Dose-response assessment of dipentyl phthalate effects on testosterone production and morphogenesis of late-gestation fetal rat testis

Dipentyl phthalate (DPeP) is a potent male reproductive toxicant that reduces fetal testicular testosterone production and induces abnormal fetal testis morphology, including multinucleated germ cells (MNGs). We aimed to test whether testosterone production, MNG density, or gene expression would be most sensitive to DPeP exposure and to determine which transcriptomic processes are initiated at the lowest DPeP dose. Timed pregnant Sprague Dawley rats were exposed to 0, 1, 11, 33, 100, or 300 mg DPeP/kg/d by oral gavage from GD 17-21. For comparison to DPeP, additional rats were exposed to vinclozolin, prochloraz, acetaminophen, mono-(2-ethylhexyl) tetrabromophthalate, and dexamethasone. Testosterone production was measured using an ex vivo culture assay, MNGs were quantified on testis sections, and fetal testes were used for RNA-seq, immunofluorescence, and in situ hybridization. Benchmark dose (BMD) analysis was used to compare apical endpoints and gene expression. DPeP dose-dependently reduced testosterone production and increased MNG density. ED₅₀ for MNG density was lower than for testosterone production, but BMD₁₀ values were similar. The lowest BMD estimates for apical toxicity (MNGs) and gene expression (R-RNO-210991: basigin interactions) were 2.675 mg/kg/d and 2.44 mg/kg/d, respectively. DPeP altered gene sets related to steroidogenesis, gonad development, epithelial cell differentiation, and vasculature development. We conclude that inhibition of testosterone production and induction of MNGs have similar utility for quantification of phthalate dose-response in the context of risk assessment. RNA-seq data suggest that cell differentiation and patterning processes were sensitive to DPeP and may contribute to phthalate toxicity mechanisms in the fetal rat testis.