Bis(2-chloroethyl)2-chloroethylphosphphonate (B2CE2CEPP) was recently reported as a contaminant in underground water samples, however, information regarding its biotransformation and adverse effects is relatively rare. In this study, B2CE2CEPP and tris(2-chloroethyl) phosphate (TCEP
a typical organophosphate triester with a very similar structure as compared to B2CE2CEPP) were used as target contaminants to comparatively investigate their cytotoxicity, biotransformation, and alteration of lipid metabolism by use of an in vitro primary hepatocyte assay of ICR mice. This study aims to thoroughly investigate the cytotoxicity, biotransformation, and lipid metabolism effects of B2CE2CEPP and TCEP on primary hepatocytes of ICR mice, in order to reveal the potential health risks of these two organophosphorus compounds. The results showed that both B2CE2CEPP and TCEP were cytotoxic with LC50 values of 456 μM (B2CE2CEPP) and 1250 μM (TCEP), respectively. Both compounds underwent significant metabolic transformation follow exposure to primary mouse hepatocytes (PMHs), and a total of 4 potential metabolites were identified based on high-resolution orbitrap mass spectrometry technique. Exposure to TCEP and B2CE2CEPP led to lipid metabolism disorders in PMHs. TG (16:0/16:1/18:1) showed high sensitivity to B2CE2CEPP exposure and exhibited a significant upregulation trend, making it a potential biomarker significantly influenced by B2CE2CEPP exposure. Lipidomic analysis showed that B2CE2CEPP affected lipid metabolic pathways including thiamine metabolism, one carbon pool by folate, and folatesteroid hormone biosynthesis. The present study filled a knowledge gap in the current research on the biotransformation and adverse effects of B2CE2CEPP, and emphasized the importance on consistent monitoring of B2CE2CEPP and its structure-like compounds in various environmental samples.