Regulatory Applications of 3Rs

The U.S. Environmental Protection Agency (EPA) has released a detailed review of major environmental statutes that summarizes which EPA laws or regulations require vertebrate animal testing, such as laboratory testing done on rats, mice, or rabbits. The July 2024 "EPA Report on Statutory and Regulatory Requirements for Vertebrate Animal Testing and Flexibility for Implementing New Approach Methods" concludes that many statutes and regulations guiding EPA’s authority are broadly written and do not preclude the use of scientific information from NAMs, which are defined as any technology, methodology, approach, or combination that can provide information on chemical hazard and risk assessment to avoid the use of animal testing.

This report is a deliverable in EPA’s NAMs Work Plan, which was originally released in June 2020 and updated in November 2021. The Work Plan outlines the Agency’s strategies and objectives for increasing the rigor and sophistication of Agency assessments while reducing the reliance on vertebrate animals to test chemicals in regulatory, compliance, enforcement, and research activities through the use of NAMs. The assessments will remain fully protective of human health and the environment. The first objective in the Work Plan was to evaluate regulatory flexibility for accommodating NAMs, and the report accomplishes that objective.

Drug development tools are methods, materials, or measures that have the potential to facilitate drug development. FDA established the Innovative Science and Technology Approaches for New Drugs (ISTAND) pilot program to encourage innovation of drug development tools that are out-of-scope for existing qualification programs but may still be useful for drug development. Approaches that could be considered under the pilot program include:

• Using MPS to assess safety or efficacy questions.
• Developing novel nonclinical pharmacology or toxicology assays.
• Using artificial intelligence-based algorithms to evaluate patients, develop novel endpoints, or inform study design.

In the pilot phase, FDA anticipates accepting two to four submissions into the ISTAND program each year with a triage and selection process that focuses on public health impact and feasibility of implementation. There were three projects accepted into ISTAND during 2022 and 2023, including one representing both the first artificial intelligence-based and digital health technology-based project and the first project in neuroscience to be accepted into the program. The Drug Development Tools Research Grant Cycle will be open through May 13, 2025, for Fiscal Year 2025.

In a January 2024 Federal Register notice, the U.S. Environmental Protection Agency (EPA) announced a new framework for identifying eye irritation and corrosion hazards for new chemicals reviewed under the Toxic Substances Control Act (TSCA).

With this new framework, EPA will place increased weight on data from non-animal test methods that are more reproducible and provide results more relevant to humans. This will streamline the decision-making process and increase efficiency through a standard process for EPA to use each time it evaluates eye irritation or corrosion hazards test data. The new framework supports EPA’s mandate under TSCA to promote the development and implementation of alternative test methods and strategies that can provide information on chemical hazards without animal testing. This framework also supports EPA’s ongoing efforts to reduce the use of animal testing and make the Agency’s review of new chemicals more efficient, helping to bring new chemicals to market more quickly while protecting human health.

The U.S. Food and Drug Administration (FDA) has finalized the guidance document, “Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals.” The purpose of this guidance is to assist sponsors in the nonclinical evaluation of the immunotoxic potential of drug products and biopharmaceuticals. The guidance addresses evaluation of topical pharmaceuticals for skin sensitization potential. Specifically, the guidance states that, for individual chemicals, “FDA will consider a battery of studies (e.g., in silico, in chemico, in vitro) that have been shown to adequately predict human skin sensitization with an accuracy similar to existing in vivo methods.”

The U.S. Consumer Product Safety Commission (CPSC) has updated its Animal Testing web page. It has been redesigned to be more user friendly and have direct access to necessary information and documents.

One of the resources available on this page is CPSC’s “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 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 the Federal Hazardous Substances Act (FHSA). This includes clarification of CPSC informational requirements and process for evaluating new approach methodologies and integrated approaches to testing and assessment.

In a January 2023 Federal Register notice, the U.S. Environmental Protection Agency (EPA) announced availability of a draft white paper, “Availability of New Approach Methodologies (NAMs) in the Endocrine Disruptor Screening Program (EDSP).” The white paper states that certain NAMs have been validated and may now be accepted by the EPA as alternatives for certain Tier 1 assays used within the EDSP. Others NAMs may be useful for prioritization purposes and for use as other scientifically relevant information, where appropriate, in weight-of-evidence evaluations. EPA accepted comment on the draft white paper through March 20.

The purpose of Tier 1 EDSP screening is to identify chemicals that have potential biological activity in the estrogen, androgen, or thyroid hormone pathways. Recent EPA research efforts have focused on the development and evaluation of high-throughput in vitro assays and in silico methods as NAMs, including databases and computational models, for use as alternatives to the current suite of assays in the EDSP Tier 1 battery. Full implementation of NAMs in this battery is expected to accelerate the pace of screening, add efficiencies, decrease costs, and reduce animal testing.

The FDA Modernization Act 2.0, passed in December 2022, modifies provisions of the Federal Food, Drug, and Cosmetics Act addressing U.S. Food and Drug Administration (FDA) regulatory guidance that requires animal testing for drugs. Specifically, FDA 2.0:

• Specifies that the term “nonclinical tests” be used in place of “preclinical tests.”
• Replaces references to animal tests with the term “nonclinical tests.”
• Adds a new section noting that “nonclinical tests” include human-relevant testing nonanimal methods such as cell-based assays, microphysiological systems (MPS, also known as organs-on-chips), or computer models.

These changes eliminate the requirement that animal studies be performed prior to first-in-human studies, opening the door for using data from new approach methodologies (NAMs) for nonclinical evaluations. FDA encourages registrants to consult with its Center for Drug Evaluation and Research about the appropriateness of data for a specific NAM to support any drug registration.

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.

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.

• EPA has implemented activities and policies that have reduced the number of animals used in testing and saved resources for the Agency and stakeholders. A page on the EPA website provides measures of cost and animal savings following these activities.
• In addition to the dermal study waivers described above, EPA has identified a number of contexts in which waivers of required studies can be considered, reducing the number of animals required for testing.
• EPA has taken steps to advance the use of computational and non-animal approaches to provide information on chemical hazard and risk assessment. Many of these, such as the Collaborative Acute Toxicity Modeling Suite (CATMoS) and skin sensitization approaches, were developed or evaluated in collaboration with NICEATM.

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.

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.

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.

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.

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.

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.