The European Partnership for Alternative Approaches to Animal Testing (EPAA) held the “New Approach Methodology (NAMs) User Forum” at the European Chemicals Agency, Helsinki, Finland on 30 – 31 October 2024. The User Forum brought together stakeholders from regulatory agencies, industry, non-governmental organisations (NGOs) and academia, as well as European Union competent authorities. Lessons learned from applying NAMs for regulatory use were provided by the European Food Safety Authority (EFSA) and European Chemicals Agency (ECHA). Progress in the development of the developmental and neurotoxicity in vitro battery (DNT IVB) and Alternative Safety Profiling Algorithm (ASPA) were described, as well as five case studies describing uses of NAMs for chemical safety assessment. The presentations confirmed progress in NAMs and, in particular, the value of tiered testing strategies to bring together different lines of evidence. Specifically, tiered testing strategies for non-animal information are organised into three tiers, which may be relevant to hazard, exposure and toxicokinetic information. Progress into, and the needs for improvement of, the tiered strategies were discussed with a particular focus on the types of NAMs (in silico and in vitro) that may be required at each tier and the how confidence may be assigned to making a decision. • NAMs described for hazard identification and exposure assessment • Updates on application of NAMs from EFSA and ECHA • Tiered testing strategies can assist in the regulatory implementation of NAMs • Case studies demonstrate the applicability of NAMs • Learnings and needs for NAMs’ development identified

Dietary uptake is estimated using residue data from food commodities and predicting the effect of food processing, such as peeling. The aim of this study was to explore the value of the analysis of duplicate portions supplemented by the analysis of urinary metabolites. Forty-three participants, consuming organic and non-organic diets collected a duplicate portion of food and beverages for 24 hours. Urine was collected up to 36 hours to account for metabolism and prolonged excretion of metabolites. Pesticide residues were analysed in food portions and urine samples using LC-MS/MS and GC-MS/MS. Multiple pesticide residues were detected per food portion. Out of 183 pesticides, 86 were detected in the diet. Glyphosate and AMPA were detected in 42% and 30% of all urine samples, respectively, while detection in the diet was low. A positive relationship between dietary intake and urinary excretion was found for 2,4-D and MCPA. No diet-urine relationship was observed for glyphosate and AMPA, indicating contribution from external routes of exposure. Hazard index (HI) indicated no exposure above the acceptable daily intake (ADI) for all participants. This study demonstrates how DPA combined with urine analysis gives insight into contribution of diet to total pesticide exposure compared to standard monitoring. • Duplicate portion analysis includes contributions of food processing and portioning • Data was collected from seven countries, and from organic and non-organic diets • DPA combined with urinary excretion provides valuable insights into exposure to pesticides • Mismatch between dietary portions and urinary excretion suggests non-dietary intake • Hazard index calculations assuming additivity indicated no exceedance of ADI