Testing Approaches and Directives for Assessing Antimicrobial Product Efficiency against C. difficile Spores on Rigid, Non-Absorbent Surfaces (September 2022)

In the realm of public health, the U.S. Environmental Protection Agency (EPA) plays a significant role in regulating antimicrobial pesticides, particularly those claimed to control bacterial spores such as Clostridioides difficile (C. difficile) in healthcare settings.

The EPA recommends the use of contemporary versions of ASTM E2839 and ASTM E3218 for evaluating C. difficile claims. These standardized methods, approved by the EPA and organizations like AOAC International, expose surfaces or carriers inoculated with spores to the pesticide under defined conditions, followed by recovery and enumeration of surviving spores.

A minimum mean six (6.0) log reduction is required across ten carriers in viable spores for the product to meet the performance standard. Control counts should have a mean log density of 6.0-7.0 spores per carrier.

The 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. Modifications to the test procedures should be submitted to the Agency for review and evaluation prior to the initiation of the tests.

Certificates of analysis (COA's) are required to substantiate the tested concentration. The three-part soil load specified in ASTM E3218 is used for all testing.

The test strain for C. difficile is ATCC 43598. Testing typically requires Biosafety Level 2 (BSL-2) containment, use of personal protective equipment, and validated molecular or culture methods to confirm reductions in viable spores.

Recent updates emphasize more rigorous contact times and realistic soiling conditions to simulate hospital environments, evaluation of residual activity and stability, and validation of methods to ensure reproducibility and relevance to final labeled claims.

As the search did not yield direct regulatory documents or newest EPA or FDA guidance updates on this topic by August 2025, it is advised to consult the latest EPA Pesticide Registration Notices and the AOAC International website for exact test protocols and updated regulatory guidance.

In summary, the updated evaluation of antimicrobial pesticides against C. difficile spores in healthcare focuses on standardized sporicidal efficacy tests under realistic conditions, adherence to EPA-regulated protocols, and ensuring laboratory safety during testing. However, specific newly published regulatory guidance from 2025 is not found in current sources. The guidance provides a framework for registrants who seek to make a claim for antimicrobial pesticide products to control C. difficile spores on hard, non-porous surfaces.

Specific cleaning directions must be included on all products bearing C. difficile claims. C. difficile claims may be added to liquid, spray, and towelette formulations of a sterilant, sporicide, or hospital disinfectant product. AISI Type 304 stainless steel carriers are used for qualifying spores, test system controls, and product evaluations.

The webpage provides test methods for evaluating the efficacy of antimicrobial pesticides against spores of C. difficile. Three batches of the product should be tested at the lower certified limit(s) listed on the confidential statement of formula. Towelettes may be evaluated using liquid directly expressed from the towelette. A test system control using a 1,500±150 ppm (total chlorine) NaOCl solution is run concurrently with each product test.

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 test methodologies described on the page 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.

The EPA's Microbiology Laboratory Branch (MLB) standard operating procedures (SOP) MB-28 and MB-31 should be followed for testing against C. difficile. The agency recommends conducting visual and gravimetric wetness tests in an environmental chamber as described. The contact time for efficacy testing should not exceed 10 minutes. All product performance data must be developed in compliance with the Good Laboratory Practice Standards (40 CFR Part 160).

Testing Approaches and Directives for Assessing Antimicrobial Product Efficiency against C. difficile Spores on Rigid, Non-Absorbent Surfaces (September 2022)