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Regulatory Applications of 3Rs

Federal agencies take an active role in facilitating the successful adoption and use of new approach methodologies to replace, reduce, and refine animal use in testing. This page provides examples of how U.S. federal agencies have applied 3Rs approaches to testing requirements.

FDA Issues Final Guidance on Carcinogenicity Testing

In an November 2022 Federal Register notice, FDA announced availability of finalized guidance for industry, “S1B(R1) Addendum to S1B Testing for Carcinogenicity of Pharmaceuticals.” The guidance expands the testing scheme for assessing human carcinogenic risk of small molecule pharmaceuticals. It introduces an integrative approach that provides specific weight-of-evidence criteria to inform whether a 2-year rat study adds value in completing a human carcinogenicity risk assessment. The draft guidance also adds a plasma exposure ratio-based approach for setting the high dose in the rasH2-Tg mouse model.

CPSC Issues Proposed Guidance on Use of Alternative Methods

In a March 2021 Federal Register notice (download as PDF), CPSC requested comment on its “Proposed Guidance for Industry and Test Method Developers: CPSC Staff Evaluation of Alternative Test Methods and Integrated Testing Approaches and Data Generated from Such Methods to Support FHSA Labeling Requirements.” CPSC has developed this guidance, building on its Animal Testing Policy, to assist stakeholders in determining what test methods are deemed reliable for determining compliance with the labeling requirements under FHSA. This includes clarification of CPSC informational requirements and process for evaluating new approach methodologies and integrated approaches to testing and assessment. The comment period for the draft guidance closed in June.

EPA Announces Guidance to Waive Toxicity Tests on Animal Skin, New Webpages on Metrics and NAMs

In January 2021, EPA finalized guidance that allows researchers to forego testing chemicals on animal skin in certain circumstances. Based on a retrospective analysis conducted by EPA, which concluded that such studies provide little to no added value in regulatory decision-making, the proposed guidance would allow waivers for studies on single active ingredients used in pesticides. This guidance, when finalized, is expected to save up to 750 test animals annually from unnecessary testing, as well as EPA, industry, and laboratory resources. 

A page on the EPA website summarizes the Agency's strategic vision for adopting NAMs. Three webpages linked from this page have details of EPA activities in this area.

FDA Guidance on Immunotoxicity Testing Supports Use of Alternatives

In February 2020, the U.S. Food and Drug Administration (FDA) issued draft guidance on “Nonclinical Safety Evaluation of the Immunotoxic Potential of Drugs and Biologics.” This guidance supplements previously issued recommendations on nonclinical evaluations of immunotoxic potential and is intended to assist sponsors in such evaluations. The guidance includes several specific recommendations on assessing potential for dermal sensitization:

  • FDA no longer recommends that sponsors conduct the murine local lymph node assay to assess the sensitization potential of topical drug products due to the limitations of the assay.
  • As an alternative to accepted guinea pig tests, FDA will consider a battery of in silico, in chemico, and in vitro studies that have been shown to adequately predict human skin sensitization with an accuracy similar to existing in vivo methods
EPA Designation of TSCA Low-priority Substances

In February 2020, EPA announced the designation of 20 chemical substances as low priority under TSCA. The designation is the third and final step in the prioritization process for reviewing chemical substances under the Frank R. Lautenberg Chemical Safety for the 21st Century Act amendments to TSCA. A low-priority designation means that risk evaluation for these substances is not warranted at this time. Designation of each substance as low priority reduces the likelihood that animals will be required for future testing of these substances.

For each chemical substance designated as low priority, EPA published a document describing the information, analysis, and basis for the designation. Information used to support the designation for each chemical included 3Rs approaches:

  • Data from in vitro high-throughput screening assays used in the EPA ToxCast program
  • Predictions of toxicity generated using quantitative structure-activity relationship models
  • Predictions of toxicity generated using read-across, a computational technique that uses toxicity data from a tested chemical to predict toxicity for an untested chemical

NICEATM has complied a summary of 3Rs approaches used to designate these substances as low priority. 

The 2018 Strategic Plan for TSCA required EPA to maintain and regularly update a list of alternative test methods or strategies (new approach methodologies, or NAMs) that do not require new vertebrate animal testing that would be acceptable for TSCA data requirements. The list was last updated in December 2019. More information about EPA's efforts to reduce vertebrate animal testing under TSCA is available on the EPA website.

EPA Evaluation of the Avian Acute Toxicity Tests for Pesticide Registration

In a February 2020 news release, EPA announced that it had finalized a science policy to reduce testing of pesticides on birds when registering conventional outdoor pesticides. The policy describes the results and implications of a retrospective study conducted by EPA and People for the Ethical Treatment of Animals. The study explored the quantitative and qualitative contributions of risk assessment methods using single oral dose and subacute dietary toxicity endpoints to the overall conclusions of acute avian risk. The analysis indicated that, in most cases, the subacute dietary results had little impact on risk conclusions arrived upon by use of acute oral data alone. This finding is expected to reduce the number of animals tested by a total of 60 birds per test, for a total projected animal savings of over 700 animals per year.

EPA Draft Science Policy on Non-animal Methods for Skin Sensitization Testing

In an April 2018 news release, EPA announced a draft Science Policy to reduce animal use in testing strategies to evaluate chemicals for their ability to cause an allergic reaction, inflammation, or sensitization of the skin. The draft policy permits the use of in vitro, in silico, and in chemico tests to identify potential skin sensitizers, an assessment that is required for registration of pesticides. The EPA action was the result of national and international collaboration among ICCVAM, NICEATM, Cosmetics Europe, the European Union Reference Laboratory for Alternatives to Animal Testing, and Health Canada’s Pest Management Regulatory Agency.

FDA Medical Device Development Tools Qualification Program

The FDA Center for Devices and Radiological Health is continuing to expand acceptance of alternative information and non-animal testing to support biocompatibility evaluations of medical devices. The Center’s guidance on Qualification of Medical Device Development Tools explains how new tools can be developed and qualified for a specific context of use, so that qualified tools can be used to support regulatory submissions to the Center. The policy outlined in the guidance is applicable to in vitro models to replace animal testing where appropriate. Tools qualified under the Center’s Medical Device Development Tool program will be published in the Medical Devices section of the FDA website.

USDA Reduction of Animal Use for Required Leptospira Vaccine Potency Testing

Vaccines used in controlling animal disease frequently undergo testing in animal models to ensure they are effective. The USDA Center for Veterinary Biologics (CVB) enforces the Virus Serum Toxin Act, which requires animal vaccines to be safe, potent, and effective. From 2013-2018, CVB developed alternatives to the codified Leptospira vaccine potency test to help reduce the number of hamsters used for this test. Approximately 40 total hamsters per serogroup are required for potency testing each leptospiral fraction. In order to reduce the number of animals used in this testing, CVB provided options to veterinary biologics manufacturers. An ELISA was developed by CVB to eliminate live animal potency testing after appropriate validation for a particular product line. In cases where the ELISA was not yet a reasonable option for a product line, CVB allowed back-titration hamsters to be removed from the codified test.

A separate group of hamsters is also required to propagate and maintain virulent strains for the codified test and developmental needs associated with regulated vaccines. Over 2,500 hamsters per year per facility are estimated to be used for propagation of virulent Leptospira. As a result, the CVB developed a cryopreservation protocol for the commonly used leptospiral strains and provided cryopreserved virulent Leptospira upon request.

From 2013 to 2018, the USDA Animal Care program monitored the number of hamsters listed under Category E on the annual reports from six companies that conduct Leptospira vaccine potency testing. Category E includes instances in which pain or distress, or potential pain or distress, is not relieved with anesthetics, analgesics and/or tranquilizer drugs. In 2013, the Category E designations indicated approximately 35,767 hamsters were used in total for Leptospira vaccine potency testing. Monitoring revealed a steady decline in animal numbers over five years such that 20,099 hamsters were used in 2018 demonstrating a 38% reduction. CVB believes the findings indicate that these options significantly contributed to the downward trend in hamster use. The options remain available and can be found on the CVB website.

EPA Alternative Testing Framework for Classification of Eye Irritation Potential of EPA-regulated Antimicrobial Cleaning Products

In 2013, the EPA Office of Pesticide Programs established the use of an non-animal testing approach to assess the eye irritation potential for antimicrobial cleaning products (AMCPs). The approach uses the bovine corneal opacity and permeability (BCOP), Cytosensor microphysiometer, and EpiOcular test methods to determine EPA hazard category and labeling requirements. The approach may also be used for other classes of pesticides and pesticide products on a case-by-case basis. EPA updated the approach in 2015 to expand the applicability of the BCOP method. A guidance document is available on the EPA website.

ICCVAM evaluated the testing strategy and made recommendations in a 2010 test method evaluation report. The initial EPA guidance issued in 2013 addressed the ICCVAM recommendations.