A larval amphibian growth and development assay was performed to evaluate the potential effects of environmentally-relevant concentrations of triclosan (TCS) on amphibian development and growth. Xenopus laevis were exposed to TCS 0.0 (control), 6.3, 12.5 and 25.0 mu g l(-1) (estimated maximum tolerable concentration) until 10 weeks post-metamorphosis. At median metamorphosis time (Nieuwkoop and Faber stage 62), five larvae per replicate were collected for snout-vent length, hind limb length and body weight measurements, and histopathological examination of thyroid glands. Endpoints evaluated at test termination were based on draft guidance (USEPA, 2010) and included: survival; snout-vent length; body weight; gender; nuptial pad development (males); and liver, kidney, gonad and gonadal ducts histopathology. Exposure to TCS did not decrease survival, induce general signs of toxicity, affect median metamorphosis time or alter sex ratios. Exposure to TCS 12.5 and 25 mu g l(-1) increased growth during the metamorphic stages relative to the control, but did not influence growth during the postmetamorphic phase. Overall, several statistically significant findings were found in larvae exposed to TCS, such as a decrease in the prevalence of stage 3 Mullerian ducts in the anterior trunk sections of TCS 25.0 mu g l(-1) dose group females as compared to controls; most were not considered toxicologically relevant. Copyright (C) 2017 John Wiley & Sons, Ltd.

This work tests the mode-of-action (MOA) hypothesis that maternal and developmental triclosan (TCS) exposure decreases circulating thyroxine (T4) concentrations via up-regulation of hepatic catabolism and elimination of T4. Time-pregnant Long-Evans rats received TCS po (0–300 mg/kg/day) from gestational day (GD) 6 through postnatal day (PND) 21. Serum and liver were collected from dams (GD20, PND22) and offspring (GD20, PND4, PND14, PND21). Serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) concentrations were measured by radioimmunoassay. Ethoxy-O-deethylase (EROD), pentoxyresorufin-O-depentylase (PROD) and uridine diphosphate glucuronyltransferase (UGT) enzyme activities were measured in liver microsomes. Custom Taqman® qPCR arrays were employed to measure hepatic mRNA expression of select cytochrome P450s, UGTs, sulfotransferases, transporters, and thyroid hormone-responsive genes. TCS was quantified by LC/MS/MS in serum and liver. Serum T4 decreased approximately 30% in GD20 dams and fetuses, PND4 pups and PND22 dams (300 mg/kg/day). Hepatic PROD activity increased 2–3 fold in PND4 pups and PND22 dams, and UGT activity was 1.5 fold higher in PND22 dams only (300 mg/kg/day). Minor up-regulation of Cyp2b and Cyp3a expression in dams was consistent with hypothesized activation of the constitutive androstane and/or pregnane X receptor. T4 reductions of 30% for dams and GD20 and PND4 offspring with concomitant increases in PROD (PND4 neonates and PND22 dams) and UGT activity (PND22 dams) suggest that up-regulated hepatic catabolism may contribute to TCS-induced hypothyroxinemia during development. Serum and liver TCS concentrations demonstrated greater fetal than postnatal internal exposure, consistent with the lack of T4 changes in PND14 and PND21 offspring. These data support the MOA hypothesis that TCS exposure leads to hypothyroxinemia via increased hepatic catabolism; however, the minor effects on thyroid hormone metabolism may reflect the low efficacy of TCS as thyroid hormone disruptor or highlight the possibility that other MOAs may also contribute to the observed maternal and early neonatal hypothyroxinemia.

In a previously reported study, we used a standard metamorphosis anuran model to assess potential effect of the antibacterial agent triclosan (TCS) on normal prometamorphic Xenopus laevis. Results indicated that environmentally relevant TCS concentrations did not alter the normal course of thyroid-mediated metamorphosis in this standard anuran model. However, to examine potential effects of TCS exposure during premetamorphosis and to distinguish between effects on metamorphosis and effects on growth, a longer term TCS exposure study was conducted. Standard Nieuwkoop and Faber (NF) stage 47 X. laevis larvae were exposed for 32 days (ca. NF stage 59-60) via flow-through to four different concentrations of TCS: < 0.2 (control), 0.8, 3.1, 12.5, or 50.0 μg TCS/l. Primary endpoints were survival, hind limb length, body length (whole; snout-to-vent), developmental stage, wet whole body weight, thyroid histology, plasma thyroid hormone (TH) concentrations, TH receptor beta (TRβ), and type II and III deiodinase (DI-2 and DI-3) expression. Endpoints measured to evaluate effects on thyroid-mediated metamorphosis including developmental stage, thyroid histology, TRβ expression, DI-2 and DI-3 expression, and thyroid gland 3,5,3',5'-tetraiodothyronine (T4) and plasma T4 and 3,5,3'-triiodothyronine (T3) levels were not affected by TCS exposure. However, increased larval growth based on whole body length (0.78, 12.5, and 50 μg TCS/l), snout-vent length (3.1 and 12.5 μg TCS/l), and whole body weight (0.8, 12.5, and 50.0 μg TCS/l) was observed following 32-day TCS exposure. These results indicated that TCS exposure during pre- and prometamorphosis increased larval growth but did not alter the normal course of metamorphosis in X. laevis. The increased growth associated with TCS exposure was not unexpected and is generally consistent with the presence of reduced bacterial stressors in culture.

Nieuwkoop and Faber stage 51 Xenopus laevis larvae were exposed for 21 days to four different concentrations of triclosan (TCS): <0.2 (control), 0.6, 1.5, 7.2, or 32.3 microg TCS/l. Primary endpoints were survival, hind limb length, body length (whole; snout to vent), developmental stage, wet whole body weight, and thyroid histology. Thyroid hormone (TH) concentrations were determined in whole thyroid and plasma samples from stage-matched exposure day 21 specimens. TH receptor-beta (TRbeta) expression was measured in stage-matched tail fin tissue samples collected at exposure days 0 and 21. Reduced larval growth occurred at exposure day 21 with 1.5 microg/l treatment. Larval developmental stage at exposure day 21 was not significantly different from controls based on observed parameters. Thyroid histology was not affected by TCS, and thyroxine (T4) levels in thyroid glands or plasma were not different from controls. A concentration-dependent increase in TRbeta expression in exposure day 21 larvae was not detected. However, increased expression was found in stage-matched larvae exposed to 1.5 or 7.2 microg TCS/l. Our study indicates that environmentally relevant TCS concentrations do not alter the normal course of thyroid-mediated metamorphosis in this standard anuran model.