United States Office of Chemical Safety and EPA 712-C-17-003

Environmental Protection Pollution Prevention February 2018

Agency (7510P)

Product Performance Test

Guidelines
OCSPP 810.2100:
Sterilants, Sporicides, and
Decontaminants
Guidance for Efficacy Testing
 NOTICE

This guideline is one of a series of test guidelines established by the United States Environmental
Protection Agency’s 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 its 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 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, see
EPA’s Test Guidelines for Pesticides and Toxic Substances site at https://www.epa.gov/test-guidelines-
pesticides-and-toxic-substances. You may also access the guidelines at http://www.regulations.gov and
searching by Docket ID #: EPA-HQ-OPP-2015-0276.

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 Table of Contents

(A) Scope ................................................................................................................................................... 1

(1) Applicability. ............................................................................................................................. 1
(2) Background. .............................................................................................................................. 1
(B) Purpose ................................................................................................................................................ 1
(C) General Considerations ....................................................................................................................... 1
(1) Claims........................................................................................................................................ 1
(2) Soil Load, Water Hardness, Lower Certified Limit (LCL), and Neutralization
Confirmation. ............................................................................................................................ 1
(3) Confirmatory Data. .................................................................................................................... 1
(4) Verification Tests. ..................................................................................................................... 1
(5) Suture Loops. ............................................................................................................................ 2
(6) Alternate Materials. ................................................................................................................... 2
(7) Entire Room or Large Chamber Application. ........................................................................... 2
(8) Repeat Testing. .......................................................................................................................... 2
(D) Sterilant Claim .................................................................................................................................... 4
(1) Liquid Products (Ready to Use, Dilutable Concentrates, and Water Soluble Powder
Formulations), Gases, and Vapors............................................................................................. 4
(2) Sprays, Mists, and Foams. ......................................................................................................... 5
(3) Gas and Vapor with Use of a Device. ....................................................................................... 5
(4) Confirmatory Testing. ............................................................................................................... 6
(E) Sporicide Claim................................................................................................................................... 6
(1) Liquid Products (Ready to Use, Dilutable Concentrates, and Water Soluble Powder
Formulations), Gases, and Vapors............................................................................................. 6
(2) Sprays, Mists, and Foams. ......................................................................................................... 7
(3) Gas and Vapor with Use of a Device. ....................................................................................... 7
(4) Confirmatory Testing. ............................................................................................................... 7
(F) Additional Spore Claims ..................................................................................................................... 7
(1) Liquids (Ready to Use, Dilutable Concentrates, and Water Soluble Powder
Formulations), Gases, and Vapors............................................................................................. 7
(G) Bacillus anthracis (B. anthracis) Decontaminant Claim .................................................................... 8
(1) Liquids (Ready to Use, Dilutable Concentrates, and Water Soluble Powder
Formulations), Gases, and Vapors............................................................................................. 8
(H) Commercial Sterilant for Aseptic Packaging of Low Acid Food Claim ............................................. 9
(I) Hospital or Healthcare Disinfectant with Sporicidal Activity against Clostridium difficile
Claim ................................................................................................................................................. 10
(J) Simulated Use Testing for Gas and Vapor Products ......................................................................... 11
(K) Data Collection and Reporting .......................................................................................................... 12
(L) References ......................................................................................................................................... 12

List of Tables

Table 1. Summary of Testing for Spore Claims* ......................................................................................... 2

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OCSPP 810.2100: Sterilants, Sporicides, and Decontaminants - Guidance for Efficacy
Testing

(A) Scope

(1) Applicability. This guideline describes test methods that EPA believes will generally
satisfy testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) (7 U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA)
(21 U.S.C. 346a). It addresses testing to demonstrate the effectiveness of antimicrobial
pesticides bearing claims for use as a sterilant, sporicide, Bacillus anthracis (B. anthracis)
decontaminant, commercial sterilant for aseptic packaging of low acid food, and/or
disinfectant against Clostridium difficile (C. difficile) spores. Refer to OCSPP guideline
810.2000: General Considerations for Testing Public Health Antimicrobial Pesticides for
additional guidance.

(2) Background. This document provides an update to the 2012 OCSPP 810.2100 –
“Sterilants – Efficacy Data Recommendations.”

(B) Purpose

This guideline addresses efficacy testing for antimicrobial pesticides intended to be used on
inanimate, environmental, and food packaging surfaces, which bear label claims for use as a
sterilant, sporicide, decontaminant against Bacillus anthracis (B. anthracis) spores, commercial
sterilant for aseptic packaging of low acid food, and/or disinfectant against Clostridium difficile
(C. difficile) spores.

(C) General Considerations

(1) Claims. This guideline provides guidance for developing data to support a sterilant,
sporicide, B. anthracis decontaminant, commercial sterilant for aseptic packaging of low
acid food, and/or additional spore claims. Furthermore, guidance is provided for
applicants seeking only a disinfectant with sporicidal activity against C. difficile claim.

(2) Soil Load, Water Hardness, Lower Certified Limit (LCL), and Neutralization
Confirmation. Refer to OCSPP guideline 810.2000: General Considerations for Testing
Public Health Antimicrobial Pesticides.

(3) Confirmatory Data. Refer to OCSPP guideline 810.2000: General Considerations for
Testing Public Health Antimicrobial Pesticides for information on confirmatory testing.
Specific information related to confirmatory testing is provided in the description for each
efficacy claim.

(4) Verification Tests. Data submitted to support sterilant, decontaminant, and/or sporicide
claims are subject to independent verification testing. Verification testing can be done in
a second laboratory or in the same laboratory with a new set of inoculated carriers,
separate study director, technical staff, and Quality Assurance auditor.

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 (5) Suture Loops. Where this guidance specifies testing with suture loops, silk suture loops
should be used, except when testing products with oxidative chemistries. Dacron loops
(also termed braided polyester) should be used instead of silk suture loops, for testing
products with oxidative chemistries such as peracetic acid, peroxide, chlorine dioxide, or
sodium hypochlorite containing products.

(6) Alternate Materials. When an antimicrobial product is intended to be effective in

treating a type of material not specified in a standard method, a method modification may
be necessary, which specifies the use of that material or an acceptable alternative material
as the carrier in the test protocol. In addition, control carrier count data (log10 spores per
carrier), neutralization confirmation data, and sterility controls should be developed to
assure the validity of the test results when alternative carriers are utilized. Method
modifications along with justification supporting such requests should be submitted to
the agency in writing for approval prior to data generation and collection.

(7) Entire Room or Large Chamber Application. A simulated use test is needed when an
antimicrobial product is intended for treating an entire room or large chamber. For general
guidance, see section (J)(2) of this document. A testing protocol specific to the product
should be developed and submitted to the agency for approval prior to data generation
and collection.

(8) Repeat Testing. Refer to OCSPP guideline 810.2000: General Considerations for
Testing Public Health Antimicrobial Pesticides for guidance on repeat testing of products.

Table 1. Summary of Testing for Spore Claims*

Test No. Batches/
Claim Formulation Test Method Organisms Carriers
AOAC Sporicidal Activity For each organism:
Liquid, water
(966.04). Ref.1 3 batches at the LCL;
soluble
(Method I – silk loops and 60 carriers per batch,
powders, gases Bacillus subtilis
Method II – penicylinders) per carrier type -
(ATCC 19659)
Sprays, mists Agency Consultation and Porcelain penicylinder
and
Sterilant and foams Protocol Review and silk suture loops
Clostridium
Verification testing
In addition to laboratory sporogenes
for each organism:
Gas/Vapor with studies, see section (J). (ATCC 3584)
1 batch at the LCL
use of device Agency Consultation and
30 carriers per batch
Protocol Review
and per carrier type
AOAC Sporicidal Activity
Liquid, water
(966.04). Ref.1
soluble
(Method I – silk loops and Bacillus subtilis
powders, gases
Sporicide: Method II – penicylinders) (ATCC 19659) For each organism:
Indicate surface type Sprays, mists Agency Consultation and and 3 batches at the LCL;
(e.g. hard non- and foams Protocol Review Clostridium 60 carriers per batch
porous, hard porous) In addition to laboratory sporogenes and one type of carrier
Gas/Vapor with studies see section (J). (ATCC 3584)
use of device Agency Consultation and
Protocol Review

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 Test No. Batches/
Claim Formulation Test Method Organisms Carriers
AOAC Sporicidal Activity
(966.04) Ref.1 (Method I –
Liquid, water silk loops and Method II –
soluble penicylinders) or the specific
Additional Spores
powders, gases quantitative method cited for
(e.g. Bacillus Spores claimed For each organism:
the test organism (e.g.
anthracis, Bacillus on the label in 2 batches at the LCL;
C. difficile, B. anthracis).
cereus, Bacillus addition to the 30 carriers per batch
Spray, mists, Agency Consultation
stearothermophilus, base claims and carrier type
foams and Protocol Review
Clostridium difficile)
In addition to laboratory
Gas/Vapor with studies see section (J).
use of device Agency Consultation and
Protocol Review

Qualitative option - AOAC

Sporicidal Activity 966.04
(Method II) Ref.1 modified for
Bacillus anthracis
Gases and vapors – In addition
to laboratory studies see
section (J).
Agency Consultation and
Protocol Review
Quantitative option - AOAC
2008.05 Efficacy of Liquid
Liquid, water Sporicides Against Spores of
Bacillus anthracis soluble Bacillus subtilis on Hard
Decontaminant powders, gases, Nonporous and Porous
and vapors Surfaces (Quantitative Three
Step Method) Ref. 2 or ASTM
E2197 Standard Quantitative
Disk Carrier Test Method
(QCT II) Ref. 3- with
modifications for Bacillus
anthracis
Gases and vapors – In addition
to laboratory studies, see
section (J).
Agency Consultation and
Protocol Review
3 batches at the LCL;
60 carriers per batch,
per carrier types—
Porcelain penicylinder
and silk suture loops
Bacillus
anthracis-
virulent strain or 3 batches at the LCL;
acceptable The number of
surrogate carriers will vary
species depending on the test
method. The coupon
material(s) should be
representative of those
found at the site(s)
that appear on the
product’s labeling,
and be acceptable to
EPA

Modification of AOAC Hydrogen For each organism:

Sporicidal Activity (966.04) peroxide based 3 batches at the LCL;
Commercial Sterilant Water-soluble Ref.1 sterilants: 60 carriers per batch –
for Aseptic powders and (Method I – silk loops and Bacillus subtilis The carrier material(s)
Packaging of Low non-volatile Method II – penicylinders) (ATCC 19659), should be
Acid Food liquid Clostridium representative of
Exposure time: ≤ 30 seconds sporogenes equipment and
Exposure Temp: 60-80˚C (ATCC 3584) packaging material

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 Test No. Batches/
Claim Formulation Test Method Organisms Carriers
Peroxyacid surface(s) that appear
Agency Consultation and based sterilants: on the product’s
Protocol Review Bacillus subtilis labeling, and be
(ATCC 19659), acceptable to EPA
Clostridium
sporogenes
(ATCC 3584),
Bacillus cereus
(ATCC 14579)
Other classes-
consult
EPA/FDA.

Refer to Methods and

Liquid, water Guidance for Testing the
soluble Efficacy of Antimicrobial
powders, and Products against Spores of
sprays Clostridium difficile on hard
Non-Porous Surfaces.
Test according to Methods and
Clostridium 3 batches at the LCL;
Guidance for Testing the
Hospital/Healthcare difficile (ATCC 10 test carriers per
Efficacy of Antimicrobial
Disinfectant with 43598) batch
Products against Spores of
Sporicidal Activity
Clostridium difficile on hard
against Clostridium Towelettes
Non-Porous Surfaces, using
difficile
liquid expressed directly from
the towelette and conduct a
wetness determination test
(see section (I)(1)(i)).
Gas, vapor,
fogger, and
Agency Consultation and Protocol Review
foam
formulations
*Note: Table 1 does not include verification or confirmatory testing. For guidance on conducting verification and
confirmatory testing, see the respective section for each claim.

(D) Sterilant Claim

This section provides testing guidance for products with sterilant claims to inactivate spores of
Bacillus subtilis and Clostridium sporogenes on both hard (testing carrier: porcelain penicylinders)
and soft (testing carriers: silk suture loops) inanimate surfaces.

(1) Liquid Products (Ready to Use, Dilutable Concentrates, and Water Soluble Powder
Formulations), Gases, and Vapors.

(i) Test Method. The agency recommends use of the AOAC International (AOAC)
Official Method 966.04 Sporicidal Activity of Disinfectants test (Method I for silk
loops; Method II for penicylinders, revised 2013, see Ref. 1). To demonstrate
efficacy of a product, testing against spores of Bacillus subtilis [American Type
Culture Collection (ATCC) 19659] and Clostridium sporogenes (ATCC 3584),
each inoculated on two types of carriers (porcelain penicylinders and silk suture
loops), should be conducted. Furthermore, neutralizer confirmation should be

4
 demonstrated using AOAC 966.04, method II (section C, subsection h) using
growth media and incubation conditions suitable for each microbe.

(ii) Batches. Three batches of the product at the LCL of the active ingredient(s) (A.I.)
listed on the confidential statement of formula (CSF) for the product should be
tested.

(iii) Number of Carriers and Related Requirements. Sixty carriers of each type
should be tested against spores of both B. subtilis and C. sporogenes (240 carriers
per batch, or a total of 720 carriers for all three batches). A mean control count of
1 × 105 to 1 × 106 spores per carrier should be attained for both microbes on both
carrier types. The inoculated carriers should meet the level of acid resistance
outlined in the AOAC 966.04 method.

(iv) Spore Production. Use AOAC 966.04 (Method I) for producing spores of
C. sporogenes in cooked meat medium amended with MnSO4 for spore generation
and inoculation of penicylinders and silk suture loops. Follow AOAC 966.04
(Method II) for producing spores of B. subtilis (nutrient agar amended with
MnSO4).

(v) Evaluation of Success. The product should kill all of the test spores on all of the
720 carriers with no positives (i.e., no growth of test organism in the subculture
medium).

(vi) Verification Testing. For verification tests, follow the above testing guidance
(D)(1)(i)-(iv), except that one batch of the product at the LCL of the A.I.(s) should
be tested against the spores of both B. subtilis and C. sporogenes on thirty carriers
representing each of the two surfaces types (porcelain penicylinders and silk suture
loops). For verification testing, the product should kill all of the test spores on all
120 carriers with no positives (i.e., no growth of test organism in the subculture
medium).

(2) Sprays, Mists, and Foams. A liquid product applied as a spray using a pump or trigger
sprayer should be tested as a liquid (see section (D)(1) above). For all other spray
formulations types (aerosols) and other formulations such as mists, foams and gels, the
applicant should submit a testing protocol consistent with the anticipated application and
use pattern of the product for agency review and approval prior to testing. Use AOAC
method 966.04 for spore production, control counts, and neutralization testing.

(3) Gas and Vapor with Use of a Device. Any sporicide which is a vapor or gas and is
recommended for use in a specific device should be tested using the AOAC Method
966.04 modified for the application. Treat the prepared carriers using the device
according to the instruction manual of that device. To address this application,
modifications to the AOAC Method 966.04 may be necessary. Method modifications
along with justification supporting such requests should be submitted to the agency for
review and approval prior to conducting the tests.

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 (4) Confirmatory Testing. For confirmatory testing, follow the above testing guidance in
sections (D)(1)-(3), with the exception that two different batches of product (at the LCL
of the A.I.(s)) should be tested against B. subtilis and C. sporogenes, on 30 carriers of
each surface type. For all methods, the product should kill all of the test microorganisms
on all of the carriers (i.e., no growth of test organism in the subculture medium).

(E) Sporicide Claim

This section provides testing guidance for products with surface-specific sporicide claims to
inactivate spores of Bacillus subtilis and Clostridium sporogenes on that specific inanimate
surface.

(1) Liquid Products (Ready to Use, Dilutable Concentrates, and Water Soluble Powder
Formulations), Gases, and Vapors.

(i) Test Method. The agency recommends use of the AOAC International (AOAC)
Official Method 966.04 Sporicidal Activity of Disinfectants test (Method I for silk
loops; Method II for penicylinders, revised 2013, see Ref. 1). To demonstrate
efficacy of a product, testing against spores of Bacillus subtilis (ATCC 19659) and
Clostridium sporogenes (ATCC 3584), each inoculated on one type of carrier
(stainless steel penicylinders, porcelain penicylinders, or silk suture loops), should
be conducted. The same carrier type should be used for both microbes.
Furthermore, neutralizer confirmation should be demonstrated using AOAC
966.04, method II (section C, subsection h) using growth media and incubation
conditions suitable for each microbe.

(ii) Batches. Three batches of the product at the LCL of the A.I.(s) listed on the CSF
for the product should be tested.

(iii) Number of Carriers and Related Requirements. Sixty carriers of each type
should be tested against spores of both B. subtilis and C. sporogenes (120 carriers
per batch, or a total of 360 carriers for all three batches). A mean control count of
1 × 105 to 1 × 106 spores per carrier should be attained. The inoculated carriers
should meet the level of acid resistance outlined in the AOAC 966.04 method.

(iv) Spore Production. Use AOAC 966.04 (Method I) for producing spores of
C. sporogenes in cooked meat medium amended with MnSO4 for spore generation
and inoculation of carriers. Follow AOAC 966.04 (Method II) for producing spores
of B. subtilis (nutrient agar amended with MnSO4).

(v) Evaluation of Success. The product should kill all of the test spores on all of the
360 carriers with no positives (i.e., no growth of test organism in the subculture
medium).

(vi) Verification Testing. For verification tests, follow the above testing guidance
(E)(1)(i)-(iv), except that one batch of the product at the LCL of the A.I.(s) should
be tested against the spores of both B. subtilis and C. sporogenes on thirty carriers
for each type of surface for which a claim is desired. For verification testing, the

6
 product should kill all of the test spores on all 60 carriers with no positives (i.e., no
growth of test organism in the subculture medium).

(2) Sprays, Mists, and Foams. A liquid product applied as a spray using a pump or trigger
sprayer should be tested as a liquid as described in section (E)(1). For all other spray
formulations types (aerosols) and other formulations such as mists, foams and gels, the
applicant should submit a testing protocol consistent with the anticipated application and
use pattern of the product for agency review and approval prior to testing. Use AOAC
method 966.04 for spore production, control counts, and neutralization testing.

(3) Gas and Vapor with Use of a Device. Any sporicide which is a vapor or gas and is
recommended for use in a specific device should be tested using the AOAC Method
966.04 modified for the application. Treat the prepared carriers using the device
according to the instruction manual of that device. To address this application,
modifications to the AOAC Method 966.04 may be necessary. Method modifications
along with justification supporting such requests should be submitted to the agency for
review and approval prior to conducting the tests.

(4) Confirmatory Testing. For confirmatory testing, follow the above testing guidance in
sections (E)(1)-(3), with the exception that two different batches of product (at the LCL
of the A.I.(s)) should be tested against B. subtilis and C. sporogenes, on 30 carriers. For
all methods, the product should kill all of all the test microorganisms on all of carriers
(i.e., no growth of test organism in the subculture medium).

(F) Additional Spore Claims

This section addresses efficacy tests for all sterilant or sporicide products with claims to inactivate
additional spores on inanimate surfaces. The agency recommends the following:

(1) Liquids (Ready to Use, Dilutable Concentrates, and Water Soluble Powder
Formulations), Gases, and Vapors.

(i) Test Method. The agency recommends use of the AOAC Method 966.04 (Method
I for silk loops; Method II for penicylinders, revised 2013, see Ref. 1) to conduct a
test using the proposed spores (or a surrogate species acceptable to EPA) inoculated
on thirty carriers representing the appropriate type(s) of surface(s) (stainless steel
penicylinders, porcelain penicylinders, and/or silk suture loops) against two
different batches of the product at the LCL of the A.I.(s) (60 carriers per surface
type). Control carrier counts should be 1 x 105 to 1 x 106 spores per carrier.

(a) Evaluation of Success. The product should kill all of the test spores on all of
the carriers with no positives (i.e., no growth of test organism in the subculture
medium).

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(G) Bacillus anthracis (B. anthracis) Decontaminant Claim

This section addresses efficacy tests for a product with claims to inactivate B. anthracis spores on
inanimate surfaces. The agency recommends two possible approaches as described in paragraphs
(G)(1)(i) through (G)(1)(ii) of this guideline.

(1) Liquids (Ready to Use, Dilutable Concentrates, and Water Soluble Powder
Formulations), Gases, and Vapors.

(i) Test Method – Qualitative Option. The agency recommends use of the AOAC
International Official Method 966.04 Sporicidal Activity of Disinfectants test
(Method I for silk loops; Method II for penicylinders, revised 2013, see Ref. 1) to
conduct a test using virulent B. anthracis spores (or a surrogate species acceptable
to EPA) inoculated on sixty carriers for each surface type (porcelain penicylinders
and silk suture loops). Three different batches of the product should be tested at the
LCL of the A.I.(s) (a total of 360 carriers). Control carrier counts should be 1 x 105
to 1 x 106 spores per carrier.

(a) Evaluation of Success. The product should kill all of the test spores on all of
the carriers (360 carriers) without any positives (i.e., no growth of test
organism in the subculture medium).

(b) Verification Testing. For verification tests, follow the above testing guidance
(G)(1)(i), except 30 carriers representing each of the two types of surfaces
(porcelain penicylinders and silk suture loops) should be tested against the
spores of B. anthracis (or acceptable surrogate) on two samples of the product
at the LCL of the A.I.(s), representing two different batches of the product. For
verification testing, the product should kill all of the test spores on all of the
carriers with no positives (i.e., no growth of test organism in the subculture
medium).

(c) Confirmatory Testing. For confirmatory testing, follow the testing guidance
in section (G)(1)(i), with the exception that thirty carriers for each of two
different batches of product (at the LCL of the A.I.(s)) should be tested against
B. anthracis (or acceptable surrogate). The product should kill all of the test
microorganisms on all of the carriers.

(ii) Test Method - Quantitative Option. The agency recommends the use of a well-
developed, quantitative sporicidal test method acceptable to EPA such as AOAC
Method 2008.05 or ASTM method E2197-11 using virulent B. anthracis spores (or
a surrogate acceptable to EPA) on porous and/or nonporous coupons acceptable to
EPA (agency consultation is recommended). The inoculum employed should
provide a mean target count of 1 × 107 to 1 × 108 spores per carrier. Three different
batches of product should be tested at the LCL of the A.I.(s). The number of carriers
should be a minimum of three for AOAC Method 2008.05 and a minimum of five
for ASTM method E2197-11. The coupon material(s) should be representative of

8
 those found at the site(s) that appear on the product’s labeling, and be acceptable to
EPA.

(a) Evaluation of Success. Each product batch should achieve a mean log10
reduction of ≥6 based on the number of viable spores recovered.

(b) Verification Testing. For verification tests, follow the above testing guidance
in section (G)(1)(ii), except that the product should be tested against the spores
of B. anthracis (or acceptable surrogate) on two samples of the product at the
LCL of the A.I.(s), representing two different batches of the product.

(c) Confirmatory Testing. For confirmatory testing, follow the above testing
guidance in section (G)(1)(ii), with the exception that two different batches of
product (at the LCL of the A.I.(s)) should be tested against B. anthracis (or
acceptable surrogate). Each product batch should achieve a mean log10
reduction of ≥6 based on the number of viable spores recovered.

(H) Commercial Sterilant for Aseptic Packaging of Low Acid Food Claim

This section addresses efficacy tests for water-soluble powders and non-volatile liquid products
with claims to inactivate spores on non-porous, inanimate surfaces of equipment and materials
used for aseptic processing and packaging of low-acid food.

(1) Test Method. The agency recommends modifications of the AOAC International Official
Method 966.04 (Method II for penicylinders, revised 2013, see Ref. 1) as described above
to demonstrate the efficacy of commercial sterilants for aseptic packaging of low acid
foods. The carriers used in testing should represent the equipment and packaging material
surface. For example, stainless steel penicylinders may be used to represent hard, non-
porous packaging surfaces. Sixty (60) carriers should be tested against spores on three
samples representing three different batches of the product (i.e., 180 carriers per
organism). The inoculum employed should provide a count of 1 × 105 to 1 x 106 viable
spores per carrier. Products should demonstrate effectiveness in 30 seconds or less at
temperatures between 60-80˚C. If a longer contact time or a different temperature is
required for the product, the applicant should provide justification supporting such
requests to the agency for approval prior to testing. To address this use, modifications to
the AOAC Method 966.04 should be submitted to the agency for review and approval
prior to conducting the tests. The type of spore former to be used is dependent on the type
of chemical sterilant as follows:

(i) Hydrogen peroxide based sterilants should be tested against B. subtilis (ATCC
19659), C. sporogenes (ATCC 3584).

(ii) Peroxyacid based sterilants should be tested against B. subtilis (ATCC 19659),
C. sporogenes (ATCC 3584) and B. cereus (ATCC 14579). B. cereus has been
shown to be more resistant to peroxyacid based biocides than B. subtilis (ref. 6).

(iii) For sterilant classes other than those listed above, consultation with EPA and the
Food and Drug Administration (FDA) is recommended prior to generation of

9
 product data to identify the organisms for supporting aseptic packaging of low acid
food label claims.

(iv) Evaluation of Sterilant Success. The product should kill all of the test spores on
all of the carriers without any failures.

(2) Confirmatory Testing. For confirmatory testing, follow the above testing guidance in
section (H)(1), with the exception that thirty carriers for each of two different batches of
product (at the LCL of the A.I.(s)) should be tested against each microorganism. For all
methods, the product should kill all of the test microorganisms on all of the carriers.

(I) Hospital or Healthcare Disinfectant with Sporicidal Activity against Clostridium difficile
Claim

This section addresses efficacy tests for a hospital or healthcare disinfectant product with claims
to inactivate Clostridium difficile spores on hard, nonporous, inanimate surfaces.

(1) Test Method. Refer to the current “Guidance for Efficacy Evaluation of Products with
Sporicidal Claims Against Clostridium difficile.” For towelette products, in addition to
the efficacy results, data should be generated to assess the amount of liquid remaining on
a hard nonporous surface treated (wiped) per the label instructions. The agency
recommends conducting visual and gravimetric wetness tests in an environmental
chamber.

(i) Gravimetric and Physical Wetness Determination. For each batch, use three pre-
cleaned 150 × 20 mm glass Petri plates to represent the surface to be treated. Prior
to use, pre-clean each carrier surface with 70% ethanol, rinse in deionized water
and allow to air dry. Place each untreated carrier on a balance and record the weight
(weight one – dry and untreated). Distribute the liquid in the canister or package by
gentle rotation. Remove the first 3-5 towelettes from the container and discard. For
each carrier, follow the label instructions and remove one towelette from the
container, unfold if necessary, and wipe the carrier surface in a circular fashion
without lifting the towelette – treat up to the edge of the carrier. Carefully place the
treated carrier on the balance are re-weigh, record the results (weight two – wet and
treated). Allow carriers to sit horizontally for the contact time in an environmental
chamber set at 35±5% relative humidity and 20-25°C. Do not use a fume hood or
biological safety cabinet. Document the residual wetness by photograph or video.
Record the final weight of each carrier (weight three – post contact time). Use a
single dry sheet of Kim Wipe (e.g., 11 x 21cm) in the visualization of wetness and
record the observations. The data must show the presence of free-liquid on the
treated surface by weight and physical observations (presence of wetness).

(2) Confirmatory Testing. For confirmatory testing, follow the above testing guidance in
section (I)(1), with the exception that five carriers for each of two different batches of
product (at the LCL of the A.I.(s)) should be tested against C. difficile. Each product batch
should achieve a mean log10 reduction of ≥6 based on the number of viable spores
recovered.

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(J) Simulated Use Testing for Gas and Vapor Products

(1) Test Method. Simulated-use testing should be conducted for vapor and gas products with
sterilant/sporicide/decontaminant claims. Protocols for the simulated-use test should be
submitted to the agency for review and approval prior to conducting the test. The testing
should be conducted under conditions that are representative of the uses specified on the
product’s labeling, and in a setting that is representative of the label use site(s). For
example, a product intended for use in a room or a large warehouse should be tested in
an empty room or large chamber. The purpose of the test is to assure that key parameters
for efficacy (chemical concentration, temperature, relative humidity and contact time) are
accurately monitored and maintained throughout the enclosed space, and establish
product generation rate (lbs/hr) and rate/volume (lbs/hr/ft3).

(2) Additional Considerations. Important issues to consider in developing the protocol for
this test include:

(i) The test should be conducted in a sealed enclosure at least the size of a typical office
or other room that simulates the intended use pattern. The test should be designed
to measure the distribution of the product and conditions needed to meet the
measure of success in the laboratory efficacy test. All possible items that might be
treated (e.g., dressers, upholstered furniture, carpet, etc.) during an actual
fumigation should be included in this test.

(ii) The protocol should specify the dimensions of the enclosure, number and location
of monitoring devices (e.g., for gas or vapor concentration, total mass of gas or
vapor injected into the enclosure, temperature, relative humidity), product
application equipment, heaters and fans, contact time, etc. The equipment used to
monitor and maintain these test parameters should be described.

(iii) All recorded test results pertaining to the test conditions/parameters should be
submitted to the agency. Additionally, the maximum volume of space that can be
treated with a particular unit should be reported to the agency as well as the
minimum total mass of gas or vapor required to maintain the required concentration
and contact time per cubic foot of space to be treated.

(iv) Appropriate controls should be employed to assess both the sterility of the test
system and viability of the spore inoculum. Uninoculated carriers and/or
uninoculated biological indicators should be placed in the test chamber to assess
sterility of the test environment. Unexposed inoculated carriers and/or biological
indicators should be used to determine the suitability of the growth medium
designed for the recovery of viable spores.

(v) Evaluation of Sporicidal Success. Measurements for the simulated-use test should
show that the same concentration, temperature, and relative humidity, can be
maintained for the required contact time needed to achieve 100% kill (i.e., no
growth of the test organism on any of the carriers) in the qualitative laboratory test,
or a ≥6 log reduction in the quantitative test. In addition, measurements of the

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 fumigant mass injection/generation rate (e.g., pounds/hour), divided by the volume
of the simulated use test bed used to arrive at the required generation rate/volume
(e.g., pounds per hour/cubic foot) for the fumigation, should be included with the
data and listed on the product label.

(K) Data Collection and Reporting

Refer to OCSPP guideline 810.2000: General Considerations for Testing Public Health
Antimicrobial Pesticides for guidance. The applicant is encouraged to use the EPA’s Standardized
Efficacy Study Report and Efficacy Study Summary.

(L) References

The references in this paragraph may be consulted for additional background information.

(1) Official Methods of Analysis of the AOAC International, Chapter 6, Disinfectants,

Official Method 966.04 Sporicidal Activity of Disinfectants, 2013. AOAC
International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD 20877-2417.

(2) Official Methods of Analysis of the AOAC International, Chapter 6, Disinfectants,

Official Method 2008.05 Quantitative Three Step Method (Efficacy of Liquid
Sporicides Against Spores of Bacillus subtilis on a Hard Nonporous Surface), 2013.
AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg, MD
20877-2417.

(3) Annual Book of ASTM Standards, Standard Quantitative Disk Carrier Test Method for
Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal
Activities of Chemicals, Designation E 2197-11. ASTM International, 100 Barr Harbor
Drive, West Conshohocken, PA 19428-2959.

(4) Annual Book of ASTM Standards, Standard Test Method for Production of Clostridium
difficile Spores for Use in Efficacy Evaluation of Antimicrobial Agents, Designation
E2839-11. ASTM International, 100 Barr Harbor Drive, West Conshohocken, PA
19428-2959.

(5) Environmental Protection Agency, Biological and Economical Analysis Division,

Office of Pesticide Programs. Antimicrobial Testing Methods & Procedures Developed
by EPA’s Microbiology Laboratory.

(6) Hilgren, J., Swanson, KM., Diez-Gonzalez, F and B. Cords. “Susceptibilities of Bacillus
subtilis, Bacillus cereus and Avirulent Bacillus anthracis Spores to Liquid Biocides.”
Journal of Food Protection. 2009. Vol. 72 (2); 360-364.

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