Methods and Guidance for Testing the Efficacy of Antimicrobial Products Against Biofilms on Hard, Non-Porous Surfaces

This webpage provides test methods for evaluating the efficacy of antimicrobial pesticides against two biofilm bacteria, Pseudomonas aeruginosa and Staphylococcus aureus, 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 these bacteria 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.

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Biofilms and Their Public Health Significance

Bacteria may grow and persist in the environment as free floating (planktonic) cells in liquids or attached to surfaces as biofilms. Biofilms form when bacteria adhere to environmental surfaces, especially those located in the presence of high moisture. Biofilms typically persist in a matrix containing slimy, glue-like substances (extracellular polymeric substances) which facilitate their attachment to many hard surfaces. The biofilm matrix provides embedded bacteria with protection from dehydration and other environmental stresses, and may interfere with the action of chemical disinfectants. Thus, bacteria in biofilms have the capacity to survive environmental stress and may serve as a reservoir of human pathogenic bacteria. EPA considers claims to control human pathogenic bacteria in biofilms to be public health claims for which the Agency must receive, review, and approve appropriate efficacy data. 

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Products that May Be Eligible for Biofilm Claims

Biofilm claims may be added to a liquid or spray product provided that the product meets the performance criteria for testing against P. aeruginosa and S. aureus using tests outlined in the most current version of Antimicrobial Efficacy Test Guideline - 810.2200 - Disinfectants for Use on Hard Surfaces – Efficacy Data Recommendations.

The biofilm test methodologies described below may be used for testing the efficacy of water soluble powders or liquid formulations against biofilm; spray formulations may be evaluated as a liquid by removing/dispensing an appropriate amount of liquid from the spray device. Applicants should consult with the Agency for other formulation 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.

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Test Procedures for Developing Efficacy Data Supporting Biofilm Claims

To evaluate biofilm claims, EPA recommends the use of two methods presented here as Standard Operating Procedures (SOP):

  • EPA Microbiology Laboratory Branch (MLB) SOP MB-19, “Growing a Biofilm using the CDC Biofilm Reactor,” and
  • EPA MLB SOP MB-20, “Single Tube Method for Determining the Efficacy of Disinfectants against Bacterial Biofilms.”

The two methods are adapted from ASTM standard methods E2562-12 and E2871-13, respectively.1 EPA MLB SOP MB-19 is utilized to generate the Pseudomonas aeruginosa or Staphylococcus aureus biofilm on coupons. The coupons are removed from the reactor and evaluated using the procedure described in EPA MLB SOP MB-20 to determine the effectiveness of the antimicrobial product. The Single Tube Method is designed to measure the reduction of bacteria in biofilm following treatment for water soluble powders or liquid formulations.

Note: The biofilm test methods are not suitable for use sites associated with water systems due to differences in nutritional status, flow rate, water treatment processes (e.g., chlorination), and other environmental conditions found in water systems (e.g., drinking water distribution systems, cooling tower systems). Therefore, the Agency does not expect that the biofilm test methods would accurately demonstrate the efficacy of antimicrobial products against public health biofilms in water systems. Proposed protocols for evaluating the efficacy of antimicrobial products against biofilms in water systems may be evaluated by the agency on a case-by-case basis.

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Test Criteria 

Applicants should apply the following product specific test criteria when evaluating products against Pseudomonas aeruginosa and Staphylococcus aureus biofilms:

Acceptable Test Strains and Generation of Test Cultures: Pseudomonas aeruginosa (ATCC 15442) and Staphylococcus aureus (ATCC 6538). Produce stock cultures and mature biofilms following EPA MLB SOP MB-19. 

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 against both test microbes for a total of six tests; see the product performance guidelines for LCL requirements. Conduct each of the three tests per organism on independent test days (P. aeruginosa and S. aureus tests may be conducted on the same day). Evaluate a minimum of five coupons per microbe against the product and three coupons as controls. Conduct testing at a single laboratory.

Neutralizer Confirmation: Conduct neutralization testing to confirm and document the neutralizer’s effectiveness for the batch(es) of product. Refer to EPA MLB SOP MB-20.

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

Diluent: For dilutable concentrates, dilute the product using the diluent consistent with that used for the evaluation of the Pseudomonas aeruginosa and Staphylococcus aureus claims based on the most current version of EPA’s Antimicrobial Efficacy Test Guideline 810.2200 – Disinfectants for Use on Hard Surfaces – Efficacy Data Recommendations.

Control Counts: Attain a mean log density of 8.0-9.5 (colony forming unit (CFU) per coupon) for coupons inoculated with P. aeruginosa, with each coupon exhibiting a log density of 8.0-9.5. Attain a mean log density of 7.5-9.0 for coupons inoculated with S. aureus, with each coupon exhibiting a log density of 7.5-9.0.

Product Performance Criteria: Attain a minimum six log reduction in viable bacteria in biofilm. Meet the performance standard in each of six individual tests. Retesting guidance is provided in EPA MLB SOP MB-20. 

Additional Bacteria: The Agency will evaluate claims against additional bacteria in public health biofilms on a case-by-case basis, provided that the product meets the performance criteria for testing against P. aeruginosa and S. aureus using tests such as those outlined in the most current version of OCSPP 810.2200 for liquid and spray formulations, and the performance criteria for public health biofilm claims for P. aeruginosa and S. aureus. Applicants should consult with the agency prior to developing an alternative biofilm growth protocol or efficacy data to support approval of such claims.

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.

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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).

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Labeling Guidance

Identification of Specific Use Sites for Public Health Biofilm Claims

Include labeling directions specific for treating hard, non-porous surfaces upon which public health biofilms are expected to form. Examples of these use sites include hard, non-porous surfaces associated with restrooms, floors, shower stalls, sink basins, drain covers, walls, countertops, instrument trays in patient care areas of hospitals, healthcare, clean room, and residential environments, excluding food contact surfaces.

The Agency will evaluate additional use sites on a case-by-case basis. Applicants should consult with the agency prior to developing efficacy related registration data for such use sites.

Examples of acceptable label claims against public health biofilms:

  • Kills 99.9999% of bacteria* in biofilms on a hard, non-porous surface
  • Kills a minimum of 99.9999% of bacteria* in biofilms
  • Reduces at least 99.9999% of bacteria* growing in biofilms
  • Formulated to kill 99.9999% of bacteria* in biofilms
  • Other related claims:
    • Kills biofilm bacteria*
    • Penetrates biofilms, killing the bacteria* living there

*[List of bacteria “tested as a biofilm”; at a minimum, Pseudomonas aeruginosa and Staphylococcus aureus]

Example of Label Language for Biofilms on Hard, Non-Porous Surfaces

Pre-clean surfaces to remove soil and filth. Wipe dry. Thoroughly wet pre-cleaned surface with product. Allow surface to remain wet for [contact time]. Rinse thoroughly.

The Agency will evaluate other label claims against biofilms on a case-by-case basis. 

EPA plans to harmonize its standard operating procedures (EPA MLB SOPs MB-19 and MB-20) with ASTM standard methods. EPA is currently working with ASTM to generate a new standard method for the CDC biofilm reactor to grow standardized biofilms of P. aeruginosa and S. aureus (ASTM workgroup WK55878). EPA is also currently working with ASTM to update ASTM E2871, the Single Tube Method (ASTM workgroup WK55813).

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