New Approach Methods In Practice: Where Are We Today?

By Jordana Andrade Santos, Ph.D.

The reduction and replacement of animal use in research and safety assessment have evolved from a purely ethical imperative into a central scientific, technological, and regulatory challenge of 21^st century toxicology.^1,2 Although concerns related to animal welfare have historically driven this movement, the current landscape requires a substantial broadening of this discussion.¹

It has become increasingly evident that the extensive use of animal models does not fully meet the demands of a science aimed at the reliable prediction of human risk, particularly in a context characterized by complex chemical exposures, highly targeted therapies, and rapid advances in biotechnology.^2,3 In this scenario, new approach methods (NAMs) emerge not only as ethical alternatives but as strategic tools to improve human relevance, data reproducibility, and the quality of regulatory decision-making.^4,5

Advances In Drug Discovery And The Pressure For Human-Relevant Models

Advances in drug discovery, particularly in the development of advanced therapies, complex biological products, and medicines targeting highly specific human molecular targets, have intensified the limitations of animal models.^2,3,6 Many of these products rely on precise interactions with human receptors, specific signaling pathways, molecular biomarkers, and tissue microenvironments that are not adequately reproduced in non-human species. ^3,6

These limitations have become even more apparent as cell-based, gene-based, RNA-based therapies and highly specific antibodies have assumed central positions within development pipelines.^3,4 In this context, even animal models traditionally regarded as closer to human biology frequently fail to capture critical mechanistic responses, thereby compromising clinical translation and the prediction of safety outcomes. ^2,4

From The 3Rs To NAMs: Conceptual And Operational Evolution

The conceptual foundations of NAMs trace back to the seminal work of Russell and Burch in 1959, which introduced the principles of replacement, reduction, and refinement, collectively known as the 3Rs.¹ In the context of NAMs, replacement refers to the complete substitution of animal tests with non-animal methods; reduction denotes strategies that minimize the number of animals used while maintaining scientific validity; and refinement involves modifications that reduce pain, suffering, or distress when animal use cannot yet be avoided. These principles continue to guide scientific and regulatory policies, although their practical implementation has evolved significantly over recent decades.⁴

The term new approach methods gained formal regulatory relevance in 2016 during a workshop organized by the European Chemicals Agency (ECHA) within the framework of its 3Rs program.⁷ Since then, the term has been used to describe approaches that include in silico, in chemico, and in vitro methods; high-throughput and high-content techniques; omics-based approaches; and the structured use of exposure data.^6,7 These methodologies are increasingly being integrated into regulatory processes, not merely as alternatives but as central tools in human risk assessment.^3,8

Evidence Of The Limitations Of Animal Models

Numerous studies have demonstrated that animal-based tests exhibit substantial variability and limited reproducibility, even when conducted under standardized guidelines. Analyses of large data sets frequently reveal lower-than-expected concordance for fundamental endpoints, such as acute oral toxicity, skin irritation, and eye irritation.^5,9-11

These findings directly challenge the historical status of animal studies as a so-called gold standard and reinforce the need for models grounded in human biological mechanisms that can offer greater consistency and predictability.^2,9

Rethinking The Gold Standard: NAMs, AOPs, And Human Risk Prediction

Traditionally, new methods have been validated through direct comparison with animal data using classical statistical metrics, such as sensitivity, specificity, and accuracy.⁹ However, if animal data are not highly predictive of human outcomes, alignment with such data does not necessarily ensure improved protection of human health.^2,4,9

Accordingly, there is growing consensus that new methods should be evaluated based on their mechanistic relevance, alignment with adverse outcome pathways (AOPs), reproducibility, and ability to support correct risk management decisions, rather than simple equivalence with variable animal study designs.^9,10 Strategies such as in vitro to in vivo extrapolation (IVIVE) and the integration of human data from clinical, epidemiological, or accidental exposure sources are becoming central elements of this emerging paradigm.¹⁰

The Role Of Regulatory Agencies: Europe, The United Kingdom, And The FDA

Europe: ECHA And EMA

Within the European Union, the European Medicines Agency (EMA) has adopted an active stance in promoting and accepting NAMs as an integral component of assessing the safety and efficacy of new medicinal products in alignment with the principles of the 3Rs and Directive 2010/63/EU.^3,11-13 Directive 2010/63/EU establishes the legal framework for the protection of animals used for scientific purposes in the EU, with its core element being the mandatory application of the 3Rs principle, requiring that animal use is permitted only when no scientifically satisfactory non-animal alternative is available and actively encouraging the development, validation, and regulatory acceptance of alternative methods, including NAMs. Accordingly, the EMA recognizes that methods such as in vitro assays, in silico approaches, and computational modeling can reduce or replace animal use during non-clinical development, provided that they are scientifically robust, mechanistically relevant, and appropriate for the proposed regulatory context.^11-13

To facilitate this transition, the EMA offers multiple formal regulatory interaction pathways for NAM developers, including briefing meetings within the Innovation Task Force (ITF), which enable:

• early dialogue between developers and regulators regarding scientific maturity and regulatory applicability
• scientific advice procedures, where specific questions related to the development and use of NAMs can be discussed in the context of clinical trials or marketing authorization applications (MAA)
• qualification procedures, through which the EMA may issue an opinion on the acceptability of a NAM for a specific context of use in medicinal product development
• voluntary submission of data mechanisms, operating as a safe harbor that allows the agency to review NAM data outside a formal MAA process in order to build regulatory confidence and refine future acceptance criteria.¹¹

These mechanisms are guided by core principles that include a clear definition of context of use, robustness, reproducibility, and biological relevance of the data, as well as the suitability of the proposed methodology for regulatory decision-making. The EMA also encourages early and transparent discussions on technical readiness and the potential contribution of NAMs within weight of evidence approaches.^3,11

In addition, the EMA has established dedicated platforms and expert working groups that bring together expertise in non-clinical methods and NAMs, supporting regulatory coordination, conceptual harmonization, and dissemination of best practices across the European regulatory network. ^3,11-13

United Kingdom: MHRA And The Non-Clinical Road Map

The United Kingdom has advanced in a particularly structured manner with the launch of the non-clinical road map led by the Medicines and Healthcare Products Regulatory Agency (MHRA). Unlike more conceptual initiatives, this road map defines concrete goals, timelines, and operational actions for the regulatory adoption of NAMs, reflecting a strategic and forward-looking approach to non-clinical transition.^3,11–14

Key objectives of the road map include technical capacity building for regulatory assessors in NAMs and mechanistic approaches; development and integration of digital tools and in silico approaches for regulatory science; accelerated validation of alternative methods supported by the U.K. Centre for the Validation of Alternative Methods (UKCVAM); and periodic publication of accepted methods, regulatory priorities, and target areas for animal test replacement.¹⁴

The road map foresees the progressive replacement of specific animal tests, including pyrogen and irritation assays, with targets extending over the coming decade. It also explicitly signals the potential acceptance of non-animal data to support first-in-human studies, particularly in cases where no pharmacologically relevant animal species are available.^3,11–14

United States: FDA And The Regulatory Shift

In the United States, the FDA has assumed an increasingly prominent role in the transition toward NAM-based approaches. A decisive regulatory milestone occurred in 2022 with the approval of the FDA Modernization Act 2.0, which removed the explicit legal requirement for animal testing as a prerequisite for the submission of new medicinal products.¹⁶ This change does not prohibit the use of animals but formally opens the pathway for the acceptance of data generated using NAMs in medicinal product development and regulatory evaluation.^14,15

Since then, the FDA has intensified efforts to integrate in vitro and in silico methods, computational modeling, and microphysiological systems (MPS) into its assessment processes. Initiatives such as the predictive toxicology road map and programs focused on MPS qualification reflect this structural shift in regulatory posture.^15,16

In practice, the FDA has emphasized that NAM integration should occur in a contextualized, risk-based manner, with integrated consideration of the mechanistic relevance of the method, its ability to address specific regulatory questions, the robustness and reproducibility of the generated data, and its integration within weight of evidence strategies.^15,16

Incorporating These Changes Into Regulatory Practice And Drug Discovery

The effective adoption of NAMs requires a structural shift in the design of safety programs. Rather than replacing an animal test with a single alternative assay, regulatory agencies encourage integrated strategies that combine multiple NAMs, exposure data, and mechanistic knowledge.

For developers of medicinal products and chemicals, this implies aligning studies with well-characterized AOPs, investing in IVIVE and computational modeling, generating data tailored to specific regulatory questions, and engaging early with regulatory agencies to discuss method acceptance and qualification.

Where Are We Today?

NAMs have moved beyond a technological promise to become strategic components of modern toxicology and regulatory science. Europe, the United Kingdom, and the United States continue to advance, albeit at different paces, toward a model in which human relevance, mechanistic understanding, and risk management occupy a central role.

The transition is underway. The current challenge is no longer to justify the need for NAMs but to operationalize their adoption in a consistent, scientifically robust, and regulatorily acceptable manner, thereby redefining evidence-based safety assessment in the 21^st century.

References

1. RUSSELL, William M. S.; BURCH, Rex L. The principles of humane experimental technique. London: Methuen, 1959. Disponível em:
   https://caat.jhsph.edu/principles/the-principles-of-humane-experimental-technique.
2. HARTUNG, Thomas. The (misleading) role of animal models in drug development. Frontiers in Drug Discovery, v. 4, p. 1355044, 2024.
   https://doi.org/10.3389/fddsv.2024.1355044
3. EDWARDS, Mariana; BLANQUIE, Oriane; MORIARTY, Orla; BEKEN, Sonja; EHMANN, Falk. New approach methodologies: EU regulatory horizons. Nature Reviews Drug Discovery, v. 24, p. 571–572, 2025.
   https://doi.org/10.1038/d41573-025-00053-7
4. SUN, Danni et al. Clinical translation of animal models: challenges and opportunities. Nature Reviews Drug Discovery, v. 21, p. 691–707, 2022.
   https://doi.org/10.1038/s41573-022-00493-2.
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   https://doi.org/10.1093/toxsci/kfab149.
6. LUECHTEFELD, Thomas et al. Analysis of public acute toxicity data reveals limited reproducibility across multiple animal studies. ALTEX, v. 33, n. 2, p. 103–115, 2016. https://doi.org/10.14573/altex.1510051.
7. LUECHTEFELD, Thomas et al. Analysis of Draize eye irritation testing reveals poor reproducibility and calls for alternative approaches. Regulatory Toxicology and Pharmacology, v. 80, p. 1–7, 2016. https://doi.org/10.1016/j.yrtph.2016.06.001.
8. LUECHTEFELD, Thomas et al. Population-based validation of Draize eye irritation test data from the ECHA database. Toxicology, v. 355–356, p. 15–22, 2016. https://doi.org/10.1016/j.tox.2016.05.008.
9. WORTH, Andrew P. et al. Reconsidering animal data as the gold standard for regulatory toxicology. ALTEX, v. 41, n. 1, p. 1–15, 2024. https://doi.org/10.14573/altex.2303221.
10. OUEDRAOGO, G. et al. Applying adverse outcome pathways and NAMs to improve human-relevant risk assessment. Toxicological Sciences, v. 195, n. 1, p. 1–14, 2025. https://doi.org/10.1093/toxsci/kfad012.
11. EUROPEAN MEDICINES AGENCY (EMA). Regulatory acceptance of new approach methodologies (NAMs) to reduce animal use in medicine testing. 2023.
    https://www.ema.europa.eu/en/human-regulatory-overview/research-development/ethical-use-animals-medicine-testing/regulatory-acceptance-new-approach-methodologies-nams-reduce-animal-use-testing
12. EUROPEAN CHEMICALS AGENCY (ECHA). New approach methodologies in regulatory science. Helsinki: ECHA, 2016. https://echa.europa.eu/documents/10162/13639/nams_workshop_report_en.pdf.
13. EUROPEAN CHEMICALS AGENCY (ECHA). New approach methodologies (NAMs) in regulatory science. Helsinki: ECHA, 2023. https://echa.europa.eu/chemicals-in-our-life/new-approach-methodologies.
14. PAREXEL. UK’s nonclinical roadmap: MHRA joins global regulatory shift toward NAMs with detailed goals and timelines. 2024.
    https://www.parexel.com/insights/blog/uks-nonclinical-roadmap-mhra-joins-global-regulatory-shift-toward-nams-with-detailed-goals-and-timelines
15. FOOD AND DRUG ADMINISTRATION (FDA). FDA Modernization Act 2.0. Public Law No. 117–328, 2022. https://www.congress.gov/117/plaws/publ328/PLAW-117publ328.pdf
16. FOOD AND DRUG ADMINISTRATION (FDA). Predictive Toxicology Roadmap. Silver Spring, MD: FDA, 2023. https://www.fda.gov/science-research/predictive-toxicology-roadmap.

About The Author

Jordana Andrade Santos, Ph.D., is a toxicologist with a doctoral degree in pharmaceutical sciences and robust experience in regulatory toxicology, non-clinical safety assessment, and the application of new approach methodologies (NAMs). Her expertise spans vitro and in vivo toxicology, ISO 10993 biological evaluations, CPSR support, and the development of human-relevant models, including 3D cultures and microphysiological systems. She has worked across academia, industry, and consultancy, contributing to regulatory dossiers, R&D programs, and NAM-based innovation for medical devices, cosmetic products, and injectable materials. With international experience in organ-on-chip research at TissUse GmbH in Germany and a background in clinical research coordination, Andrade Santos bridges mechanistic science, regulatory strategy, and translational decision-making in modern safety assessment.