Comparison of rat and chick limb bud micromass cultures for developmental toxicity screening.

ABSTRACT

This study compares the responses of rat and chick limb bud micromass cultures to chemical treatment. Eight chemicals, of diverse structure, potency and mechanism, were tested, using two endpoints extractable alcian blue stain as a measure of differentiation to chondrocytes, and extractable neutral red stain as an index of proliferation. Each chemical reduced differentiation and proliferation in a concentration-related manner. IC(50)s, concentrations that reduced staining by 50%, ranged from 10 nm (colchicine) to 4 mm (acetazolamide). Rat and chick responses to acetazolamide, colchicine and diazepam were indistinguishable. For diphenhydramine and sulphisoxazole, concentration-response curves were very similar, but rat IC(50)s were half that of chick. For two chemicals, concentration-response slopes were markedly steeper for chick; in the case of beta-aminopropionitrile, IC(50)s were similar, but rat cultures were three-fold more sensitive than chick to cytosine arabinoside. 6-Aminonicotinamide gave a U-shaped response curve, for both endpoints and both species, so IC(50)s may be misleading, but the IC(50) for proliferation was lower for chick (0.6 mum) than rat (4 mum). In vivo and in vitro parameters for validation of developmental toxicity screens are contentious. Diphenhydramine apart, these chemicals can be teratogenic in vivo, although their 'hazard' can be debated. An IC(50)-proliferation/IC(50)-differentiation ratio > 2 has been suggested to predict specific developmental toxicity. Only sulphisoxazole and 6-aminonicotinamide had significantly different IC(50)s for proliferation and differentiation, with ratios of 4.4 (both species), and 10.4 for rat and 1.9 for chick, respectively. All other ratios were close to 1. The general consistency of this ratio, and the concentration-responses, in the two species suggests that the chick is a viable alternative to laboratory mammals, but the predictive ability of micromass remains to be determined.