Amphibian metamorphosis assays- biological & histopathological data and range finding studies

Five chemicals [2-ethylhexyl 4-hydroxybenzoate (2-EHHB), 4-nonylphenol-branched (4-NP), 4-tert-octylphenol (4-OP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) were subjected to a 21-day Amphibian Metamorphosis Assay (AMA) following OCSPP 890.1100 test guidelines. The selected chemicals exhibited estrogenic or androgenic bioactivity in high throughput screening data obtained from US EPA ToxCast models. Xenopus laevis larvae were exposed nominally to each chemical at 3.6, 10.9, 33.0 and 100 µg/L, except 4-NP for which concentrations were 1.8, 5.5, 16.5 and 50 µg/L. Endpoint data (daily or given study day (SD)) collected included: mortality (daily), developmental stage (SD 7 and 21), hind limb length (HLL) (SD 7 and 21), snout-vent length (SVL) (SD 7 and 21), wet body weight (BW) (SD 7 and 21), and thyroid histopathology (SD 21). 4-OP and BBP caused accelerated development compared to controls at the mean measured concentration of 39.8 and 3.5 µg/L, respectively. Normalized HLL was increased on SD 21 for all chemicals except 4-NP. Histopathology revealed mild thyroid follicular cell hypertrophy at all BBP concentrations, while moderate thyroid follicular cell hypertrophy occurred at the 105 µg /L BBP concentration. Evidence of accelerated metamorphic development was also observed histopathologically in BBP-treated frogs at concentrations as low as 3.5 µg/L. Increased BW relative to control occurred for all chemicals except 4-OP. Increase in SVL was observed in larvae exposed to 4-NP, BBP and DBP on SD 21. With the exception of 4-NP, four of the chemicals tested appeared to alter thyroid axis-driven metamorphosis, albeit through different lines of evidence, with BBP and DBP providing the strongest evidence of effects on the thyroid axis.