Inefficacy of dietary test substance administration in Amphibian Metamorphosis Assay (AMA) studies

Traditionally, the Amphibian Metamorphosis Assay (AMA; OECD TG 231) is performed by exposing Xenopus laevis tadpoles to test substances dissolved in laboratory water. Recently, the use of dietary administration has been proposed to combat poorly soluble test substances in ecotoxicologically-based regulatory endocrine disruption (ED) studies, specifically the AMA warranting an investigation into the efficacy of dietary administration. An efficacy study comprised of two phases: 1) evaluation of the physical influence of the loading process via solvent and 10, 1, and 0.1 mg/l test substance or surrogate (sunflower oil, SFO) on the Sera® Micron Nature (SMN) diet, and 2) performance of a modified AMA in which Nieuwkoop and Faber (NF) stage 51 X. laevis larvae were exposed to dechlorinated tap water using one concentration of the SFO in the diet for 21 days, was performed. In phase 1, the addition of acetone or acetone with bis(2-propylheptyl) phthalate (DPHP) or SFO to SMN with subsequent solvent purge altered the diet reducing the density of the liquified diet and dietary pellet size following centrifugation indicative of alteration of the physical properties of the diet. Treatments used in the modified AMA were acetone alone and 0.1 mg/l SFO dissolved in acetone. These treatments were evaluated against an SMN benchmark using standard AMA endpoints. Both the acetone-treated SMN and 0.1 mg/l SFO-treated diets significantly reduced survival rates, 67 and 70% relative to the SMN benchmark (100%), decreased developmental stage distribution and snout-vent length-normalized hind limb length relative to the SMN benchmark, and slightly increased the prevalence and severity of thyroid follicular cell hypertrophy. Although the acetone-treated diets may have impacted the hypothalamo-pituitary-thyroid axis, clinical signs of gastrointestinal impaction and tail flexure were also observed in the acetone-treated diets, but not the SMN diet alone. Ultimately, test substance exposure via the diet in an AMA study can produce results that may confound data interpretation, which suggests that the traditional aqueous exposure route is generally more appropriate.