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Petroleum substances are highly complex materials made up of hundreds to millions of different hydrocarbons.Heavy petroleum substances, such as heavy fuel oil (HFO) and untreated lubricating oils, contain relatively high concentrations of 3-to 7-ring PAHs and may induce prenatal developmental toxicity (PDT) in experimental animals.In contrast, gas-to-liquid (GTL) products, the modem synthetic analogues of petroleum substances, are totally devoid of aromatic compounds and do not induce PDTin vivo.This has led to the hypothesis that the PDTobserved with heavier petroleum substances is due to the presence of 3-to 7-ring PAHs in these products.Consequently, it is assumed that light petroleum substances with noPAHs or low concentrations of PAHswill not induce PDT.PDT studies are animal intensive and testing this hypothesis in vivo would require large numbers of experimental animals.The application of anin vitro assay, such as the embryonic stem cell test (EST), may reduce animal experimentation and resources needed to study the PDT potencies of petroleum substances and GTL products.The present study aims to test thehypothesis that PAHs, which are present in heavy petroleum substances, are responsible for the observed PDT using the EST.To this end, DMSO-extracts of a range of petroleum substances, containing different amounts of PAHs and two GTL products, which are devoid of PAHs, were tested in the ES-D3 cell proliferation and ES-D3 cell differentiation assay of the EST.The obtained results showed that petroleum substances were able to inhibit the differentiation of ES-D3 cells in a concentration-dependent manner at non-cytotoxic concentrations, and that this potencywas related to their PAH content.In contrast, GTL samples did not decrease viability and also did not inhibit differentiation of the ES-D3 cells.The results support the hypothesis that the PDT observed with some petroleum substances is correlated with their high concentration of PAHs.The EST showed a great promise to evaluate the in vitroembryotoxicity of petroleum substances.Yet, future studies are needed to determinethelevel and type of PAHs that are responsible for PDT.