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.

Short-term embryo-larval, limb development, and a battery of toxicity identification studies using sediment and water collected from selected ponds found that developmentally toxic samples contained elevated levels of petroleum hydrocarbons and As, Cu, Pb, Cr, Cd, Hg, Fe, and Zn. Sediments spiked with diesel fuel or metals confirmed that the levels of petroleum hydrocarbons and metals found in the sediment were capable of inducing developmental toxicity individually. However, joint mixture interaction studies indicated that the metals and petroleum hydrocarbons acted synergistically. To more rigorously evaluate exposure pathways and the effects induced, chronic laboratory-based microcosm experiments were performed using B. marinus from early embryo to metamorphosis. Results from these microcosm studies indicated that reasonably consistent frequencies of malformations were induced by exposure to contaminated media samples from four contaminated sites compared with frequencies found in field-collected specimens. The frequencies of specific types of deformities in newly metamorphosed organisms from the microcosm studies and in free-roaming field-collected specimens were also similar. Exposure to contaminated media from three of the four sites decreased the frequency of metamorphic completion and at one site reduced the metamorphic rate, compared to exposure to laboratory controls or reference media. Elevated whole body petroleum hydrocarbons and metals tissue residues were detected in organisms from the laboratory microcosms, as well as in the free-roaming specimens collected. To evaluate the impact of maternal contaminant transfer as an exposure route, cross-over exposure studies in which reference organisms were raised, from embryos through metamorphosis, in contaminated media and organisms from a contaminated site were cultured in reference media were conducted. Results from these studies indicated that environmental exposure and maternal toxicant transfer were the potential exposure routes. Overall, the present studies suggested the petroleum hydrocarbons and metals that accumulated in B. marinus during development in microcosms corresponded with increased frequencies of malformation.

Currently, no standardized and well-validated alternative models exist for screening for progesterone-responsive endocrine disrupting chemicals (EDCs). Because of this, a rapid assay for evaluating progestin/antiprogestin activity using Xenopus oocyte germinal vesicle breakdown (GVBD) as a model was evaluated. Five compounds, including progesterone (P), ethinyl estradiol (EE), ethylene glycol monomethyl ether (EGME), cadmium (Cd), and boric acid (B) were used to validate the model on a preliminary basis. Each test material was tested for progestin/anti-progestin activity. The binding affinity of each test material to the oocyte plasma membrane receptor (OPMR) relative to progesterone was then determined. Results from the present studies suggested that both EE and EGME were capable of inhibiting GVBD in a concentration-dependent manner. Cd had a subtle inhibitory effect at high concentrations. B had no effect on GVBD even at concentrations of 100 mg B/L, and thus appears to have no EDC activity in this model. The binding capacity of the test substances to the OMPR relative to progesterone was low. Thus, the relative inhibitory potential of the test materials study was EE > EGME >> Cd > B. However, the relative binding affinity of the toxicants to the OMPR can be expressed as P >> EGME > EE > Cd > B. Although EE was more effective in inhibiting GVBD, EGME appeared to bind more tightly to the OMPR.

As a formal recommendation from an Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) workshop review of the Frog Embryo Teratogenesis Assay--Xenopus (FETAX) developmental toxicity model, the use of Xenopus tropicalis as an alternative test species for this model was evaluated. Three test substances with varying developmental toxicity potentials were evaluated using FETAX modified to accommodate the use of X. tropicalis. Two separate definitive concentration-response tests were performed with isoniazid, methotrexate, and 6-aminonicotinamide. Historical FETAX results with X. laevis were compared to the results from FETAX assays with X. tropicalis. Test with X. tropicalis indicated that each of the compounds possessed teratogenic potential with varying degrees of potency: 6-aminonicotinamide > methotrexate > isoniazid. Based on overt teratogenicity, but not embryo-lethality, results from these studies indicated that these two species responded similarly to the test compounds. Malformation syndromes induced in both species were similar in X. tropicalis and X. laevis. These results suggested that X. tropicalis should be further evaluated as an alternative test organism for the FETAX model.