**Guidance on Test Methods for Determining the Efficacy of Antimicrobial
Products for Inactivating *Bacillus anthracis* Spores on Inanimate
Surfaces**

**6/18/07**

**[Presented To The FIFRA Scientific Advisory Panel By:]{.underline}**

U.S. EPA Office of Pesticide Programs

**[Presented On:]{.underline}**

July 17-19, 2007

![](media/image1.png){width="1.9979166666666666in"
height="1.9979166666666666in"}

Office of Prevention, Pesticides & Toxic Substances

U.S. Environmental Protection Agency

Washington, D.C. 20460

**Table of Contents**

**Preface** Page 3

**1. Introduction** Page 4

**2. Test Methods for Determining the Efficacy of Antimicrobial
Sterilants/Sporicides** Page 5

2.1 AOAC Sporicidal Activity of Disinfectants Test Page 5

2.2. Modifications to the AOAC Official Method 966.04 Page 5

**3. Options for Supporting Registration of Anthrax-Related Products**

3.1 Test procedure for Sterilant/Sporicide plus

*B. anthracis* Claim Page 7

3.2 Test Procedure for Sporicidal Decontaminant\--Qualitative Testing
Page 7

3.3 Test Procedure for Sporicidal Decontaminant\--Quantitative Testing
Page 7

**4.** **Simulated Use Test for Gas and Vapor Products** Page 8

**5.** **References** Page 8

**Preface**

Following the "anthrax attacks" in late 2001, the EPA Office of
Pesticide Programs (EPA/OPP) has played a critical role in regulating
the use of antimicrobial chemicals for decontaminating facilities and in
conducting research on the sporicidal efficacy of available chemicals.

Because no antimicrobial products were registered at that time
specifically for inactivation of *Bacillus anthracis* spores , EPA had
to issue crisis exemptions under Section 18 of FIFRA to permit the use
of these products to treat contaminated facilities and their contents.
Altogether, EPA received 63 requests for crisis exemptions, of which it
approved 28 and denied 35.

Rather than have to issue crisis exemptions in the future if another
bioterrorism attack occurs, EPA/OPP plans to allow for the registration
of products that have claims to inactivate *Bacillus anthracis* spores
(hereafter referred to as "anthrax-related products") so that they will
be readily available to trained, qualified persons for decontaminating
buildings and their contents.

Toward this end, EPA is proposing guidance on the product performance
(i.e., efficacy) data needed to support the registration of
anthrax-related products. This proposal is set forth in this document
and in a draft insertion to the document entitled "OPPTS 810.2100A.
Products for Use on Hard Surfaces---Basic Efficacy Data
Recommendations."

On July 17-19, 2007, the FIFRA Scientific Advisory Panel (SAP) will meet
publicly to consider the scientific basis for the efficacy test methods
and associated criteria described in the cited document.

Following is background information on the proposed efficacy test
methods that would support the registration of anthrax-related products.
Details are available in the cited references, which are being provided
to the SAP.

**1. Introduction**

EPA regulations require that product performance (efficacy) studies be
submitted to support registration of an antimicrobial product for which
public health claims are made such as "disinfect" or "sterilize" (40 CFR
Part 158.640). In addition, any claim of inactivation of a specific
microorganism should be supported by valid data that demonstrate the
efficacy of the product against that particular microorganism. At the
July 17-19 SAP meeting, the EPA will present draft guidance concerning
what efficacy testing should be conducted to support the registration of
an anthrax-related product.

EPA's draft guidance describes three basic options for a registrant to
follow in pursuing such a registration:

1.  For any liquid, gas or vapor "sterilant/sporicide" product, conduct
    the full AOAC Official Method 966.04 on silk suture loops and
    porcelain penicylinders, which represent porous and nonporous
    surfaces, respectively. In addition, conduct "confirmatory testing"
    using AOAC 966.04, but instead of *B. subtilis* or *C. sporogenes*,
    use virulent *B. anthracis* spores on carriers made of porcelain and
    silk. If both tests are passed (by showing no growth on any
    carrier), then the product may be registered as a
    "sterilant/sporicide" with a claim that the product inactivates *B.
    anthracis* spores on inanimate surfaces. \[Note: Any gas or vapor
    product would also have to undergo a "simulated use test" (see
    section 4. below).\]

2.  For any liquid, gas or vapor "sporicidal decontaminant\" product,
    conduct the *Bacillus* component of AOAC Official Method 966.04 on
    silk suture loops and porcelain penicylinders using virulent *B.
    anthracis* spores (instead of *B. subtilis* or *C. sporogenes*). The
    test would be conducted without the *Clostridium* requirement. If
    the product passes this test, it could be registered as a
    "sporicidal decontaminant" with a claim that the product inactivates
    *B. anthracis* spores on inanimate surfaces. \[Note: Any gas or
    vapor product would also have to undergo a "simulated use test" (see
    section 4. below).\]

3.  For any liquid, gas or vapor "sporicidal decontaminant\" product,
    conduct a well developed, quantitative sporicidal test method
    acceptable to EPA using virulent *B. anthracis* spores on porous
    and/or non-porous surfaces (with coupon materials acceptable to
    EPA). If the product achieves at least a 6 log reduction of target
    spores on nonporous and/or porous surfaces, it could registered as a
    "sporicidal decontaminant" with a claim that the product inactivates
    *B. anthracis* spores on inanimate surfaces. \[Note: Any gas or
    vapor product would also have to undergo a "simulated use test" (see
    section 4. below).\]

An issue that applies to all three testing options is the possible use
of surrogate *Bacillus* spores in place of virulent *Bacillus anthracis*
spores. Also, the issue of whether various coupon materials may be used
for quantitative sporicidal tests applies to the third testing option.

Lastly, EPA's draft guidance proposes to require "simulated use testing"
to support registration of gas and vapor sterilants/sporicides and
sporicidal decontaminants. This testing would demonstrate a product's
ability to be applied in large, enclosed spaces and to achieve the same
concentration, temperature, and relative humidity that was necessary to
achieve 100% kill (i.e., no growth on any of the 720 carriers) in the
qualitative laboratory test, or a 6 log reduction in the quantitative
test.

**2. Available Sterilant/Sporicides Efficacy Test Methods**

> **2.1 AOAC Official Method 966.04\--Sporicidal Activity of
> Disinfectants Test (Method I)**

The original AOAC Official Method 966.04 (Method I) was developed in the
1960s and adopted by EPA and the FDA as a validated product performance
test that demonstrates that a product is a "sterilant" or "sporicide"
(Reference 1). The AOAC procedure is qualitative and determines the
ability of an antimicrobial product to inactivate two spore types
(*Bacillus subtilis* and *Clostridium sporogenes*) on porcelain and silk
carriers. A sterilant or sporicide is a substance that destroys all
microorganisms on inanimate surfaces, including vegetative and spore
forms of bacteria and fungi, as well as viruses. All of EPA's registered
sterilants/sporicides were tested with this method and were shown to
pass the requirement of no growth on any carrier.

Until or unless the AOAC Official Method 966.04 is formally replaced by
another method, EPA and FDA will continue to require it for making
regulatory decisions about sterilants. However, EPA, FDA and other
agencies have been working collaboratively to make improvements to this
test and to compare the performance of relatively new quantitative tests
to the qualitative AOAC Method 966.04.

> **2.2. Modifications to the AOAC Official Method 966.04 (Method II)**

EPA led a multi-laboratory collaborative study, facilitated by AOAC
International, to officially modify and improve the AOAC Official Method
966.04 (Reference 3). These modifications/improvements included:

1.  Replacement of the current sporulation medium (soil extract nutrient
    broth) with a synthetic, standardized sporulation medium (nutrient
    agar amended with manganese sulfate).

2.  Replacement of the porcelain carriers currently being used with
    stainless steel penicylinders. (This change is not currently
    recommended for use until the *Clostridium* component is
    collaboratively modified for stainless steel.)

3.  Addition of a carrier count procedure for enumerating spores.

4.  Establishment of a target spore titer per carrier as a minimum of 1
    10^5^ and a maximum of approximately 1 10^6^ spores/carrier.

5.  Addition of a neutralization confirmation protocol.

6.  Editorial changes to the procedure.

The collaborative study, initiated in June 2005, involved five
laboratories. These laboratories had existing microbiology programs, had
conducted the AOAC method 966.04 in the past, and were suitable testing
labs based on an EPA quality assurance readiness-review process. The OPP
Microbiology Laboratory was the lead laboratory; others included the
U.S. Food and Drug Administration (FDA) in Denver, CO; FDA in
Winchester, MA; MICROBIOTEST, Inc. in Sterling, VA; and Advanced
Sterilization Microbiology Lab in Irvine, CA.

The OPP laboratory provided the following: The study design and the
necessary protocols; test forms; data sheets; media preparation sheets
for test chemicals, media, and reagents; test chemicals; garden soil;
and porcelain penicylinders. Using a step-wise comparative process,
modifications involving the new sporulation medium and the new carrier
type were evaluated against the current method using three parameters:

> 1\. Spore counts on carriers
>
> 2\. HCl resistance
>
> 3\. Efficacy against common sporicidal agents

The study design required that three carrier-medium combinations be
tested against one chemical (a high and low treatment) on the same day
(six 30-carrier tests). An acceptable randomization method was decided
by the lead laboratory before experimentation was initiated. This
involved a randomized order of testing of chemicals and carrier-medium
combinations for each participating laboratory.

The data evaluation was qualitative (i.e., how many positives versus
negatives), which was acceptable to the AOAC. The data were collected
and summarized by August 12, 2005. Statistical analysis was performed by
Dr. Martin Hamilton (Montana State University).

The final, validated Method II has been published in the Journal of AOAC
International (Reference 3).

The most significant concerns associated with method 966.04 are the
qualitative nature of the method, the use of raw garden soil extract as
a source of minerals for spore production, the lack of a standardized
procedure for enumeration of spores, and the lack of a target spore
load. An official AOAC collaborative study was conducted to modify and
improve selected aspects of the method. The proposed modifications were
limited to liquid formulations, *B. subtilis*, and the hard surface
carrier (porcelain penicylinder) components of the method.

The study included the evaluation of a replacement for soil extract
nutrient broth and an establishment of a minimum spore titer per
carrier, both considered crucial for the improvement and utilization of
the method. Additionally, an alternative hard surface material and a
neutralization confirmation procedure were evaluated. Based on the
results of this study, it was determined that nutrient agar amended with
5 µg/mL manganese sulfate as a sporulation medium, the spore enumeration
procedure, a target carrier count (10^5^ to approximately 10^6^
spores/carrier) and the neutralization confirmation procedure were
suitable modifications.

The modifications have been approved by AOAC-International (AOACI), and
the manuscript describing the study has been accepted for publication.
Additional studies are planned in 2007-2008 to assess the impact of the
modifications when applied to suture loops and with gaseous
formulations.

**3. Options for Supporting Registration of Anthrax-Related Products**

**3.1** Sterilant/Sporicide plus *B. anthracis* Claim

The AOAC Sporicidal Activity of Disinfectants Test (AOAC Official Method
966.04, Methods I and II) should first be conducted for any liquid, gas
or vapor product to demonstrate that it is a sterilant/sporicide on
porous and non-porous environmental surfaces. A product must inactivate
viable spores on all 720 carriers in the AOAC sporicidal test. Second,
"confirmatory testing" needs to be conducted on two samples
(representing two batches of product) using virulent *B. anthracis*
spores, or a surrogate acceptable to EPA, to show that the product
inactivates these spores on all 120 carriers (porcelain penicylinders
and silk suture loops). If both tests are passed, then the product may
be registered as a "sterilant" or "sporicide" with a claim that the
product inactivates *B. anthracis* spores on inanimate surfaces. \[Note:
Any gas or vapor product would also have to undergo a "simulated use
test" as well (see section 4. below).\]

> **3.2** Sporicidal Decontaminant (Qualitative Testing) plus *B.
> anthracis* Claim

A product may be registered as a "sporicidal decontaminant" if it is
successfully tested using the AOAC Official Method 966.04 to demonstrate
the product's effectiveness on porcelain or silk carriers only tested
against virulent *B. anthracis* spores, or a surrogate acceptable to
EPA. Sixty carriers representing either or both of two types of carriers
(porcelain penicylinders and silk suture loops) should be tested on
three samples representing three different batches of product, one of
which should be at least 60 days old. If one surface type is tested,
then there are 60 carriers per sample, or a total of 180 carriers; if
two surfaces types are tested, then the total number of carriers is 360.
\[Note: Any gas or vapor product would also have to undergo a "simulated
use test" as well (see section 4. below).\]

> **3.3.** Sporicidal Decontaminant (Quantitative Testing) plus *B.
> anthracis* Claim

A product may be registered as a "sporicidal decontaminant" if it is
successfully tested using a well established, quantitative sporicidal
test method acceptable to EPA using virulent *B. anthracis* spores (or a
surrogate acceptable to EPA) on porous and/or nonporous surfaces. The
product should be tested on three samples representing three different
batches of product, one of which should be at least 60 days old. The
number of carriers will depend upon the method that is used. The product
needs to achieve at least a 6 log reduction of virulent *B. anthracis*
spores (or a surrogate) on either nonporous or porous surfaces. \[Note:
Any gas or vapor product would also have to undergo a "simulated use
test" as well (see section 4. below).\]

**4. Simulated Use Testing for Gas and Vapor Products**

> Simulated use testing is needed to support registration of gas and
> vapor sterilants/sporicides and sporicidal decontaminants. This
> testing would demonstrate a product's ability to be applied
> effectively in large, enclosed spaces rather than just in small spaces
> (such as glove boxes). The purpose of the test would be 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).

> Generally, the test chamber or space should be at least the size of a
> typical office. Key parameters such as ambient temperature, relative
> humidity, gas concentration, gas injection rate and/or total mass
> usage, and contact time should be monitored throughout the test.

Measurements should show that the same concentration, temperature, and
relative humidity, can be maintained for the required contact time that
were necessary to achieve 100% kill (i.e., no growth on any of the
carriers) in the qualitative laboratory test, or a 6 log reduction in
the quantitative test is demonstrated in the simulated-use test. In
addition, measurements of the fumigant mass injection/generation rate
(e.g., pounds/hour), divided by the volume of the simulated use test
bed, that was 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.

**5. References are listed in the Charge Memorandum dated 6/7/07)**