In mammals, exposure to antiandrogenic chemicals during sexual differentiation can produce malformations of the reproductive tract. Perinatal administration of AR antagonists like vinclozolin and procymidone or chemicals like di(2-ethylhexyl) phthalate (DEHP) that inhibit fetal testicular testosterone production demasculinize the males such that they display reduced anogenital distance (AGD), retained nipples, cleft phallus with hypospadias, undescended testes, a vaginal pouch, epididymal agenesis, and small to absent sex accessory glands as adults. In addition to DEHP, di-n-butyl (DBP) also has been shown to display antiandrogenic activity and induce malformations in male rats. In the current investigation, we examined several phthalate esters to determine if they altered sexual differentiation in an antiandrogenic manner. We hypothesized that the phthalate esters that altered testis function in the pubertal male rat would also alter testis function in the fetal male and produce malformations of androgen-dependent tissues. In this regard, we expected that benzyl butyl (BBP) and diethylhexyl (DEHP) phthalate would alter sexual differentiation, while dioctyl tere- (DOTP or DEHT), diethyl (DEP), and dimethyl (DMP) phthalate would not. We expected that the phthalate mixture diisononyl phthalate (DINP) would be weakly active due to the presence of some phthalates with a 6-7 ester group. DEHP, BBP, DINP, DEP, DMP, or DOTP were administered orally to the dam at 0.75 g/kg from gestational day (GD) 14 to postnatal day (PND) 3. None of the treatments induced overt maternal toxicity or reduced litter sizes. While only DEHP treatment reduced maternal weight gain during the entire dosing period by about 15 g, both DEHP and DINP reduced pregnancy weight gain to GD 21 by 24 g and 14 g, respectively. DEHP and BBP treatments reduced pup weight at birth (15%). Male (but not female) pups from the DEHP and BBP groups displayed shortened AGDs (about 30%) and reduced testis weights (about 35%). As infants, males in the DEHP, BBP, and DINP groups displayed femalelike areolas/nipples (87, 70, and 22% (p < 0.01), respectively, versus 0% in other groups). All three of the phthalate treatments that induced areolas also induced a significant incidence of reproductive malformations. The percentages of males with malformations were 82% (p < 0.0001) for DEHP, 84% (p < 0.0001) for BBP, and 7.7% (p < 0.04) in the DINP group. In summary, DEHP, BBP, and DINP all altered sexual differentiation, whereas DOTP, DEP, and DMP were ineffective at this dose. Whereas DEHP and BBP were of equivalent potency, DINP was about an order of magnitude less active.

Phthalate diesters are chemicals to which humans are ubiquitously exposed. Exposure to certain phthalates during sexual differentiation causes reproductive tract malformations in male rats. In the fetal rat, exposure to the phthalates benzylbutyl phthalate (BBP), di(n)butyl phthalate (DBP), and diethylhexyl phthalate (DEHP) decreases testicular testosterone production and insulin-like 3 hormone mRNA levels. We characterized the dose-response effects of six individual phthalates (BBP, DBP, DEHP, diethyl phthalate [DEP], diisobutyl phthalate [DiBP], and dipentyl phthalate [DPP]) on gestation day (GD) 18 testicular testosterone production following exposure of Sprague-Dawley rats on GD 8-18. BBP, DBP, DEHP, and DiBP were equipotent (ED50 of 440 +/- 16 mg/kg/day), DPP was about threefold more potent (ED50 = 130 mg/kg/day) and DEP had no effect on fetal testosterone production. We hypothesized that coadministration of these five antiandrogenic phthalates would reduce testosterone production in a dose-additive fashion because they act via a common mode of toxicity. In a second study, dams were dosed at 100, 80, 60, 40, 20, 10, 5, or 0% of the mixture. The top dose contained 1300 mg of total phthalates/kg/day including BBP, DBP, DEHP, DiBP (300 mg/kg/day per chemical), and DPP (100 mg DPP/kg/day). This mixture ratio was selected such that each phthalate would contribute equally to the reduction in testosterone. As hypothesized, testosterone production was reduced in a dose-additive manner. Several of the individual phthalates and the mixture also induced fetal mortality, due to pregnancy loss. These data demonstrate that individual phthalates with a similar mechanism of action can elicit cumulative, dose additive effects on fetal testosterone production and pregnancy when administered as a mixture.

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