New advanced models (NAMs) for risk assessment of bisphenol A alternatives.

The safety of bisphenol A (BPA) due to its adverse effects on the immune system has led to an increasing concern and a significant regulatory shift. The European Food Safety Authority (EFSA) proposed a reduction in the tolerable daily intake (TDI) of BPA in food in their 2023 scientific opinion, highlighting the need for stricter regulations compared to their previous assessment in 2015. This regulatory action has spurred the production of BPA alternatives, raising concerns about their safety due to insufficient toxicological data. Addressing this gap is crucial for ensuring human and environmental health. In this project, multiple genotoxicity endpoints were applied for testing of two regulatory relevant BPA alternatives, bisphenol E (BPE) and bisphenol P (BPP), in different human models: 2D HepG2 liver cells, 3D liver spheroids and primary human peripheral blood lymphocytes. DNA strand breaks and oxidised base lesions were evaluated by the enzyme-modified version of the comet assay, while clastogenicity and aneugenicity were analysed by the in vitro micronucleus assay (OECD TG 487, 2016), together with cytotoxicity. Development of new advanced models (NAMs), as 3D spheroids, are essential for next-generation risk assessment (NGRA) in line with the 3R's to replace, reduce or refine animal experiments. In this aspect, validation and standardisation of NAMs are needed to reach regulatory readiness level and development of OECD Test Guidelines. Therefore, a standardisation and pre-validation of the advanced 3D liver spheroid model was performed by using multiple genotoxicity endpoints and by comparing the obtained results with standard genotoxicity models.