil y (e.g., hepatitis C virus), Herpesviridae family ( e.g., herpesvirus), Poxviridae family ( e.g., vaccinia), Hepadnaviridae family ( e.g., hepatitis B virus), Orthomy xoviridae family

( e.g., In fluenza), Paramy xoviridae famil y (e.g., parainfluenza) and Retroviridae family

(e.g., human immunodeficiency virus). (B) Ten carriers for each of two samples, representing two batches of disinfectant, should be tested against a recoverable dried virus titer of at least 10 4 viral particles from the test surface for a specified exposure period at room temperature. Instead of spraying the inoculated surface of the glass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. One towelette should be used to wipe a minimum of 10 inoculated slides. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The protocol for the viral assay should provide the information in paragraphs (f)(5)(ii)(B) (1

) through (f)(5)(ii)(B) (7) of this guideline : (1 ) The virus recovery

(titer) should include a minimum of four determinations for e ach dilution in the assay

s ystem (e.g., cell culture, embryonated egg, animal infection)

. (2 ) Cy totoxicity c ontrols. The effect of the test substance on the viral assay s ystem should include a minimum of four determinations for each dilution. (3 ) The activity

o f the test substance against the test virus should include a minimum of four determinations for each dilution in the assay system. (4 ) Neutralization controls. N eutralization controls should be performed (see reference (i )( 6)) and should include a minimum of four determinations per each dilution. (5 ) Any special methods which are used to increase the virus titer and to detoxify the residual test substance should be described. (6 ) The LD 50 values calculated for each assay should be provided

. (7 ) The test results should be reported as the reduction of the virus titer by the activity

of the test substance (LD 50 of the virus control less the LD 50 of the test system) expressed as the logarithm to the base 10 and calculated by a statistical method (e.g., Reed and Munch, Most Probable Number)

. (C) Evaluation of virucidal success. The product should demonstrate complete inactivation of the virus at all dilutions. I f cy totoxicity i s present, at least a 3

- log reduction in viral titer should be demonstrated beyond the cy totoxic level recovered from the carrier surface.21

(g) Disinfectants with tuberculocidal claims

. This section addresses efficacy testing for broad

- spectrum or hospital disinfectant products which bear label claims of effectiveness as tuberculocides. In the Agency ’s “Data Call

- In Notice for Tuberculocidal Effectiveness for All Antimicrobial Pesticides with Tuberculocidal Claims ,” dated June 13, 1986 (Ref. 8) , applicants were given the option of choosing from one of three test methods (AOAC Tuberculocidal Activity of Disinfectants test, a modified AOAC Tuberculocidal Activity of Disinfectants test, or the Quantitative Tuberculocidal Activity T est) for conducting tuberculocidal efficacy tests. In general, the Agency

does not beli eve that a suspension test is appropriate for disinfectant formulations used on hard surfaces. An exception to this is for glutaraldehyde

-based products, which have never been validated in the AOAC Tuberculocidal Activity o f Disinfectants test (a carrier based test) . Therefore, the Quantitative Tuberculocidal Activity Test should only

be used for glutaraldehyde -based products. The Agency strongl y recommends all other formulations to use the carrier

-based AOAC Tuberculocidal Activity of Disinfectants test. (1) Water
- soluble powders and non -volatile liquid products test procedures

. The Agency recommends the test procedures in paragraphs (g)(1)(i) through (g)(1)(iv) of this guideline. (i) AOAC International Tuberculocidal Activity o f Disinfectants test. The AOAC International Tuberculocidal Activity o f Disinfectants test (see reference (i )(8 ) of this guideline) employing a 10 minute contact time and 20  ̊C temperature. Ten carriers for each of two samples representing two different batches of the product should be tested against Mycobacterium bovis (BCG)( M.bovis )

. If the product is intended to be represented as tuberculocidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the b acterial inoculum

. (ii) AOAC International Tuberculocidal Activity o f Disinfectants test with modifications. The AOAC International Tuberculocidal Activity of Disinfectants test with modifications to the 10 minute contact time and/or 20  ̊C temperature (see reference (i )( 7) of this guideline). Ten carriers for each of two samples representing two different batches of the product should be tested against M

. bovis (BCG). If the product is intended to be represented as tuberculocidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (iii) Evaluation of tuberculocide success. For the AOAC International Tuberculocidal Activity o f Disinfectants test, all organisms on all carriers should be killed, and there should be no growth in any of the inoculated subculture media. (iv) Validation testing for Quaternary Ammonium Compounds. Products formulated solely with quaternary

ammonium compounds as the active ingredient(s) should be supported with validation testing to confirm their tuberculocidal label claim. O ne additional product sample should be tested in a different laboratory f rom the original one, using the same test22

procedure and condition s as used in the first laboratory test. (2) Glutaraldehyde formulations (i) Test Procedure. For glutaraldehyde formulations, the Agenc y recommends the Quantitative Tuberculocidal Activity T est. This test has been published in the Agency ’s “Data Call

- In Notice for Tuberculocidal Effectiveness for All Antimicrobial Pesticides with Tuberculocidal Claims”, dated June 13, 1986 (Ref. 8 ). Two samples, representing two different batches of the product should each be uti lized in at least four separate studies (a total of at least eight studies), against M. bovis, so that upper 95 percent confidence limits can be determined for each point on the survival curve. If the product is intended to be represented as tuberculocidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (ii) Evaluation of tuberculocide success. For the Quantitative Tuberculocidal Activity Method, survival curves should be constructed from the average of four separate replicates so that the upper 95% Confidence Limit can be determined for each point on the curve. The minimum time claimed for efficacy i s determined by

f inding the point where the average survival curve intersects the probability o f one survivor. If the data show a four

- log reduction, but the survivor curve does not intersect the one - survivor line, the minimal time is found by extrapolating the upper 95% confidence limit curve such that the value where it intersects the one sur vivor line is not 50% greater that when the survivor curve intersects the one survivor line. (3) Germicidal spray products and volatile liquid products

—(i) Test procedure. The Agency recommends the AOAC International Germicidal Spray Products as Disinfectants test (Ref. 3 ), using the media, test culture, and other elements described in the AOAC International Tuberculocidal Activity

o f Disinfectants test. Ten carriers for each of two samples representing two different batches of the product should be tested against M

. bovis (BCG). If the product is intended to be represented as tuberculocidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. (ii) Evaluation of tuberculocide success. When using the AOAC International Germicidal Spray

Products as Disinfectants test, all organisms on all carriers/slides should be killed, and there should be no growth in any of the inoculated subculture media. (4) Single
-Use Towelettes (i) Test Procedure. The Agency recommends the use of a modified AOAC Germicidal Spray Products as Disinfectants test. T en carriers for each of two samples representing two different batches of the product should be evaluated against M

. bovis (BCG). If the product is intended to be represented as tuberculocidal in the presence of organic soil (one

- step) , an appropriate organic soil, such as 5 percent blood serum, should be included with the bacterial inoculum. Instead of spraying the inoculated surface of the gl ass slide, the product should be tested by wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. The towelette should be removed from its container23

and subse quently

handled with sterile gloves. One towelette should be used to wipe a minimum of 10 inoculated slides. Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. The area of the towelette used for wiping should be rotated so as to expose a maximum amount of its surface in the course of wiping a set of slides

. Note: Adetailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency for review and approval prior to conducting the test. (ii) Evaluation of tuberculocidal towelette success. All organisms on all carriers/slides should be killed, and there should be no growth in any of the inoculated subculture media. (5) Bridging for disinfectant towelettes. In some cases, disinfectant towelette formulations are identic al to registered liquid formulations. In order to bridge efficacy data from the EPA registered bulk liquid disinfectant used to saturate a towelette or other related product form, the studies in paragraphs (g)(5)(i) and (g)(5)(ii) of this guideline should be conducted and submitted to EPA for review

. (i) Chemical Testing
- Comparison of Expressed Liquid from the Towelette(s) to the EPA Registered Liquid Disinfectant Formulation to which it is being bridged: All active ingredients in the expressed liquid should be within the certified limits of the Confidential Statement of Formula of the liquid formula being referenced/bridged. The disinfectant towelettes package should be filled according to the manufacturing specifications. Excess liquid in the bulk towelette containers can not be poured off for use in the chemical testing for bridging of the efficacy data. The liquid used in the chemical testing should only be that expressed from the towelettes. Three batches (one of which is 60 days old) should be tested. Analy tical data for the active ingredients in the expressed liquid should be submitted for review. (ii) Efficacy Testing: Efficacy testing should be conducted under the same testing conditions (e.g. soil load, contact time, temperature) as used for the bulk liquid testing. This testing allows bridging of data from the registered bulk liquid used to saturate the towel for each ty pe of organism in paragraph (g)(5)(ii)(A) of this guideline

. Note: A detailed description of the wiping procedure, including the towelette folding and rotation process should be included in the test protocol and documented in the raw data and final report. The applicant should submit their towelette protocol to the Agency for review and approval prior to conducting the test

. (A) Test Procedure. The Agency recommends the use of the AOAC Germicidal Spray Products as Disinfectants test, modified for towelette testing. The test should be modified to conform to appropriate elements (e.g., media, growth conditions, etc) in the AOAC International Tuberculocidal Activity

o f Disinfectants test. Ten carriers for each of two samples, representing two batches of the product , should be tested against M

. bovis BCG

. Instead of spraying the inoculated surface of the glass slide, the prod uct should be tested by

wiping the surface of the glass slide with the saturated towelette, and then subculturing the slides after the specified holding time. One towelette should be used to wipe a minimum of 10 inoculated slides.24

Alternatively, one carrier with a surface area equivalent to ten 1 x 1 inch carriers can be wiped using one towelette per carrier set per batch. (B ) Evaluation of tuberculocidal towelette success. All organisms on all carriers/slides should be killed, and there should be no growth in any of the inoculated subculture media

. (C) This testing is intended to support bridging of all mycobacteria listed on the EPA registered liquid disinfectant used to saturate the towel to the EPA registered towelette product

. (h) 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 information in paragraphs (h)(1)(i) through (h)(i)(xiv) of this guideline : (i) Study title; (ii) Product i dentity ; (iii) Guideline number; (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) 40 CFR Part 160 Good Laboratory Practice compliance; (viii) Purpose of the study; (ix) Date and time of the start and end of the test; (x) Statistical treatment of the data; (xi) Conclusions; (xii) References; (xiii) Appendices ; (xiv) Certification The applicant is encouraged to use the EPA’s standard efficacy report format, which may25

be found at http://www.epa.gov/oppad001/efficacystudystandards.htm

. (2) Data for recommended methods. When recommended methods from the Official Methods of Analysis of AOAC International; the Annual Book of Standards of the American Society f or Testing and Materials; or, EPA methods are used to develop efficacy data, certain minimal information, in addition to that described in this guideline, should be provided in the test report. The report should include, at the least, the information in paragraphs (h)(2)(i) through (h)(2)(xii) of thi s guideline : (i) Test employed, and any s ignificant modifications thereto (e.g., organic load, hard water); (ii) Test microorganisms employed, including identification of the specific strain (ATCC or other); (iii) Description of the test substance, including the percent of active ingredient; (iv) Concentration or dilution of the product tested and how prepared; (v) Number of samples, batches and replicates tested; (vi) Preparation dates of each product batch (individually f ormulated preparation of the product); (vii) Identification of all material or procedural options employed, where such choice is permitted or recommended in the test method selected (e.g., growth media, drying time for inoculated carriers, neutralization confirmation and/or subculture media, secondary subculturing); (viii) Test exposure conditions (e.g., contact time, temperature, and relative humidity ); (ix) Complete reports of results obtained for each replication; (x) Any control data essential to establish the validity of the test; (xi) Carrier counts; (xii) Any additional data pertinent for specific tests described in this guideline. (3) Data for modifications of recommended methods . When recommended methods are modified to support specific claims and/or use patterns for a product, the protocol, identifying and describing each modification, should be provided with the study report. The applicant should submit the proposed modification to the Agency f or review and evaluation prior to initiation of the test. (4) Data for oth er methods . When recommended methods, or modifications thereto, are26

not employed to develop efficacy data (such as actual in

-use or many kinds of simulated

-use testing), complete testing protocols should be submitted with the test reports. All materials and procedures employed in testing should be described in a manner consistent with original research reports published in technical or scientific journals. Where references to published reports or papers are made, copies or reprints of such references should be provided with the test reports. The applicant should submit the proposed testing protocols for in

-use or simulated

-use studies (with a proposed label to show the claims to be supported by the protocol) to the Agency f or review and evaluation prior to initiation of the test. (i) References. The references in this paragraph may b e consulted for additional background information : (1) Official Methods of Analysis of the AOAC International, Chapter 6, Disinfectants, Use

- Dilution Methods (955.14, 955. 15, & 964.02) , Eighteenth edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417. (2) Official Methods of Analysis of the AOAC International, Chapter 6, Disinfectants, Hard Surface Carrier Test Methods, Eighteenth edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417. (3) Official Methods of Analysis of the AOAC International, Chapter 6, Disinfectants, Official Method 961.02 Germicidal Spray Products as Disinfectants, Eighteenth edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417. (4) Official Methods of Analysis of the AOAC International, Chapter 6, Disinfectants, Official Method 955.17 Fungicidal Activity o f Disinfectant s. Eighteenth edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417. (5) Annual Book of ASTM Standards , Test Method for Efficacy o f Virucidal Agents Intended for In animate Environmental Surfaces, Designation E10 53

- 97. American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition. (6) Annual Book of ASTM Standards , Standard Test Method for Neutralization of Virucidal Agents in Virucidal Efficacy Evaluations, Desi gnation E1483

- 04. American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428, current edition. (7) Official Methods of Analysis of the AOAC International, Chapter 6, Disinfectants, Official Method 965.12 Tuberculocidal Activity of Dis infectants. Eighteenth edition. AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877

- 2417. (8) Environmental Protection Agency, Data Call

- in Notice for Tuberculocidal Effectiveness Data for All Antimicrobial Pesticides with Tuberculocidal Claims (Registration Division, Office of Pesticide Pro grams, June 13, 1986).