A Decision Tree for Acute Oral Toxicity: Implementing the Last Resort Requirement in Practice.

Acute oral toxicity (AOT) testing is widely used for hazard identification in chemical safety assessment, but traditional animal methods are still implemented despite their scientific limitations and ethical implications. Despite the availability of non-animal methods, regulatory uptake has been inconsistent, also due to a lack of structured guidance. To address this, the Animal-Free Safety Assessment Collaboration has developed a science-based Decision Tree (DT) aligned with the Organisation for Economic Cooperation and Development (OECD) Integrated Approaches to Testing and Assessment (IATA) framework. IATA integrate existing information in a Weight of Evidence (WoE) assessment with the possible generation of new data appropriate to the decision context. The DT is built as a specific IATA to provide a transparent, modular workflow integrating in silico tools, in vitro assays, and WoE strategies to support AOT hazard identification for regulatory decision-making, such as European Union's Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) Regulation, ensuring that animal testing is used only as a last resort. The DT aims to inform regulatory guidance updates and promote acceptance of non-animal approaches by formalising decision-making to bridge the gap between legal mandates to reduce animal testing and current regulatory practice, still heavily reliant on animals, supporting the transition toward improved chemical safety evaluations.