Search

There is a call for a paradigm shift in developmental neurotoxicity (DNT) evaluation, which demands the implementation of faster, more cost-efficient, and human-relevant test systems than current in vivo guideline studies. Under the umbrella of the Organisation for Economic Co-operation and Development (OECD), a guidance document is currently being prepared that instructs on the regulatory use of a DNT in vitro battery (DNT IVB) for fit-for-purpose applications. One crucial issue for OECD application of methods is validation, which for new approach methods (NAMs) requires novel approaches. Here, mechanistic information previously identified in vivo, as well as reported neurodevelopmental adversities in response to disturbances on the cellular and tissue level, are of central importance. In this study, we scientifically validate the Neurosphere Assay, which is based on human primary neural progenitor cells (hNPCs) and an integral part of the DNT IVB. It assesses neurodevelopmental key events (KEs) like NPC proliferation (NPC1ab), radial glia cell migration (NPC2a), neuronal differentiation (NPC3), neurite outgrowth (NPC4), oligodendrocyte differentiation (NPC5), and thyroid hormone-dependent oligodendrocyte maturation (NPC6). In addition, we extend our work from the hNPCs to human induced pluripotent stem cell-derived NPCs (hiNPCs) for the NPC proliferation (iNPC1ab) and radial glia assays (iNPC2a). The validation process we report for the endpoints studied with the Neurosphere Assays is based on 1) describing the relevance of the respective endpoints for brain development, 2) the confirmation of the cell type-specific morphologies observed in vitro, 3) expressions of cell type-specific markers consistent with those morphologies, 4) appropriate anticipated responses to physiological pertinent signaling stimuli and 5) alterations in specific in vitro endpoints upon challenges with confirmed DNT compounds. With these strong mechanistic underpinnings, we posit that the Neurosphere Assay as an integral part of the DNT in vitro screening battery is well poised for DNT evaluation for regulatory purposes.

Introduction: The Adverse Outcome Pathway (AOP) concept facilitates rapid hazard assessment for human health risks. AOPs are constantly evolving, their number is growing, and they are referenced in the AOP-Wiki database, which is supported by the OECD. Here, we present a study that aims at identifying well-defined biological areas, as well as gaps within the AOP-Wiki for future research needs. It does not intend to provide a systematic and comprehensive summary of the available literature on AOPs but summarizes and maps biological knowledge and diseases represented by the already developed AOPs (with OECD endorsed status or under validation).Methods: Knowledge from the AOP-Wiki database were extracted and prepared for analysis using a multi-step procedure. An automatic mapping of the existing information on AOPs (i.e., genes/proteins and diseases) was performed using bioinformatics tools (i.e., overrepresentation analysis using Gene Ontology and DisGeNET), allowing both the classification of AOPs and the development of AOP networks (AOPN).Results: AOPs related to diseases of the genitourinary system, neoplasms and developmental anomalies are the most frequently investigated on the AOP-Wiki. An evaluation of the three priority cases (i.e., immunotoxicity and non-genotoxic carcinogenesis, endocrine and metabolic disruption, and developmental and adult neurotoxicity) of the EU-funded PARC project (Partnership for the Risk Assessment of Chemicals) are presented. These were used to highlight under- and over-represented adverse outcomes and to identify and prioritize gaps for further research.Discussion: These results contribute to a more comprehensive understanding of the adverse effects associated with the molecular events in AOPs, and aid in refining risk assessment for stressors and mitigation strategies. Moreover, the FAIRness (i.e., data which meets principles of findability, accessibility, interoperability, and reusability (FAIR)) of the AOPs appears to be an important consideration for further development.