Mapping disease-linked AOPs to inform bioassay selection for chemical hazard assessment

Modern societies synthesize, distribute, and utilize thousands of chemicals, but their contribution to the global burden of non-communicable diseases remains insufficiently characterized. Improved availability of robust hazard data would enable more systematic assessment, prioritization, and management of chemical-related health risks. New Approach Methodologies (NAM) are promising because they provide high-throughput, mechanistically informative, and animal-free testing that improves current regulatory toxicity testing. However, selecting biologically relevant in-vitro assays capable of predicting chemical-induced effects remains a challenge. Here, we present a systematic strategy for assembling relevant in-vitro assays. We identified critical molecular and cellular key events of Adverse Outcome Pathways (AOPs) across six clusters of diseases that can be triggered by chemical exposure. Curation and filtering of 487 AOPs resulted in 266 AOPs, which were manually mapped to the AOP networks of disease clusters containing 25-84 AOPs each. The three most frequently occurring key events across all clusters were cytotoxicity (6/6 clusters), oxidative stress (5/6 clusters), and mitochondrial dysfunction (4/6 clusters). This prioritization aligns with oxidative stress as an overarching "key characteristic of toxicants". Receptor-mediated key events were also frequent, with aryl hydrocarbon and estrogen receptor activation in multiple clusters. Other key events were cluster-specific, such as triglyceride accumulation in metabolic diseases. Building on this analysis, we propose high-throughput cell-based assays covering critical endpoints by integrating the key characteristics of toxicants framework to ensure coverage beyond current AOP limitations. A panel of 15 multiplexed assays meeting criteria for simplicity, robustness, and high-throughput could enable efficient screening of single chemicals or chemical mixtures.