From Mechanisms to Decisions: Adverse Outcome Pathways Reshaping the Future of Risk Assessment

The ongoing transition toward Next-Generation Risk Assessment (NGRA) is reshaping regulatory toxicology and the risk assessment of regulated products by promoting exposure-driven, human-relevant and mechanistically informed approaches to evaluate potential risks without relying on animal testing. In this context, Adverse Outcome Pathways (AOPs) serve as a transparent and structured framework to organize mechanistic evidence and support the integration of New Approach Methodologies (NAMs) into risk assessment processes. By linking molecular initiating events to key events across biological levels and, ultimately, to adverse outcomes, AOPs provide biologically plausible and regulatorily meaningful mechanistic information. This work critically examines how AOPs and AOP networks developed by the European Food Safety Authority (EFSA) are being integrated into pesticide risk assessment. Two regulatory-relevant case studies are analyzed: (i) pesticide-induced mitochondrial dysfunction leading to Parkinson’s disease, and (ii) endocrine-disrupting mechanisms leading to uterine adenocarcinoma. For both cases, we reconstructed the AOP event flows and associated NAM batteries, assessing their scientific robustness, applicability domains, and regulatory readiness. The analysis shows that NAM-based evidence can comprehensively cover multiple key events—such as mitochondrial dysfunction, proteostasis impairment, ERα activation and epigenetic alterations—while also revealing persistent limitations, including gaps in toxicokinetic considerations, limited quantitative understanding of key event relationships, and challenges in characterizing uncertainties. Overall, our findings illustrate how AOPs can strengthen mechanistic integration, improve evidence transparency and support regulatory prioritization, while also highlighting the critical need for enhanced toxicokinetic data. In particular, further improvements —such as qAOPs and TK–TD integration— are essential to fully enable NGRA in pesticide risk assessment.