BPA, a widely-studied environmental endocrine disruptor, has been identified to affect cardiac differentiation and produce cardiotoxicity in heart tissue.Great numbers of BPA analogs in blooming are expected to work as safe replacement to BPA.However, the example of BPS indicates that BPA analog(s) still represents adverse effect on cardiac differentiation and cardiotoxicity; the teratogenicity and cardiotoxicity of other BPA analogs are tremendously unknown.In advocating animal welfare, the 3Rs principle (refinement, reduction and replacement) isoutlined and alternative tests to developmental and reproductive toxicity tests using in vitro methods were developed, for both economical and ethical reasons.Embryonic stem cells (ESC) utilize the advantages of pluripotency, self-renewal ability and tissue-specific differentiation potential, which can mimic organogenesis in vitro, and allows accurate determination of chemically induced adverse effects during embryo development.Embryonic stem cell test (EST),which is established based on the cardiac differentiation of ESC, functions as a prediction model for embryotoxicity.The parameters required include 50% inhibition of cellular proliferation (IC50) on 3T3 fibroblasts and ESC, together with 50% inhibition of differentiation (ID50) of ESC into cardiomyocytes.Besides, gender specific effect is an inherent property of chemicals, characterized by variations caused by chemical-biology interaction.Even though the cardiotoxicity of BPA and BPS are reported to be female-susceptive, it is still unclear if other BPA analogs have gender specific effect.To address this, a "double-objects in unison" (DOU)-EST, which consisted of male ESC and female ESC, are established to discriminate the potential gender-specific effect existed in BPA and its analogs.Therefore, to indicate differences in the teratogenic potency among BPA and its 12 analogs which differ in substituent group or side chain length, here, QSAR were proposed for embryotoxicity prediction based on a 7-day protocol of flow cytometery determination of intracellular marker proteins.The embryotoxicity of BPA and its analogs were evaluated and differences in their teratogenic potency were suggested.Preliminary information regarding gender-specific effect among BPA analogs in vitro are provided for further test in future.