Advancing the Neurosphere Assay as a Regulatory-Ready NAM: Multiparametric Assessment of CPF and PFAS Mixtures Across Key Neurodevelopmental Endpoints

Background: Developmental neurotoxicity (DNT) arises from disruption of key neurodevelopmental processes, including proliferation, differentiation, migration, and synaptogenesis. Traditional in vivo testing is costly and lacks mechanistic resolution, prompting OECD and EFSA to endorse new approach methodologies (NAMs) such as the neurosphere assay (NSA). Methods: We validated a mouse-derived NSA over a three-week differentiation period using multiparametric endpoints—proliferation, neuronal and glial differentiation, radial migration, synaptogenesis, and astrocytic maturation. Baseline characterization was established by flow cytometry, confocal immunofluorescence, and qPCR. Toxicants were applied under two exposure scenarios: (i) chronically during the 7-day proliferation phase, and (ii) chronically after three days of proliferation and maintained throughout the three-week differentiation period. Chlorpyrifos (CPF) served as a DNT-positive reference, and a biomonitoring-relevant mixture of PFAS (PFOS, PFOA, PFUnDA, PFHxS) was designed from the French Esteban study. Results: Baseline analyses confirmed progressive neuronal and glial differentiation, synaptic maturation, and growth factor responsiveness. CPF induced biphasic effects: early enlargement, subsequent growth arrest, migration impairment, overt cytotoxicity at 250 µM, and GFAP downregulation. PFAS mixtures produced modest viability effects but consistently reduced migration and downregulated GFAP and SYP at low-nM levels, consistent with epidemiological and experimental evidence linking PFAS to neurodevelopmental disorders. Migration emerged as a more sensitive endpoint than bulk viability, revealing functional impairments below overt toxicity thresholds. Conclusions: The NSA faithfully recapitulates corticogenesis and detects toxicant-specific liabilities, with CPF impairing astrocytic maturation and PFAS mixtures disrupting astrocytic and synaptic programs at environmentally relevant levels. By integrating complementary endpoints under chronic exposure conditions across proliferation and differentiation, this study advances the NSA as a mechanistic, human-relevant, and regulatory-ready NAM for DNT assessment.