NAMs for NGRAs

Traditionally, toxicological risk assessment has involved identifying a point of departure (PoD) such as a NOAEL or LOAEL in a study of small creatures, adjusting it to be relevant to humans (to derive, for example, a TI or DNEL), and then calculating the margin of safety to a measured or estimated external exposure. As part of this process, toxicologists have to account for the uncertainties that arise in moving from the species, route and duration of the laboratory animal study to the real human world. 

The current great hope is that New Approach Methodologies (NAMs) may remove some of these uncertainties. These are test methods based on a mechanistic understanding of the pathways that might follow chemical exposure. They typically make use of human cells, and compare PoDs to estimated internal exposures (i.e. plasma concentrations) in Next Generation Risk Assessment (NGRA).

The following models are tailored to specific toxicological endpoints designed to replace, reduce and refine animal-based test methods, as part of the “3Rs” principle. 

ToxTracker is a validated NAM which uses a panel of stem cell lines that incorporate fluorescent reporters representing four distinct biological responses associated with carcinogenesis: cytotoxicity, DNA damage, oxidative stress and protein damage. It can also provide an insight into the mechanisms of toxicity. Cells are exposed to test chemicals both in the presence and absence of metabolic activation (S9). Increases in Green Fluorescent Protein (GFP) expression are analysed and PoDs, such as No-Observed-Effect Levels (NOELs), Lowest-Observed-Effect Levels (LOELs), No-Observed-Genotoxic-Effect Levels (NOGELs) and Lowest-Observed-Genotoxic-Effect Levels (LOGELs), are assigned. 

High-throughput transcriptomics (HTTr) was developed to enable bulk characterisation of effects on RNA expression using human hepatoblastoma (HepG2/HepaRG) or breast cancer (MCF-7) cells. A “global PoD”, defined as the overall lowest value for any individual probe-level response, is used to identify a gene-level No-Observed-Transcriptional-Effect Level (NOTEL). This NOTEL is used to estimate the minimum effect concentration that induces perturbations of gene expression. 

The Cell Stress Panel monitors biomarkers across key stress pathways, including mitochondrial toxicity, cell stress and cell health, and can shed light on a wide range of parameters and biomarkers through a number of different assays in HepG cells. A PoD is calculated from concentration-response models, from which a “global PoD” can be derived, representing the minimum effect concentration across the cellular stress assays. 

In vitro Pharmaceutical Profiling (IPP) can be used in the early assessment of pharmaceuticals and provide information on their effects on specific organs or organ systems to identify molecular interactions that may cause Adverse Drug Reactions (ADRs) in humans. It does this by screening compounds against a wide range of “targets” (73 in a standard panel) including G-protein-coupled receptors, ion channels, enzymes and transporters. Different technical approaches, including binding or functional assays, have been adopted in establishing IPP. Binding assays use a fluorescent probe or radioisotope to measure a compound’s ability to displace the binding of said probe, while functional assays measure the activation, inhibition or modulation of the activity of the target organ or system. EC­50­­ (the concentration of a drug that gives half-maximal response) and LC­50­ values (the concentration of an inhibitor where response is reduced by half) greater than 50% are considered to represent significant effects of the test compound. 

CALUX is a panel of bioassays that evaluates transcriptional activity of chemicals in the presence and absence of metabolic activation. Bioassays comprise human bone cell lines (U2-OS) and a light-emitting reporter gene coupled to responsive elements. CALUX pathways are downstream from the IPP markers and can be used as a follow-up to IPP “positives” to evaluate whether the binding identified in the IPP assay actually results in a perturbation of the pathway. The PoDs here are the Lowest-Effect-Concentration (LEC) and the PC­50, defined as the concentration where the activity of the sample equals 50% of the maximum activity of the reference compound. 

ReproTracker is a validated NAM, but this focuses on the developmental toxicity endpoint. The most recent version of the assay, ReproTracker 2.0, measures the disruption of stem cell differentiation into heart, liver and neural lineages, and early embryonic development through expression of specific biomarker genes. Significant expression of biomarkers will lead to the classification of a compound as a teratogen. 

DevTox quickPredict is a human embryonic stem cell assay used for human developmental toxicity screening. It measures the changes in ornithine and cysteine, which are involved in the metabolic pathways important for normal cell proliferation and differentiation during development. An o/c ratio is calculated and used to predict the developmental and general toxicity potential of test chemicals. Results are reported in a metabolic response curve which gives the change in o/c metabolism following exposure to a test compound. 

The Zebrafish developmental toxicity test is used to explore effects of a test chemical on vertebrate development. It can shed light on the mechanisms associated with specific chemicals and determine teratogenicity. Embryos are harvested and incubated in an embryo media until they reach the specified stage for each assay. The media is then replaced with the test item, incubated and analysed post fertilisation for the presence of gross developmental defects and incidence of lethality. Embryos are then euthanised before statistical analysis. The percentage of morphological alterations and mortality are used to calculate EC­50­ and ­­LC­50­­­ values, of which the ratio is then used to calculate the tolerable intakes (TIs) at different developmental stages. A test compound that induces developmental defects, and has a single or combined TI ≥2 would be considered teratogenic. An indicative parameter is selected in order to compare data from the zebrafish assay to in vivo toxic effects in mammals.

Running a comprehensive set of NAMs to cover all systemic human health endpoints – such that NGRA offers an equivalent level of protection to the more familiar animal-based approach – is clearly a significant undertaking, requiring extensive expertise and a significant budget. It is unlikely that regulators will adopt NGRA as a wholesale replacement for risk assessments based on laboratory animal data any time soon. However, there is growing engagement with industry and academics to better understand these new assays and how they might form part of a future chemical assessment framework. 

Key papers: 

Middleton AM et al. (2022). Are Non-animal Systemic Safety Assessments Protective? A Toolbox and Workflow. Toxicological Sciences, 189(1), 124-147. https://doi.org/10.1093/toxsci/kfac068 

Wood A et al. (2024). Next generation risk assessment for occupational chemical safety – a real world example with sodium 2-hydroxyethane sulfonate.
https://doi.org/10.1016/j.tox.2024.153835

ECHA (2023). New Approach Methodologies workshop: Towards an animal-free regulatory system for industrial chemicals. https://echa.europa.eu/-/new-approach-methodologies-workshop-towards-an-animal-free-regulatory-system-for-industrial-chemicals