Phthalates are ubiquitous environmental contaminants widely used as plasticizers in poly vinyl chloride products, and growing evidence implicates them in metabolic activity reduction and epigenetic dysregulation. However, their direct effects on non-transformed human cells remain incompletely characterized. The present in vitro study evaluated the reduction in cell viability and DNA methylation–modulating effects of three widely distributed phthalates, di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DnBP), and di-isononyl phthalate (DINP), on normal human fibroblast (NHF) cells. Cells were exposed to serial concentrations (31.25–1000 μg/mL) for 72 h, and the viability was assessed by MTT assay, followed by IC50 determination via nonlinear regression. Global DNA methylation was subsequently quantified at the IC50 concentration by ELISA-based measurement of 5-methylcytosine (5-mC). All three phthalates produced significant concentration-dependent reductions in cell viability. Constraining the regression plateaus to 100% and 0% viability yielded estimated IC50 values of 19.2 μg/mL for DEHP, 43.4 μg/mL for DnBP, and 13.1 μg/mL for DINP; as viability fell below 50% at the lowest concentration tested, these values represent extrapolation below the sampled range, their confidence intervals overlap, and no potency ranking is asserted. At fixed high-effect concentrations, DEHP and DINP significantly elevated global 5-mC levels to 189.18% and 159.56% of control, respectively, whereas DnBP induced only a non-significant increase (120.06%). These findings indicate that phthalates compromise both the viability and epigenetic integrity of NHF cells in a congener-specific manner, underscoring their potential contribution to methylation-associated human pathologies.
