To assess the potential reproductive effects over multiple generations, Diethylhexylphthalate (DEHP), CAS No. 117-81-7, was administered in the diet at concentrations of 1.5 (control), 10, 30, 100, 300, 1,000, 7,500, and 10,000 ppm to groups of 17 male and 17 female Sprague-Dawley rats. The control dose level was set at 1.5 ppm as this was the amount of DEHP found in the control feed. Animals in the F0 generation began exposure as adults and were bred to produce the F1 generation (F1a, 1b, 1c), the F1 adults were bred to produce the F2 generation (F2a, 2b, 2c), and the F2 adults were bred to produce the F3 generation (F3a, 3b, 3c). Additional non-mating males (up to three per litter) were selected from the F1c, F2c, F3c litters, and were maintained following similar procedures as those for mating males, except they were not cohabited with females. The 10,000 ppm animals only completed the F1 generation and were terminated due to the inability to produce any F2 generation animals. Parameters evaluated over the course of the study included body weights, feed consumption, clinical observations, reproductive performance, anogenital distance, pup survival, sexual development, estrous cyclicity, sperm endpoints, gross pathology, organ weights, and limited/selected histopathology. Based on measured feed consumption, mg/kg daily doses were calculated to be 0.12, 0.78, 2.4, 7.9, 23, 77, 592, and 775 mg/kg/day in the F0 animals; 0.09, 0.48, 1.4, 4.9, 14, 48, 391, and 543 mg/kg/day in the F1 animals; and 0.1, 0.47, 1.4, 4.8, 14, 46, 359 mg/kg/day in the F2 animals.
Reductions in terminal body weights were noted at 7500 ppm in the F1 and F2 males (10% and 14%, respectively), and at 10,000 ppm in the F0 and F1 males (6% and 21%, respectively) and females (12% and 19%, respectively). Feed consumption was generally comparable in all groups in all generations on a g/animal/day basis, but was increased at 7500 and 10,000 ppm on a g/kg body weight/day basis. Clinical signs were generally comparable among all groups in all generations.
Reproductive effects were noted in the 7500 ppm and 10,000 ppm groups. The total number of males per litter was decreased at 10,000 ppm in the F1a litter and at 7500 ppm across all F1 litters combined (F1a + F1b + F1c). The total number of F1a pups per litter was decreased at 7500 and 10,000 ppm. The total number of pups per litter across all F1 (F1a + F1b + F1c) litters combined was also decreased at 7500 ppm. At 10,000 ppm, male and female pup weights, unadjusted and/or adjusted for litter size, were decreased in the F1a and F1b litters on PND 1 and in the F1c litters on PND 1, 4, 7, 14, and 21. Male anogenital distance (AGD) was decreased at 10,000 ppm in the F1a, F1b, and F1c pups and at 7500 ppm in the F1a and F1b pups. Testes descent, vaginal opening, and preputial separation were delayed at 10,000 ppm and 7500 ppm in the F1c pups. None of the F1 mating pairs produced offspring at 10,000 ppm. Male and female pup weights, unadjusted and adjusted for litter size, were decreased at 7500 ppm in the F2c litter and combined F2a, b, c litters. Male and female pup weights were decreased at 7500 ppm throughout the lactation period (PND 1-21) of the F2c pups. Male anogenital distance (AGD) was decreased at 7500 ppm in the F2a and F2c pups. There was also a decrease in the pregnancy index for the F2 mating pairs (45%) at 7500 ppm. Male anogenital distance (AGD) was decreased at 7500 ppm in the F3a pups. Testes descent, vaginal opening, and preputial separation were delayed at 7500 ppm in the F3c pups. Retained nipples were observed in the F3c male pups at 7500 ppm. No changes were noted in the female AGD throughout all the Mating Trials.
Crossover matings were conducted using the 7500 and 10,000 ppm males and females. At 7500 and 10,000 ppm, when treated males were crossed with nulliparous naive females, there were decreased numbers of implantation sites, and decreased indices of mating, pregnancy, and fertility. At 7500 and 10,000 ppm, when treated females were crossed with naive males there was a decrease in AGD in the male pups. Also at 7500 ppm, male, female, and combined pup weights were decreased, both when unadjusted and adjusted for litter size.
At terminal necropsies, various sperm end-points were found to be decreased at 7500 ppm in the F1, F2, and F3 males and at 10,000 ppm in the F0 and F, males. Density (sperm/mg cauda) (F2, and F3 males only), sperm/cauda, spermatids/testis, and spermatids/mg testes were decreased at 7500 ppm in the F1, F2, and F3 males. Spermatids/testis were decreased at 10,000 ppm in the F0 males and no sperm or spermatids were noted in the F1 males.
Organ weight changes were seen in the liver, kidney, and male accessory sex organs. The absolute and/or relative liver weights were increased at 1,000 ppm in the F1 males at 7500 ppm in the F0, F1 F2 and F3 males and at 10,000 ppm in the F0 males. Absolute and relative liver weights were also increased at 7500 ppm in the females in all generations; in some cases these increases in liver weights extended to the 300 and 1,000 ppm groups. Absolute and/or relative kidney weights were increased at 7500 ppm in the F0, F1, and F2 males, and F0 females and at 10,000 ppm in the F0 males and F0 females. The absolute kidney weight at 10,000 ppm in the F1 females was increased. The absolute and/or relative cauda, epididymis, and testis weights were decreased at 7500 ppm in the F1, F2, and F3 males and at 10,000 ppm in the F0 and/or F1 males.
Treatment-related gross necropsy observations at 7500 and 10,000 ppm in the F1 and F2 mating and non-mating males included small testes, epididymis, and caudal epididymis. In the non-mating males selected from the F1 and F2 male pups, aplastic testes and epididymis, and small testes, seminal vesicles, and prostates were noted in 1-3 animals at 300 ppm. While the incidence of these findings is low, they are consistent with the syndrome of effects seen with other phthalate-induced male reproductive toxicity. The number of animals with small testes is greater than that of TherImmune historical control. The toxicological significance of male reproductive abnormalities seen at 300 ppm may be questionable because the incidence is low and no other reproductive effects were noted. These effects, however, are consistent with phthalate-induced male reproductive toxicity and represent sampling of only a small number of animals (1 male/litter); therefore, they are considered as potentially treatment-related.
Treatment-related Histopathologic abnormalities were noted at 7500 and 10,000 ppm in the testes, epididymis, liver, adrenal glands, and kidney in the F0, F1, and F2 animals and at 1,000 ppm in the liver, kidneys, and adrenal glands of F1 animals and in the liver of F2 animals. In the testes, minimal to marked atrophy of the seminiferous tubules characterized by loss of germ cells and the presence of Sertoli cell-only tubules, as well as occasional failure of sperm release, were noted at 10,000 ppm in the F1 males and at 7500 ppm in the F1 and F2 males. The epididymis was observed to have sloughed epithelial cells and residual bodies at 7500 and 10,000 ppm in the F0 males (10,000 ppm only) and F1 males and also in the F2 males at 7500 ppm. Minimal to mild hepatocellular hypertrophy was noted at 10,000 ppm in the F0 and F1 animals, at 7500 ppm in the F0, F1 and F2 animals, and at 1,000 ppm in the F1 and F2 animals. Dilation of the renal tubules and mineralization occasionally associated with chronic pyelonephritis was observed at 1,000 ppm in the F1 animals, at 7500 ppm in the F1 and F2 animals, and at 10,000 ppm in the F1 animals. Cortical vacuolization of the adrenals was noted at 7500 ppm in the F1 animals, and at 10,000 ppm in the F0 and F1 animals.
The data obtained in this study indicate that DEHP is a reproductive toxicant at 7500 and 10,000 ppm with the presence of toxicity in the liver, kidneys, and adrenals. Other than the hepatocellular toxicity at 1,000 ppm, there was no general toxicity observed at dose levels below 1,000 ppm. There was no reproductive toxicity observed at doses lower than 7500 ppm except for a possible increase of small testes and prostates which may represent an increased incidence of developmental abnormalities in the male reproductive organs at 300 and/or 1000 ppm.
NTIS # PB2004-104000