Use of the alternative test R-FETAX (Refined-Frog Embryo Teratogenicity Assay-Xenopus) to evaluate the Fetal Alcohol Spectrum Disorders (FASD).

Fetal Alcohol Spectrum Disorders (FASD) refer to a range of conditions in children caused by alcohol consumption during pregnancy, including morphological defects, developmental delays, and neurobehavioral impairments. Ethanol (EtOH) at high concentrations (1-3 % v/v) was shown to induce malformations and lethality in Xenopus laevis embryos exposed throughout the FETAX test (from the mid-blastula stage to the final pre-feeding larval stage). The aim of this work was to evaluate multiple morphological and neurobehavioral effects of EtOH exposure (0.1-3 % v/v) using the R-FETAX protocol. Embryos obtained through natural mating were exposed during specific developmental windows: the organogenetic period (sensitive to morphological abnormalities) and the neurodevelopmental window (sensitive to behavioral alterations). Additional groups were exposed either throughout the entire test duration (classical FETAX exposure) or for a brief 4-hour period before the end of the test (acute exposure). Lethality was monitored over the six-day test period. At the conclusion of the test, a functional deglutition test was performed, and external gross morphology as well as developmental delays (FETAX-score method) were assessed. Neurobehavioral swimming test was conducted only on tadpoles considered normal at gross morphological evaluation. Dose-response relationships were modeled using PROAST software to derive benchmark dose levels, with response set at levels used as points of departure for risk assessment. The findings demonstrated dose- and stage-specific effects that mimic FASD symptoms observed in humans. These results emphasize that no amount of alcohol exposure can be considered safe during development.