Functional Mapping of Neurodevelopmental Disease Pathways to Key Neurodevelopmental Processes Represented in the Developmental Neurotoxicity In Vitro Testing Battery

ABSTRACT The Developmental Neurotoxicity (DNT) in vitro battery (IVB) enables efficient and human‐relevant evaluation of chemicals for DNT potential. To expand its biological applicability domain toward human disease, this study maps neurodevelopmental disorder (NDD)‐relevant signaling pathways to key neurodevelopmental processes (KNDPs) using primary human fetal neural progenitor cells (NPCs). Using pharmacological intervention, eighteen NDD pathways are assessed for their impact on seven KNDPs, namely NPC proliferation, radial glia migration, neuronal and oligodendrocyte differentiation and migration, and neurite outgrowth. In total, modulation of sixteen pathways is associated with changes in at least one KNDP. Oligodendrocyte differentiation shows the highest sensitivity (13 pathways), followed by radial glia migration (11 pathways) and NPC proliferation (9 pathways), whereas neuronal migration remains unaffected. Perturbation of the RhoA and mitochondrial complex I pathways is associated with the broadest phenotypic responses, influencing five KNDPs each, while STAT3‐ and TrkB‐related modulation falls outside the assay's applicability domain. Pathway‐KNDP associations are integrated into an exemplary interactive physiological map of human oligodendrocyte development, linking mechanistic perturbations to human‐relevant biology. Defining which NDD pathways can be functionally probed refines the DNT IVB's biological applicability domain, increases confidence in its protective power, and supports mechanistic interpretation of new approach methodology‐based DNT assessment.