Evaluating Thiram-Induced Embryotoxicity Using Integrated In Silico, In Vitro, and Transcriptomic Approaches

Long-term exposure to low-dose food contact materials (FCMs) has raised concerns regarding developmental toxicity. In the present study, we prioritized FCMs with potential developmental toxicity using a weight-of-evidence computational model, which predicted 127 chemicals to be of high concern. From these, we selected 7 chemicals-representing both high and low concern categories-and evaluated their potential embryotoxicity using the mouse embryonic stem cell test (mEST). Among the selected chemicals, thiram most strongly inhibited cardiac differentiation in mEST. We further examined the effects of thiram on morphology and expression of differentiation-related genes in mouse embryonic stem cells (mESCs). Treatment with thiram inhibited 50% early differentiation in mESCs, suppressed the expression of markers associated with the three-germ layers, but increased the expression of neurectoderm markers during early embryogenesis. Additionally, treatment with 20 ng/mL thiram, which was the lowest-observed-effect concentration of cytotoxicity, disrupted neuronal differentiation in both mESCs and human pluripotent embryonal carcinoma NT2 cells. Finally, based on transcriptome analysis, 20 and 30 ng/mL thiram disrupted the neural crest differentiation pathway, altering the expression of genes including homeobox A1 (HOXA1), homeobox B1 (HOXB1), Heart And Neural Crest Derivatives Expressed 1 (HAND1), Distal-Less Homeobox 5 (DLX5), and transcription factor AP-2 alpha (TFAP2A) in NT2 cells. Therefore, disruption of neural crest differentiation is one of the potential mechanisms underlying thiram-induced embryotoxicity. The integrated alternative approach adopted in the present study to identify mechanism-based biomarkers for thiram-induced embryotoxicity in a human-relevant model could facilitate safety assessment for data-poor chemicals in future.