Evaluation of temperature- and ethanol-related developmental degree variations by a new scoring system (FETAX-score) applicable to Frog Embryo Teratogenicity Assay: Xenopus.

The aim of the present work is to propose a new quantitative assessment method (FETAX-score) for determining the degree of Xenopus laevis embryo development intended for use in embryotoxicity studies. Inspired by a similar scoring system used to evaluate developmental delays (young-for-age phenotypes) in rat embryos cultured in vitro, the FETAX-score was established by considering seven morphological features (head, naris, mouth, lower jaw, tentacles, intestine, anus) that are easily evaluable in tadpoles during the late stages of development at the conclusion of the test. Given that X. laevis development is temperature-dependent and that temperatures below 14°C and above 26°C are teratogenic, the FETAX-score was tested in embryos maintained at 17, 20, 23 and 26°C. No abnormalities were observed in any group, while the total score was temperature-related, suggesting that the FETAX-score is sensitive to moderate distress that does not influence general morphology. Intestine and anus were the least sensitive structures to temperature variations. To assess the applicability of the FETAX-score in developmental toxicological studies, we evaluated FETAX-score in tadpoles exposed during the morphogenetic period to Ethanol (Eth) at concentrations of 0, 0.25, 0.5, 1, 1.5, and 2% v/v. Gross malformations were observed only in tadpoles from the Eth 2% group. By contrast, data analysis of the other Eth groups showed dose-related reductions in the FETAX-score. Tentacles were the most sensitive structures to Eth-related delays. These results support the use of the FETAX-score to quantitatively assess developmental deviations in FETAX embryotoxicity studies.

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Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Elena Menegola reports financial support was provided by University of Milan. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Competing interests The authors have no relevant financial or non-financial interests to disclose. Authors’contribution All authors contributed to the study conception and design. Material preparation was performed by M. Battistoni, F. Di Renzo, R. Bacchetta, E. Menegola; data collection and analysis were performed by M. Battistoni, F. Di Renzo, E. Menegola. Data modelling was performed by F. Metruccio, E. Menegola. The first draft of the manuscript was written by E. Menegola; all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.