An in silico protocol for endocrine activity assessment: Integrating predictions, experimental evidence, and expert reviews across estrogen, androgen, thyroid, and steroidogenesis modalities

Endocrine disruption (ED) has been introduced as a new classification, labelling and packaging (CLP) hazard category under Regulation (EC) No 1272/2008. Additionally, consideration of endocrine-disrupting properties and endocrine-related effects continues to be an important aspect of chemicals management under the Canadian Environmental Protection Act (CEPA) 1999 for the prioritization and hazard characterization of potential hormone disrupting substances. To support chemical prioritization and hazard assessment, this study presents a structured in silico protocol for assessing endocrine activity across the estrogen (E), androgen (A), thyroid (T), and steroidogenesis (S) (EATS) modalities. The protocol integrates (Quantitative) Structure–Activity Relationship ((Q)SAR) predictions with experimental data using a structured approach grounded in a hazard assessment framework (HAF) and defines principles for evaluating the reliability and confidence of predictions. Key endpoints and model development opportunities are identified for each modality. Two case studies are presented to demonstrate the application of the protocol. In the assessment of 4-Chloro-1-[2,2-dichloro-1-(4-chlorophenyl)ethenyl]-2-(methylsulfonyl)benzene, structurally similar analogs supported a medium-confidence assessment of estrogen and androgen activity. Whereas, in the assessment of chloroprene, uncertainties due to potential metabolic transformation limited confidence in negative assessments. These case studies illustrate how model outputs, experimental evidence, an analysis of analogs, and expert review can be integrated to produce transparent and reproducible assessments. The framework supports a weight-of-evidence (WOE) non-testing approach for identifying endocrine-active substances.