Limb bud cell cultures for estimating the teratogenic potential of compounds. Validation of the test system with retinoids.

Mesenchyme cells, derived from embryonic limb buds, cultured at high cell density, multiply and differentiate into chondrocytes. Using alcian blue, a stain specific for cartilage proteoglycans, the degree of chondrogenesis can be visualized in the micromass cultures as well as quantified by extraction of the stain and spectrophotometric determination of its absorbance. In the presence of active retinoids chondrogenesis is concentration-dependently inhibited. For comparison of the activity of the various retinoids the concentration needed to reduce alcian blue staining by 50% was estimated. In order to validate whether the activity in limb bud cells can predict the teratogenic potential in vivo, the in vitro activity of 25 retinoids was compared with their in vivo teratogenicity observed mainly in rodents. For retinoids which were already in the biologically active form like those with a free carboxylic acid endgroup, there was a good quantitative correlation between the in vitro and in vivo activity. In contrast, the ethylester analog etretinate was slightly active and the ethylamine analog motretinide inactive in vitro but both were teratogenic in vivo. This finding may indicate that these retinoids were not metabolized to the active form in vitro. In conclusion, these results suggest that the limb bud cell culture system may be useful for a preliminary testing to select non-teratogenic retinoids. For the risk assessment in humans, however, the in vitro result should be verified in animal studies.