Dipentyl phthalate dosing during sexual differentiation disrupts fetal testis function and postnatal development of the male Sprague-Dawley rat with greater relative potency than other phthalates

Abstract

Phthalate esters (PEs) constitute a large class of plasticizer compounds that are widely used for many consumer product applications. Ten or more members of the PE class of compounds are known to induce male fetal endocrine toxicity and postnatal reproductive malformations by disrupting androgen production during the sexual differentiation period of development. An early study conducted in the rat pubertal model suggested that dipentyl phthalate (DPeP) may be a more potent testicular toxicant than some more extensively studied phthalates. Regulatory agencies require dose-response and potency data to facilitate risk assessment; however, very little data are currently available for DPeP. The goal of this study was to establish a more comprehensive data set for DPeP, focusing on dose-response and potency information for fetal and postnatal male reproductive endpoints. We dosed pregnant rats on gestational day (GD) 17 or GD 14-18 and subsequently evaluated fetal testicular testosterone (T) production on GD 17.5 and GD 18, respectively. We also dosed pregnant rats on GD 8-18 and evaluated early postnatal endpoints in male offspring. Comparison of these data to data previously obtained under similar conditions for di (2-ethylhexyl) phthalate indicates that DPeP is approximately eightfold more potent in reducing fetal T production and two- to threefold more potent in inducing development of early postnatal male reproductive malformations. Additionally, fetal testicular T production was more sensitive to inhibitory effects of DPeP exposure than was gene expression of target genes involved in male reproductive development, supporting the use of this endpoint as a critical effect in the risk assessment process.

FIG. 1.

Fetal testicular T production ontogeny in control rats (GD 14.5–18). Data columns represent mean value of ex vivo T production of all testes from two (GD 14.5) to three litters after 3 h of incubation. Error bars are ± SEM. Asterisks (*) represent statistically significant from previous GD, determined by ANOVA, two-tailed t-test (p < 0.0001).

FIG. 2.

Comparison of fetal testicular ex vivo T production after 3 h incubation between rats exposed in utero to vehicle or 500 mg/kg DPeP for 5 h on GD 17. Columns represent mean value (± SEM) of all testes from six litters. Asterisk (*) represents statistical significance from control as determined by ANOVA, two-tailed t-test (p < 0.0001).

FIG. 3.

Ex vivo fetal testicular T production following a single maternal dose (DPeP GD 17.5) or 5 days of maternal dosing (DPeP 14–18) (Panel A). (Panel B) shows a comparison between the dose-response curves for DPeP (solid line) and 10-day maternal dosing with DEHP (GD 8–18, dashed line, data derived from Howdeshell et al., 2008). Data points represent litter means (± SEM) for two to three litters (DPeP) or three to six litters (DEHP). The ED50’s for each curve are 666.6 mg/kg/day DPeP GD 17, 47.4 mg/kg/day DpeP GD 14–18, and 388.2 DEHP 8–18.

FIG. 4.

Fetal testicular gene expression dose-response data for (A) StAR (ED50 = 45.65), (B) insl3 (ED50 = 87.46), and (C) Cyp11a (ED50 = 143.7), following 5-day DPeP exposure during sexual differentiation (GD14–18). Each data point represents the mean ± SEM of three to six litters.

FIG. 5.

Early postnatal effects of in utero DPeP exposure (GD8–18) and DEHP (Gray et al., 2009) on (A) AGD (PND 2, ED50 = 252.3 for DPeP and 521.0 for DEHP) and (B) nipple retention (number of female-like nipples per male out of 12 possible; PND 13, ED50 = 175.0 for DPeP and 784.8 for DEHP) in SD male rats. Female AGD was approximately 50% of control males. Data points represent mean (± SEM) of four to five litters.

FIG. 6.

Male AGD decline on PND 2 following in utero exposure to DPeP from GD 8 to 18 (ED50 = 252.3) versus fetal testicular T production on GD18 following in utero exposure to DPeP from GD 14 to 18 (ED50 = 47.0). Each data value represents the litter means of three to six litters.

FIG. 7.

Dose-responsive decline of percentage of males without nipples at PND 13 (100% Normal = 0 nipples vs. 0% normal = 12 nipples; ED50 = 175.0) versus decline in male AGD at PND 2 (Control male AGD = 100% vs. control female AGD = 0% [50% of control male value] ED50 = 252.3) and decline in pup survival from implantation to PND 2 (ED50 = 294.3). Data points represent litter means (± SEM) of four to five litters.