Effect of a binary mixture of ethanol and fluconazole on rat embryo development in vitro by using the double “fix and moving” protocol

The binary mixture of ethanol and of the clinically used antifungal fluconazole (FLUCO) has been tested on postimplantation rat whole embryos cultured in vitro (WEC method).The mixture model “fix and moving” has been applied: E9.5 embryos were exposed in vitro to Eth (17-42.5-85-127 mM), FLUCO (62.5-125-250-500M) or were co-exposed to the Eth NOAEL (17mM)+ the scaled concentrations of FLUCO or, vice versa, to the FLUCO NOAEL (62.5M)+ the scaled concentrations of Eth. At the end of the culture (48 h) embryos were morphologically examined to evaluate growth parameters and abnormalities. Concentration-related effects were observed after the exposure to Eth (42.5–127mM) or FLUCO (125–500M). The observed abnormalities were, for FLUCO, specifically at the level of the branchial apparatus, while Eth was able to induce multiple abnormalities, including defects at the level of branchial arches. Based on bench mark dose (BMD) and relative potency factor (RPF) approaches, the outcome of mixtures were modelled and the predicted effects, derived under the dose-additivity hypothesis, compared with the experimentally obtained results. The coexposure to Eth and FLUCO produced a significant enhancement of teratogenic effects on branchial arches in comparison to the single exposures. In addition, the mixture of the two molecules at their NOAELs resulted effective too, inducing almost 40% branchial arch abnormalities. Results from BMD approach show that, regarding branchial arches abnormalities, there are no significant deviations from concentration–additivity and suggest that FLUCO and Eth share, limited to the branchial pathogenesis, the same mode of action.