Dose addition in mixtures of compounds with dissimilar endocrine modes of action in in vitro receptor activation assays and the zebrafish sexual development test

Human exposure to pesticides is being associated with feminisation for which a decrease of the anogenital distance (AGD) is a sensitive endpoint. Dose addition for the cumulative risk assessment of pesticides in food is considered sufficiently conservative for combinations of compounds with both similar and dissimilar modes of action (MoA). The present study was designed to test the dose addition hypothesis in a binary mixture of endocrine active compounds with a dissimilar mode of action for the endpoint feminisation. Compounds were selected from a list of chemicals of which exposure is related to a decrease of the AGD in rats and completed with reference compounds. These chemicals were characterised using specific in vitro transcriptional activation (TA) assays for estrogenic and androgenic properties, leading to a final selection of dienestrol as an ER-agonist and flutamide, linuron, and deltamethrin as AR-antagonists. These compounds were then tested in an in vivo model, i.e. in zebrafish (Danio rerio), using sex ratio in the population as an endpoint in order to confirm their feminising effect and MoA. Ultimately, the fish model was used to test a binary mixture of flutamide and dienestrol. Statistical analysis of the binary mixture of flutamide and dienestrol in the fish sexual development tests (FSDT) with zebrafish supported dose addition.