Testing Strategies for Metabolite-Mediated Neurotoxicity

Compounds, which rely on metabolism to exhibit toxicity, pose a challenge for next-generation risk assessment (NGRA). Since many of the currently available non-animal new approach methods (NAMs) lack metabolic activity, their use may lead to an underestimation of the true hazard to humans (false negative predictions). We explored here strategies to deal with metabolite-mediated toxicity in assays for developmental neurotoxicity. First, we present an overview of substances that may serve as potential positive controls for metabolite-related neurotoxicity. Then, we demonstrate, using the MitoMet (UKN4b) assay, which assesses the adverse effects of chemicals on neurites of human neurons, that some metabolites have a higher toxic potency than their parent compound. Next, we designed a strategy to integrate elements of xenobiotic metabolism into assays used for (developmental) neurotoxicity testing. In the first step of this approach, hepatic post-mitochondrial fractions (S9) were used to generate metabolite mixtures ("metabolisation module"). In the second step, these were applied to a NAM (exemplified by the UKN4b assay) to identify metabolite-mediated toxicity. We demonstrate the applicability and transferability of these approaches to other assays, by an exemplary study on the basis of the cMINC (UKN2) assay, another NAM of the developmental neurotoxicity in vitro battery. Based on the experience gained from these experiments, we discuss key issues to be addressed if this approach is to be used more broadly for NAM in the NGRA context.