P22-47 Advanced in vitro and ecotoxicological models for Graphene Oxide risk assessment: Insights from the POTENTIAL project

The POTENTIAL (for Platform Optimisation To Enable NanomaTerIAL safety assessment for rapid commercialisation) project aims to provide industrial stakeholders with harmonized, accessible protocols for the characterization, toxicity testing, and risk assessment of advanced nanomaterials (Ad-NMs), with a focus on Graphene Oxide (GO) particles presenting different oxidation states. As inhalation and ingestion represent key exposure routes, pulmonary and intestinal in vitro models were selected to evaluate GO toxicity. A tiered testing strategy was implemented, ranging from simple monocultures to physiologically more relevant 3D co-culture model. Submerged monocultures of A549 alveolar epithelial and Caco-2 intestinal epithelial cells were used to assess cytotoxicity, offering cost-effective and high-throughput options for early hazard identification. In parallel, the ALIsens® 3D alveolar model was employed under Air-Liquid Interface (ALI) conditions to better mimic in vivo -like exposure scenarios. This model combines alveolar epithelial, endothelial, and immune cell lines, allowing the study of intercellular interactions and immune responses. Cytotoxicity was evaluated using different viability assays (MTS, Alamar Blue, LDH, and ATP assays), to determine IC50 values while accounting for potential assay interference due to the reactive surfaces of GO particles. Protocol reproducibility and robustness were assessed through parallel testing in two independent laboratories (LIST and IRFMN). Results indicated that submerged monocultures combined with ATP release measurement at 24 h provide a reproducible and sensitive method for toxicity assessment. Nevertheless, the use of the 3D ALIsens® co-culture model may offer enhanced insight into GO toxicity, especially where cellular interactions and immune response pathways are involved. To complement human-relevant in vitro models, ecotoxicological assessments were performed using Daphnia magna and Caenorhabditis elegans as representative environmental organisms. Toxicity endpoints included survival, reproduction, and physiological stress. OECD Guideline TG202 was adapted to evaluate acute toxicity in D. magna , with pH variation to reflect environmental conditions influencing GO toxicity. C. elegans was employed to investigate developmental and sublethal effects, contributing additional data for environmental risk evaluation. In summary, the POTENTIAL project provides a reproducible and harmonized framework for the toxicity and ecotoxicity testing of GO particles. This approach, integrating simple and advanced in vitro models with standardized ecotoxicological assays, supports regulatory decisionmaking and safe innovation in the field of nanomaterials.