Diethyl-hexyl-cyclohexane (Eco-DEHCH) is a safer phthalate alternative that does not elicit neuroendocrine disrupting effects

Alternative phthalates (APs) have been developed due to the reported adverse effects of conventional phthalates (CPs). However, whether APs are nontoxic and can replace CPs remains controversial due to their endocrine-disrupting (ED) effects. Herein, to investigate the ED potential of diethyl-hexyl-cyclohexane (DEHCH), a newly developed non-phthalate-structured AP, we employed in silico (molecular docking simulation), in vitro (cell-based assays for estrogen and androgen receptors), and in vivo (zebrafish embryo model) methods. We also compared the results with two CPs (di(2-ethylhexyl) phthalate [DEHP] and diisononyl phthalate [DINP]) and two previously proposed non-phthalate-structured APs (1,2-cyclohexane dicarboxylic acid diisononyl ester [DINCH] and di-2-ethylhexyl terephthalate [DEHTP]). DEHCH did not exhibit the highest binding affinity for any of the five receptors such as estrogen, androgen, glucocorticoid receptors, and thyroid receptor alpha and beta. None of the tested phthalates exhibited agonistic or antagonistic effects on estrogen and androgen receptors. In zebrafish larvae, DEHCH did not affect the expression of the nine endocrine-related genes and neurobehaviors, which correlates well with the lack of changes in the endogenous concentrations of the five neurosteroids. In contrast, DINCH, DEHP, and DEHTP induced hyperactivity, and except for DEHCH, four phthalates significantly upregulated at least one gene. In addition, DINCH significantly increased the expression of cortisol and DEHP increased progesterone, allopregnanolone, and cortisol. These findings demonstrate that DEHCH is safer than CPs and the previously proposed APs in terms of ED effects, including neuronal system dysregulation.