New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project

Abstract

In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools.

Keywords: DNT-IVB; adult neurotoxicity (ANT); applicability domain; developmental neurotoxicity (DNT); new approach method (NAM); zebrafish.

FIGURE 1

The DNT-IVB. Version 1.0 of the DNT-IVB (Masjosthusmann et al., 2020; Blum et al., 2022; OECD, 2023) contains endpoints for human neural progenitor cell (hNPC) proliferation and apoptosis, cell (neuronal, radial glia, oligodendrocytes) migration, hNPC-neuronal and oligodendrocyte differentiation, neurite outgrowth, neuronal maturation and synaptogenesis, and neuronal network formation (top). While most assays are conducted in human cells (blue boxes), two are performed in rat primary cells (yellow boxes). PARC 5.2.1e builds and evaluates NAMs for key battery gaps including myelination, blood-brain barrier (BBB) formation, mitochondrial function, and a suite of automated behavior-based NAMs in early life-stage zebrafish (green boxes). In addition, replacement of rodent-based NAMs with human-based test systems is also underway. Lastly, early epigenetic or sub-cellular morphological indicators of later DNT-related effects are being generated and evaluated for potential use in the DNT-IVB v2.0. Adapted from Crofton and Mundy, 2021.

FIGURE 2

Functional NAMs performed in early life stage zebrafish. A suite of automated zebrafish-embryo behavior-based NAMs are under development for potential inclusion in the DNT-IVB v2.0. All NAMs are performed using automated tracking. Most assays will be performed in 96 square well plates for a comparable throughput to in vitro assays. Based on the exposure paradigm used, these assays can be performed with protocols that predict DNT and/or ANT. For the detection of DNT, chemical exposure occurs during development and is removed prior to behavior assessments. In contrast, if exposure takes place after neurogenesis is complete, any adverse effect related to the specific signalling pathways that have reached maturation may be indicative of acute ANT. However, as the organism’s nervous system is still developing, it cannot be excluded that the nature of the effect may be considered within a DNT assessment framework.