Comparative sensitivity of Xenopus tropicalis and Xenopus laevis as test species for the FETAX model.

The use of Xenopus tropicalis as an alternative test species for the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) model was evaluated. Five test substances with varying developmental toxicity potential were evaluated using the traditional FETAX (X. laevis) and a modified assay to accommodate the use of X. tropicalis. Two separate definitive concentration-response tests were performed with ethanol, semicarbazide, copper, 6-aminonicotinamide (6-AN) and atrazine. In order to evaluate the impact of culture temperature on species sensitivity, tests with X. tropicalis were performed concurrently at 27 degrees C (optimum temperature) and 23 degrees C (traditional FETAX temperature). Tests with X. laevis were performed only at 23 degrees C (optimal for X. laevis). Regardless of culture temperature, tests with X. laevis and X. tropicalis indicated that each of the compounds possessed teratogenic potential: semicarbazide>6-AN>atrazine approximately copper>ethanol. Results from these studies indicated that these two species responded similarly to the test compounds. Xenopus tropicalis was somewhat less sensitive to 6-AN, semicarbizide and atrazine when tested at 27 degrees C than at 23 degrees C. Ethanol, copper and atrazine were reasonably equipotent in X. tropicalis and X. laevis in terms of teratogenic response (EC50 for malformation), whereas 6-AN and semicarbizide were less potent in X. tropicalis than in X. laevis. No substantial differences (order of magnitude) in potency were observed between X. laevis and X. tropicalis with any of the test materials evaluated. Malformation syndromes induced in both species were similar in X. tropicalis and X. laevis. These results suggested that X. tropicalis could be used effectively as a test organism for the FETAX model.

Effect of methoxychlor on various life stages of Xenopus laevis.

The toxicological effects of the organochlorine pesticide methoxychlor were evaluated at various life stages of the South African clawed frog, Xenopus laevis, in an effort to determine stage-specific sensitivity. A battery of four separate assays, including a short-term (4-day) early embryo-larval assay (FETAX) (NF stages 8-46 [Nieuwkoop and Faber, 1994]), 30-day hind limb development assay (NF stages 8-54), 18-day metamorphic climax assay (NF stages 58-66), and 30-day adult reproduction assay were performed. Test concentrations for the FETAX, hind limb development, metamorphic climax, and reproductive assays ranged from 0.0001-1.0 mg/l, 0.0001-0.1 mg/l, 0.0001-0.1 mg/l, and 0.001-0.1 mg/l, respectively. Results from the short-term embryo-larval assay indicated that increased embryo-lethality, malformation, and growth inhibition were not induced at /=0.01 mg/l delayed hind limb digit differentiation. Follicular hyperplasia of the thyroid glands was noted in specimens exposed to 0.1 mg/l methoxychlor. Results from the 18-day metamorphic climax assay indicated that methoxychlor inhibited the rate of tail resorption in a concentration-dependent manner. Whole body tissue triiodothyronine (T(3)) profiles showed a reduced and delayed surge during climax compared to controls. For the reproductive assessment, adult female X. laevis were super-ovulated and both female and male were then exposed to varying concentrations of methoxychlor. A concentration-dependent reduction in ovary weight and the number of viable oocytes was observed. In exposed male specimens, a concentration-dependent reduction in testis weight and sperm count was found. Methoxychlor was found to accumulate in the ovary, and to a lesser extent in the testis. Based on breeding studies in which exposed females were bred with control males and exposed males bred with control females, the frequency of amplexus, fertilization, and embryo viability was also affected by adult female methoxychlor exposure, and to a lesser extent by male exposure. Overall, these results suggested that sensitivity to methoxychlor is most dramatic during the reproductive and metamorphic phases of the life cycle and least sensitive during early embryo-larval development.

Evaluation of the developmental and reproductive toxicity of methoxychlor using an anuran (Xenopus tropicalis) chronic exposure model.

The chronic toxicity of methoxychlor to the South African clawed frog, Xenopus (Silurana) tropicalis, was evaluated using a life cycle approach. The chronic exposure period ranged from mid-cell blastula stage [NF (Nieuwkoop and Faber, 1994) stage 8] to 90 days of exposure, during which time the organisms generally completed metamorphosis and emerged as juvenile frogs. Methoxychlor concentrations ranged from 1 to 100 micrograms/l. Methoxychlor concentrations >10 micrograms/l caused delayed development. Organisms exposed to 10 micrograms/l methoxychlor for 30 days showed enlarged thyroid glands with follicular hyperplasia. No increase in mortality or external malformation was observed at any of the test concentrations during early embryo-larval development (NF stage 8 to NF stage 46; ca. 2 days exposure). A concentration-dependent increase in external malformations and internal abnormalities of the liver and gonads were noted after 90 days of exposure, however. Skewing of the sex ratio toward the female gender decreased ovary weight and number of oocytes, and increased oocyte immaturity and necrosis were noted at methoxychlor concentrations of 100 micrograms/l. Reductions in testis weight and sperm cell count were also detected at 100 micrograms/l methoxychlor. Results from these studies suggested that methoxychlor was capable of altering the rate of larval development, but did not adversely affect early embryo-larval development (2 days of exposure) as manifested in external malformations. Internal malformations, increases in the ratio of phenotypic females, were induced by chronic methoxychlor exposure. In addition, reproductive endpoints, most notably in the female specimens, were adversely affected by methoxychlor exposure. These studies add to the standardization and validation of a useful amphibian test methods capable of evaluating both reproductive and developmental effects of potential endocrine disrupting chemicals over a life cycle exposure.