Micro- and nanoplastic pollution poses a global threat due to environmental accumulation, poor reversibility, and adverse effects on humans and wildlife. Nanoplastics can cross the blood-brain barrier (BBB) and induce neurotoxicity, yet studies investigating the developmental effects of nanoplastic exposure during early life (a period vulnerable to environmental stressors) and their influence on later-life outcomes remain limited. This study examined whether early-life exposure to polystyrene nanoplastics (PS-NPs) during embryonic and larval stages has lasting effects and alters susceptibility to hexabromocyclododecane (HBCD), a brominated flame retardant globally detected in aquatic environment, in zebrafish. Developmental exposure to PS-NPs led to reduced locomotor activity in both larvae (5 dpf) and adults (157 dpf). Furthermore, early PS-NP exposure modified behavioral response to HBCD in adulthood: adult fish exposed to HBCD for the first time exhibited hyperactivity, whereas those previously exposed to PS-NPs showed no significant change. These differential responses were strongly correlated with altered expressions of neurological and neurodevelopmental genes (e.g., drd2a , glud1a , fezf2 ) and global DNA methylation levels. Our findings suggest that epigenetic processes may contribute to the observed differences in adult locomotor behavior and susceptibility to subsequent HBCD exposure. This study highlights the need for further research into locus-specific epigenetic regulation of neural genes during development and their quantitative relationship to adverse outcomes. In light of the conservation of epigenetic mechanisms across species and the widespread presence of environmental substances from industrial and consumer products that can disrupt these pathways, our study underscores the broader implications for environmental health. • Early exposure to PS-NPs reduced locomotor activity in larvae and adult zebrafish. • Developmental PS-NPs can influence adult response to HBCD-induced hyperactivity. • PS-NPs disrupted expression of neurodevelopmental and neurotransmission genes. • Distinct global DNA methylation patterns were observed across exposure scenarios. • Epigenetic regulation may contribute to persistent neurobehavioral outcomes.

The rapid transition toward animal-free chemical safety evaluation has positioned in vitro new approach methodologies (NAMs) as central components of next-generation risk assessment (NGRA). Advances in complex in vitro systems, high-content phenotypic profiling, multi-omics technologies, and AI-assisted analytics have greatly expanded the capacity to characterize human-relevant biological responses. However, despite their scientific promise, the translation of NAM-derived information into regulatory decision-making remains challenging in general. Key bottlenecks include incomplete alignment with apical regulatory endpoints, limited toxicokinetic context in conventional in vitro systems, and substantial variability across assays, data structures, and analytical pipelines. This review aims to summarize the current state of in vitro NAM technologies, evaluate the major barriers limiting their regulatory application, and discuss emerging frameworks that enable their integration into NGRA. To strengthen regulatory relevance, increasing efforts focus on integrating mechanistic NAM outputs into adverse outcome pathway (AOP) frameworks and applying high-throughput toxicokinetic (HTTK) modeling to support in vitro –to– in vivo extrapolation (IVIVE). Early NGRA case studies show that NAM-based points of departure can, in some instances, approximate or bracket traditional in vivo thresholds, although results remain heterogeneous across chemical classes and endpoint domains. Going forward, progress in NAM-based risk assessment will depend not only on advancements in assay technologies but also on decision frameworks capable of effectively incorporating existing NAM evidence. Tiered and evidence-integrated approaches will be essential, particularly in light of the varied NAM data availability across chemicals. Strengthening the iterative exchange between NAM application and method development will help guide future improvements and support a more transparent, adaptive, and human-relevant assessment paradigm.