Methods and Guidance for Testing the Efficacy of Antimicrobial Products Against Spores of Clostridioides difficile on Hard Non-Porous Surfaces (September 2022)

This update reflects revised nomenclature for Clostridioides difficile (formerly Clostridium), revised carrier specifications, and revised verification testing.

This webpage provides test methods for evaluating the efficacy of antimicrobial pesticides against spores of Clostridioides difficile (C. difficile), as well as regulatory guidance for pesticidal claims for those products. These test methods and guidance provide a framework for registrants who seek to make a claim for antimicrobial pesticide products to control C. difficile spores on hard, non-porous surfaces.

This guidance supersedes the Agency’s previous guidance for the efficacy evaluation of antimicrobial pesticides registered or proposed for use against C. difficile on hard, non-porous surfaces. This guidance is not binding on EPA or any outside parties, and EPA may depart from the guidance where circumstances warrant and without prior notice. Registrants and applicants may propose and submit alternative practices (e.g., modifications to the recommended test methodology) to the Agency for assessment. The Agency will evaluate any proposed method modifications for appropriateness on a case-by-case basis. This guidance may be updated in the future and incorporated in the OCSPP Product Performance Test Guideline 810.2200.

On this page:

C. difficile spores and their public health significance
Products that may be eligible for C. difficile claims
Test procedures for developing efficacy data supporting C. difficile claims
Test criteria
Data submission procedures for efficacy data
Labeling guidance

C. difficile Spores and Their Public Health Significance

C. difficile is an anaerobic spore-forming bacterium and a frequent cause of hospital-acquired infections. The Agency recognizes the potential public health implications of these spores in healthcare settings and the importance of having antimicrobial products with demonstrated efficacy against C. difficile. EPA considers claims to control C. difficile spores to be public health claims for which the Agency must receive, review, and approve appropriate efficacy data.

Products That May Be Eligible for C. difficile Claims

C. difficile claims may be added to liquid, spray, and towelette formulations of a sterilant, sporicide, or hospital disinfectant product provided that the product meets the performance criteria for testing against C. difficile outlined in the most current version of the Product Performance Test Guidelines – OCSPP 810.2100: Sterilants, Sporicides, and Decontaminants – Guidance for Efficacy Testing or Product Performance Test Guideline, –OCSPP 810.2200: Disinfectants for Use on Environmental Surfaces, Guidance for Efficacy Testing.

The test methodologies described below may be used for testing the efficacy of water-soluble powders or liquid formulations against C. difficile; spray formulations may be evaluated as a liquid by removing/dispensing an appropriate amount of liquid from the spray device.

Towelettes may be evaluated using liquid directly expressed from the towelette. In addition, data should be generated to assess the amount of liquid remaining on a hard, non-porous surface treated (wiped) with the towelette per the label instructions as well as chemical testing for the expressed liquid active ingredient concentration. The agency recommends conducting visual and gravimetric wetness tests in an environmental chamber as described below.

Gravimetric and Physical Wetness Determination.

For each batch, use three pre-cleaned 12-inch × 12-inch glass or stainless steel carriers or 150 × 20 mm glass Petri plates to represent the surface to be treated.
Pre-clean each carrier surface with 70% ethanol rinse in deionized water and air dry.
Record the weight (weight #1: dry and untreated).
Distribute the liquid in the canister or package; remove and discard the first 3-5 towelettes.
For each carrier, remove one towelette from the container, unfold, and wipe the carrier surface in a circular fashion without lifting the towelette. Treat up to the edge of the carrier.
Re-weigh; record the results (weight #2: wet and treated).
Allow carriers to sit horizontally with the lid off for the contact time in an environmental chamber set at 35±5% relative humidity and 20-25°C.
Record the final weight (weight #3: post contact time).
Document the residual wetness by photograph or video.
Use a single dry sheet of Kim Wipe (e.g., 11 cm × 21 cm) in the visualization of wetness, record observations.
The data must show the presence of free-liquid on the treated surface by weight and physical observations (presence of wetness).

Applicants should consult with the Agency for other formulations types prior to developing data in support of registration. The Agency may require submission of protocols for review and approval prior to commencement of testing.

Test Procedures for Developing Efficacy Data Supporting C. difficile Claims

To evaluate C. difficile claims, the EPA recommends the use of the most contemporary versions of the following two methods:1

ASTM E2839: “Standard Practice for Production and Storage of Spores of C. difficile for Use in Efficacy Evaluation of Antimicrobial Agents”, and
ASTM E3218: “Standard Test Method for Quantitative Method for Testing Antimicrobial Agents against Spores of C. difficile on Hard, Nonporous Surfaces”

ASTM E2839 is used to produce and qualify spores of C difficile. The spores are used in ASTM E3218 to determine the effectiveness of the antimicrobial product.

Test Criteria

Applicants should apply the following product specific test criteria when evaluating products against C. difficile spores:

Acceptable Test Strain and Spore Production: Use C. difficile strain (ATCC 43598). Produce spore suspensions following ASTM E2839, meeting all the qualifications outlined in the procedure before use in efficacy testing.

Number of Batches and Test Coupons per Batch: Test three batches of the product at the lower certified limit(s) (LCL) listed on the confidential statement of formula of the product; see the 810.2000 product performance guideline for LCL requirements. Certificates of analysis (COA’s) to substantiate the tested concentration should be provided in the study report. Conduct each of the three tests on independent test days. Evaluate ten carriers against the product, three test system control carriers (against sodium hypochlorite, 1500 ppm) and three unexposed carriers as controls in each test. Conduct testing at a single laboratory.

Carriers: Use AISI Type 304 stainless steel (SS) carriers for qualifying spores, for the test system controls and product evaluations.

Neutralizer Confirmation: Conduct neutralization testing to confirm and document the neutralizer’s effectiveness for the batch(es) of product. Refer to ASTM E3218.

Contact Time: Do not use a contact time for efficacy testing that exceeds 10 minutes.

Diluent: For dilutable concentrates, refer to guidance under the most current OCSPP 810.2200 guideline for Dilution of Products for Testing.

Soil Load: Use the three-part soil load specified in ASTM E3218 for all testing including product, control, and test system control carriers.

Control Counts: Attain a mean log density of 6.0-7.0 (spores per carrier), with each carrier exhibiting a log density of 6.0-7.0 spores/carrier.

Test System Control: Run a test system control using three carriers against a 1,500±150 ppm (total chlorine) NaOCl solution concurrently with each product test (per test day) using a three-minute contact time. A log reduction (LR) of <3 for the test system control ensures the quality of the test system. LR above 3 invalidates the test and test repetition will be needed.

Product Performance Criteria: Attain a minimum mean six (6.0) log reduction (spores/carrier) across ten carriers in viable spores. Meet the performance standard in each of the three individual tests. Retesting guidance is provided in EPA’s Frequent Questions for the 2018 Series 810 – Product Performance Test Guidelines: Antimicrobial Efficacy Test Guidelines, Appendix II – Revised Repeat Testing Policy.

Modifications to the Test Procedures: When recommended methods are modified by the applicant to support specific claims for a product, submit the complete testing protocol, identifying and describing each modification to the Agency for review and evaluation prior to the initiation of the tests. Describe fully all materials and procedures employed in the testing.

Data Submission Procedures for Efficacy Data

To assist in the proper review and evaluation of product performance data, submit complete descriptions of the test employed and the results obtained to the Agency following the data submission guidelines. All product performance data must be developed in compliance with the Good Laboratory Practice Standards (40 CFR Part 160).

Labeling Guidance

Example of acceptable label claims against C. difficile: Kills and/or inactivates 99.9999% of spores of Clostridioides difficile on treated hard, non-porous surfaces.

Special Label Instructions for Cleaning Prior to Disinfection against Clostridioides difficile

Include the following specific cleaning directions on all products bearing Clostridioides difficile claims:

Personal Protection: Refer to the product label for appropriate personal protective equipment.
Cleaning Procedure: Special attention is needed for high-touch surfaces; cleaning in an appropriate manner and adherence to manufacturer’s label instructions for use and contact/dwell times is necessary. Pre-clean surfaces to remove visible soil and filth. Wipe dry. Thoroughly wet pre-cleaned surface with product. Allow surface to remain wet for [contact time]. Surfaces in patient rooms are to be cleaned in an appropriate manner, such as from right to left or left to right, on horizontal surfaces, and top to bottom, on vertical surfaces, to minimize spreading of spores. Restrooms are to be cleaned last. Do not reuse soiled cloths.
Infectious Waste Disposal: Materials used in the cleaning process that may contain feces/wastes are to be disposed of immediately in accordance with local regulations for disposal of infectious materials.

¹Additionally, see EPA Microbiology Laboratory Branch (MLB) standard operating procedures (SOP) MB-28 and MB-31. These SOPs are the Agency's interpretation of ASTM E2839, respectively, and are found on the Agency's antimicrobial testing methods and procedures website (https://www.epa.gov/pesticide-analytical-methods/antimicrobial-testing-methods-procedures-developed-epas-microbiology).