Use of Frog Embryo Teratogenesis Assay-Xenopus and an exogenous metabolic activation system to evaluate the developmental toxicity of diphenylhydantoin.

The teratogenic potential of diphenylhydantoin (DPH) and hydroxylated metabolites (HPPH) was evaluated with the Frog Embryo Teratogenesis Assay--Xenopus (FETAX). Embryos of the South African clawed frog, Xenopus laevis were exposed to DPH and HPPH in two separate static-renewal experiments with and without the presence of an exogenous metabolic activation system (MAS) for 96 hr. Two separate dose-response tests were also conducted with DPH and HPPH with a MAS modulated by various mixed functional oxidase inhibitors [carbon monoxide (CO) (broad spectrum cytochrome P450), cimetidine (mainly cytochrome P450), and ellipticine (cytochrome P448)] and an epoxide hydrolase inhibitor (cyclohexene oxide). Assessment of the potential teratogenic hazard was based on teratogenic indices [TI = 96-hr LC50/96-hr EC50 (malformation)], types and severity of malformations, and embryo growth endpoints. Addition of the intact MAS to DPH increased the 96-hr LC50 and EC50 (malformation) from approximately 74.5 and 32.4 mg/liter to 126.4 and 62.9 mg/liter, respectively. The TI was reduced 1.2-fold. Both p-HPPH and m-HPPH were much less developmentally toxic than DPH. CO and cimetidine inhibition of cytochrome P450 maintained much of the developmental toxicity of DPH, whereas ellipticine inhibition of cytochrome P448 was much less effective in maintaining the developmental toxicity of DPH. Cyclohexene oxide inhibition of epoxide hydrolase markedly increased DPH-induced embryotoxicity decreasing the 96-hr LC50 from approximately 74.5 to 38.6 mg/liter. These results suggest that unmetabolized DPH and an embryotoxic epoxide intermediate may serve as the teratogenic species in FETAX.