New Methods in Human Safety Assessment: Right Here, Right Now

The use of non-animal technologies is growing, but validation and regulatory acceptance remain a major hurdle to wider application in safety assessment

For many years, the use of animals in regulatory safety assessment of chemicals and drugs has been regarded as the gold standard. However, times are changing and New Approach Methodologies (NAMs), defined as any non‐animal technology, methodology, approach, or combination thereof, can be used to provide information on chemical hazard and human risk assessment and support the 3Rs [1].

It is worth pointing out that the acronym 'NAM,' although widely used and generally understood to mean non-animal technologies, is not universally defined [2]. NAMs are sometimes referred to as New Approach Methods or Novel Alternative Methods, and harmonisation is needed to avoid confusion [2). In any case, the concept of alternative methods for animal experimentation was formally developed some time ago, 1978 to be exact, by British physiologist D.H. Smyth [3].

Today non-animal technologies are already accepted in some sectors for safety assessment e.g. cosmetic testing in the UK, Europe and Canada, where animal experimentation for these products is banned. Also, NAMs are permitted by the FDA and other regulatory authorities if validated and approved (Reviewed in [4]).

Meanwhile, wider application of NAMs is accelerating with the help of disruptive technologies like artificial intelligence (AI) and human-based in vitro microfluidic systems, such as organs-on-a-chip. Both these technologies are changing the landscape not solely because they are more ethical but because they are proving to more effective, quicker, and less costly for risk-based decision-making. For example, at the World Organoid conference held in Cambridge this year, the keynote speaker, Thomas Hartung, reported that AI had predicted the toxicity of over 4,700 food chemicals with 87% accuracy in 1 hour, which would otherwise have used 38,000 animals [5,6]. Similarly, microfluidic systems which better mimic the three-dimensional (3D) structure of cells are getting more sophisticated. Last year a team from Cortical Labs, Monash University, the University of Melbourne, and University College London demonstrated human brain organoids could play a 'game' [7]. Specifically, the team demonstrated the ability of a brain organoid to process an input and provide a measurable output, as a learned response to stimuli, and therefore the first step in models for neurodevelopment/ degenerative disorders in a dish [7,8].

In response to these scientific developments and public opinion, the FDA Modernization Act 2.0 became law in December 2022, that brought legislation into practice, and indicated support for in vitro and in silico technologies, including organ chips, sophisticated computer modelling and cell-based assays. The act replaced the word 'Animal' in the text to 'non-clinical' and defined the term 'non-clinical' to mean a test conducted in vitro, in silico, or in chemico. However, the enactment of the FDA Modernization Act 2.0 is considered too slow by democratic and republican representatives of congress and a follow-up act to speed up the validation and acceptance of NAMs was proposed this year [9]. Similarly, the European Medicines Agency shared a Concept paper updating the guidelines on the principles of regulatory acceptance of 3Rs) using NAM-based testing approaches [10].

Gaining Confidence in NAMs for Safety Assessment Studies

Regulatory acceptance of NAMs is a major hurdle to wider application in safety assessment. In response, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) have just published a report to help developers, end users and regulatory agencies build confidence in NAMs [1,10].

The report outlines a framework intended to be adaptive and based around the key concept that the extent of validation for a specific NAM will depend on the Context of Use (CoU). For example, a CoU for pre-regulatory test would require less validation/stringencies compared to a CoU requiring quantitative risk assessment. This framework moves away from 'one-test-fits-all' applications and instead allows flexibility based on the question being asked and the level of confidence needed for decision-making.

The proposed framework from ICCVAM is made up of six elements: defined context of use, biological relevance, data integrity, technical characterisation, information transparency and independent review [1]. Importantly, the guideline recognises that human relevant NAMs may provide mechanistic data for human risk assessment such that a comparison with animal data is unnecessary or not helpful. The guideline highly recommends early interactions between NAM developers and regulatory agencies to ensure the criteria for a particular CoU are understood and developed for the required regulatory need. The ICCVAM guideline lists OECD guidelines/case studies that currently replace animal experimentation, and they include skin sensitisation, endocrine disruptors, developmental neurotoxicity and inhalation toxicology (Table). Of note, the NAM-based lung organotypic studies for the inhalation toxicology OECD case study were performed at CRL [11,12] and the approach taken is aligned with the CoU framework.

Summary

The potential for NAMs to provide human-relevant risk assessment is now recognised by multiple stakeholder groups including governments and regulatory authorities and while NAMs are already used for internal decision-making their use in regulatory safety assessment is limited [4]. However, funding for developing NAMs, such as Complement ARIE, a Common Fund set up this year by the US National Institutes of Health [12], includes dollars for validation networks that will go a long way towards putting the ICCVAM framework into practice and developing the necessary confidence for human safety assessment.

References:
[1] Validation, Qualification, and Regulatory Acceptance of New Approach Methodologies, March 2024, ICCVAM

[2] M. New Acronym Mayhem (NAM): Why we need a consistent definition of NAMs. https://contemporarysciences.org/new-acronym-mayhem-why-we-need-a-consistent-definition-of-nams/
https://ntp.niehs.nih.gov/whatwestudy/niceatm/resources-for-test-method-developers/submissions

[3] Alternatives to Animal Experiments by Smyth, David Henry, 1978 https://archive.org/details/alternativestoan0000smyt/page/n11/mode/2up

[4] Stresser, D.M., Kopec, A.K., Hewitt, P. et al. Towards in vitro models for reducing or replacing the use of animals in drug testing. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01154-7

[5] https://organoidspheroid.com/word%2B-24-in-person-event, Cambridge Feb 2024.

[6] Hartung T. Predicting toxicity of chemicals: software beats animal testing. EFSA J. 2019 Jul 8;17(Suppl 1) :e170710. doi: 10.2903/j.efsa.2019.e170710. PMID: 32626447; PMCID: PMC7015478.

[7] Kagan BJ, Kitchen AC, Tran NT, Habibollahi F, Khajehnejad M, Parker BJ, Bhat A, Rollo B, Razi A, Friston KJ. In vitro neurons learn and exhibit sentience when embodied in a simulated game-world. Neuron. 2022 Dec 7;110(23):3952-3969.e8. doi: 10.1016/j.neuron.2022.09.001. Epub 2022 Oct 12. PMID: 36228614; PMCID: PMC9747182.

[8] Smirnova L, Hartung T. Neuronal cultures playing Pong: First steps toward advanced screening and biological computing. Neuron. 2022 Dec 7;110(23):3855-3856. doi: 10.1016/j.neuron.2022.11.010. PMID: 36480938.

[9] https://trackbill.com/bill/us-congress-house-bill-7248-fda-modernization-act-3-0/2510010/

[10] van der Zalm AJ, Barroso J, Browne P, Casey W, Gordon J, Henry TR, Kleinstreuer NC, Lowit AB, Perron M, Clippinger AJ. A framework for establishing scientific confidence in new approach methodologies. Arch Toxicol. 2022 Nov;96(11):2865-2879. doi: 10.1007/s00204-022-03365-4. Epub 2022 Aug 20. PMID: 35987941;

[11] Wallace Jo 2022. It's All About the Dose - How to Link In Vitro and In Vivo. https://www.criver.com/eureka/its-all-about-dose-how-link-vitro-and-vivo

[12] OECD 367: Case Study on the use of an Integrated Approach for Testing and Assessment (IATA) for New Approach Methodology (NAM) for Refining Inhalation Risk Assessment from Point of Contact Toxicity of the Pesticide, Chlorothalonil. https://one.oecd.org/document/ENV/CBC/MONO(2022)31/en/pdf

[13] https://commonfund.nih.gov/complementarie

TABLE: Examples of Endpoints where NAMs have supported regulatory applications for safety assessment (adapted Ref 1)<_o3a_p>

Endpoint<_o3a_p>	Summary <_o3a_p>	Reference/<_o3a_p>
Skin Sensitisation<_o3a_p>	Multiple non-animal testing strategies incorporating in vitro, in chemico, and in silico inputs proved equivalent or superior performance to the in vivo model when compared to both animal and human data for skin sensitization.<_o3a_p>	OECD, 2021<_o3a_p>
Endocrine Disruption<_o3a_p>	Certain NAMs have been validated and may now be accepted by the EPA as alternatives for certain endocrine Tier 1 assays while others are useful for prioritization purposes and for use as other scientifically relevant information, where appropriate, in weight of evidence evaluations.<_o3a_p>	EPA 2023<_o3a_p>
Developmental Neurotoxicity<_o3a_p>	Battery of NAMs covering critical process of human neurodevelopment have been developed.<_o3a_p>	OECD, 2022<_o3a_p> OCED, 2023<_o3a_p>
Inhalation Toxicology<_o3a_p>	Human lung complex 3d models plus computational modelling used to decide hazard for human occupational exposure by the inhalation route. <_o3a_p>	EPA, 2021<_o3a_p> OECD, 2022,<_o3a_p> OECD IATA Case Study. <_o3a_p>

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