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The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicity  in vitro . The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75   μ  g/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes:  Pnpla6 ,  Afp ,  Hdac7 ,  Vegfa , and  Nes . The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression of  Nes . These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting for  in vitro  risk assessment embryotoxicity.

Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation