Application of AOPs to assist regulatory assessment of chemical risks - Case studies, needs and recommendations.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

Interactive toxicity of copper and cadmium in regenerating and adult planarians.

In a polluted environment, metals are present as complex mixtures. As a result, organisms are exposed to different metals at the same time, which affects both metal-specific as well as overall toxicity. Detailed information about the molecular mechanisms underlying the adverse effects of combined exposures remains limited in terms of different life stages. In this study, the freshwater planarian Schmidtea mediterranea was used to investigate developmental and physiological responses associated with a combined exposure to Cu and Cd. In addition, the cellular and molecular mechanisms underlying the provoked adverse effects were studied in different exposure scenarios. Mixed exposure resulted in a decline in survival, diverse non-lethal morphological changes, neuroregenerative impairments, altered behaviour and a limited repair capacity. Underlying to these effects, the cellular redox state was altered in all exposure conditions. In adult animals, this led to DNA damage and corresponding transcriptional changes in cell cycle and DNA repair genes. In regenerating animals, changes in hydrogen peroxide and glutathione contents led to regenerative defects. Overall, our results demonstrate that (1) developing organisms are more susceptible to metal exposures, and (2) the toxicity of an individual metal increases significantly in a mixed exposure scenario. These aspects have to be included in current risk assessment strategies.