Trypan blue teratogenesis in the rat: further observations in vitro

Explanted 9 1/2- and 10 1/2-day rat conceptuses were cultured for 24 or 48 hours and 24 hours respectively in immediately centrifuged, heat-inactivated rat serum to which trypan blue was added at concentration of 150 micrograms/ml or 300 micrograms/ml or 450 micrograms/ml. The embryos were assessed for normal growth and differentiation using the following criteria: heart beat, vitelline circulation, fusion of the allantois with the chorion, normal turning, normally closed neural tube, presence of optic vesicles, presence of forelimb buds, tail, somites, somite number, yolk sac diameter, crown-rump length, and protein content. The results indicated that trypan blue is more teratogenic when 9 1/2-day conceptuses are cultured for the first 24 hours, and that its teratogenicity decreases after 10 1/2 days. Morphological abnormalities produced included neural tube defects, turning defects, and tail defects, the tail abnormalities being manifest as fluid-filled blebs. Ten and one-half-day conceptuses cultured for 24 hours in treated serum produced predominantly tail defects. The conceptuses were cultured at 11 days, when the embryo lies completely within the yolk sac. Culture of such conceptuses in serum containing 450 micrograms/ml trypan blue produced mainly tail defects. Injection of 0.5 microliters of 0.5% dye solution into the yolk sac cavity also produced tail abnormalities; sham treatment, or the injection of sterile water or a nonteratogenic dye, azo blue, did not affect embryonic development.