Adaption of a dermal in vitro method to investigate the uptake of chemicals across amphibian skin

Background: Literature data indicate that terrestrial life stages of amphibians may be more sensitive to xenobiotics than birds or mammals. It is hypothesized that dermal exposure could potentially be a significant route of exposure for amphibians, as there is evidence that their skin is more permeable than the skin of other vertebrate species. Thus, higher amounts of xenobiotics might enter systemic circulation by dermal uptake resulting in adverse effects. Heretofore, no guidelines exist to investigate dermal toxicity of chemicals to amphibians. In order to minimize vertebrate testing, this work was targeted to develop an in vitro test system as a possible model to assess the dermal uptake of chemicals across amphibian skin. Results: The dermal absorption in vitro method (OECD guideline 428), an established toxicological (mammal) test procedure, was adapted to amphibian skin, in a first approach using the laboratory model organism Xenopus laevis and reference compounds (caffeine and testosterone). Skin permeability to both reference substances was significantly higher compared to published mammalian data. Caffeine permeated faster across the skin than testosterone, with ventral skin tending to be more permeable than dorsal skin. As usage of frozen mammalian skin is accepted, frozen skin of X. laevis was tested in parallel. To the freshly excised skin, however, freezing led to increased skin permeability, in particular to caffeine, indicating a loss of skin integrity due to freezing (without additional preservation measures). Conclusions: This work has demonstrated that the chosen method can be applied successfully to amphibian skin, providing the basis for further investigations. In future, well-established in vitro test systems and a broad dataset for many chemicals may help assess potential amphibian risk from xenobiotics without the need for extensive vertebrate testing.