4.4 Summary of Reproductive Toxicity Data
4.4.2 Experimental Animal Data
Mitsubishi.Chemical.Safety.Institute,.Ltd..(92), in an unpublished report, described a 65-week oral-dose toxicity study of DEHP in marmosets. DEHP was administered by gavage in corn oil to juvenile marmosets beginning at 90 – 115 days of age until 18 months of age (young adulthood) at dose levels of 0, 100, 500, and 2500 mg/kg bw/day. Both males and females were assessed with a battery of in-life hormonal assays and with histopathology at necropsy. In the female marmosets, DEHP at 500 and 2500 mg/kg bw/day was associated with early increases in serum 17b-estradiol consistent with an early onset of puberty, manifested as a significant increase in ovarian and uterine weights at necropsy in these groups.
Male reproductive toxicity data in experimental animals are summarized in Table 34.
At dose levels of 10 and 100 mg/kg bw/day, Akingbemi.et.al..(111, 179) showed reduced Leydig cell testosterone production ex vivo, increased serum LH, testosterone, and 17b-estradiol, and Leydig cell hyperplasia. The authors concluded, and the Expert Panel concurs, that DEHP increases Leydig cell populations associated with chronically increased LH and testosterone levels, and that a decrease in testosterone and increase in estradiol synthesis (per cell) was consistent with induction of aromatase activity in Leydig cells. The authors identified 1 mg/kg bw/day as a NOAEL and 10 mg/kg bw/day as a LOAEL.
In another study, 10-week-old male mice were given 0 or 2% dietary DEHP for 16 days following which all animals were given untreated feed for an additional 50 days with vitamin supplementation (175)..
Groups of animals were killed during and after the end of exposure and assessed for testicular effects by weight and histology. By day 12 of exposure, testis weights were decreased and recovered towards control levels 20 days after cessation of exposure. Similarly, testis histopathology abnormalities were seen during treatment and recovered after the end of exposure. Groups of animals were killed during and after the end of exposure and assessed for testicular effects by weight and histology. By day 12 of exposure, testis weights were decreased and recovered towards control levels 20 days after cessation of exposure. Similarly, testis histopathology abnormalities were seen during treatment and recovered after the end of exposure.
Table 34. Summary of Male Reproductive Toxicity Data from Studies in Rats and Mice Species and Dosing Most Sensitive Outcome Effect Levels
(mg/kg bw/day) Reference Single Dose-Level Studies (excluding control)
Mice, C57Bl6 wild-type and gld mutant
MEHP 1000 mg/kg bw × 1 by gavage
TUNEL labeling greater in wild-type than fasL-deficient mutant
mice 1000 Richburg et al.
(169) Mice, C57Bl6 wild-type and gld
MEHP 1000 mg/kg bw × 1 by gavage
Apoptotic response decreased in fasL-deficient mutant compared to
wild-type mice 1000 Giammona et
al. (171) Mice, CD
DEHP 2% in the diet for 16 days
Decreased testis weight, abnormal
histopathology findings  Ablake et al.
(175) Rat, Sprague-Dawley
MEHP 1000 mg/kg bw × 1 by gavage
Increased expression of death
receptors 1000 Giammona et
al. (171) Rat, Wistar
MEHP 400 mg/kg bw × 1 by gavage
Germ cell sloughing, collapse of Sertoli cell vimentin filaments 3
hours after treatment 400 Dalgaard et al.
(172) Rat, Sprague-Dawley
DEHP 2 mg/rat/day
day].× 1 – 14 days
Decreased body and testis weight, testis histologic evidence of
apop-tosis beginning treatment day 3 [19,000.–.29,000] Park et al.
Multiple Dose Levels Rat, Wistar
DEHP 0, 1000, or 2000 mg/kg bw/day by gavage × 7 days
Decreased total glutathione, low molecular weight thiols, and
ascorbic acid in testis LOAEL 1000 Kasahara et al.
(174) Rat, Wistar
DEHP 0, 100, or 1000 mg/kg bw/day by gavage × 5 days
Decreased aromatase and
increased CYP2C11 and CYP3A2 testosterone hydroxylation in testis
LOAEL 100 a Kim et al.
(177) Rat, Long-Evans
DEHP 0, 1, 10, 100, or 200 mg/kg bw/day by gavage × 14 or 28 days
Decreased 17α-hydroxylase in testis, altered ex vivo Leydig cell
testosterone synthesis LOAEL 10
NOAEL 1 Akingbemi et al. (111) Rat, Long-Evans
DEHP 0, 10, or 100 mg/kg bw/
day by gavage × 70 or 100 days
Increased serum LH and testoster-one, decreased ex vivo Leydig cell
testosterone synthesis (PND 90) LOAEL 10 Akingbemi et al. (179)
a The use of a recovery period in the dosing schedule prevented the Expert Panel from being able to interpret the results.
22.214.171.124 Male and Female
Reproductive toxicity data in male and female rats are summarized in Table 35.
Schilling.et.al..(151) performed a 2-generation reproductive study of DEHP in Wistar rats. DEHP was administered in feed at 0, 1000, 3000, and 9000 ppm, resulting in estimated DEHP intakes of 0, 113, 340, and 1088 mg/kg bw/day. The high-dose level (9000 ppm) was associated with a decrease in feed consumption and weight gain at several intervals during the study. An extensive evaluation was conducted, including a functional observation battery, water maze, estrous cyclicity, cauda epididymal sperm evaluation, testicular spermatid head counts, presence of nipples and areolae in male pups, and day and weight at vaginal opening and preputial separation. F2 pups in the high-dose group were smaller and gained less weight from birth through the assessment of functional observation battery and water maze testing. Grip strength was reduced in males and hind-limb splay was reduced in both sexes in the high-dose group. Differential ovarian follicle counts of F0 and F1 adults showed a deficit in growing follicles and corpora lutea in the high-dose group. [The.Expert.Panel.noted.dose-related.
Mitsubishi.Chemical.Safety.Institute,.Ltd..(92), in an unpublished report, described a 65-week oral-dose toxicity study of DEHP in marmosets. DEHP was administered by gavage in corn oil to juvenile marmosets beginning at 90 – 115 days of age until 18 months of age (young adulthood) at dose levels of 0, 100, 500, and 2500 mg/kg bw/day. Both males and females were assessed with a battery of in-life hormonal assays and with histopathology at necropsy. Although there were significant limitations in the study and the reporting of findings, the results suggest little effect of DEHP exposure at 2500 mg/kg bw/day in marmosets on testicular structure and function. Mean serum testosterone levels were highly variable, but the data suggested the possibility of a delay in the onset of puberty in male marmosets with increasing DEHP dose. The authors suggested, based on an accompanying pharmacokinetic study (reviewed in Section 2.1), that the most likely explanation for the lack of testicular toxicity of DEHP in marmosets as opposed to rodents was the limited accumulation of DEHP and its metabolites in the marmoset testis. This limited accumulation may in part be a result of the short gut transit time and propensity to diarrhea. In addition, the male marmoset has an apparent relative end-organ steroid resistance compared to humans. [Because.of.the.limited.blood.levels.achieved.in.this.study,.differ-ences.between.marmosets.and.humans.in.terms.of.their.steroid.resistance,.and.the.difficulties.
The.NTP.(114) sponsored a multigeneration continuous-breeding study in rats. Sprague-Dawley rats were fed diets containing 1.5 (control group exposed to background DEHP levels), 10, 30, 100, 300, 1000, or 7500 ppm DEHP from the first day of the study until the day of necropsy. Due to a described lack of reproductive effects in the first litter produced, the study was repeated with 2 additional doses, 1.5 (control) and 10,000 ppm..Ranges of DEHP intake in the F0, F1, and F2 animals were estimated at 0.09 – 0.12, 0.47 – 0.78, 1.4 – 2.4, 4.8 – 7.9, 14 – 23, 46 – 77, 392 – 592, and 543 – 775 mg/kg bw/day.
At about 5 weeks of age, F0 rats were fed the DEHP-containing diets for 6 weeks prior to mating and were then cohabitated for 9 weeks. The first 2 litters delivered during the cohabitation period (F1a
and F1b) were counted, weighed, assessed for anogenital distance, and discarded. The third litter (F1c) was raised by the dam. Following weaning of pups, vaginal cytology was monitored in F0 females for 14 days. After completion of crossover studies described below, at least 10 F0 rats sex/group were necropsied. Sperm analyses were conducted, and organs were collected for histopathological evalu-ation. F1 pups were counted, weighed, and examined for anogenital distance and nipple retention during the lactation period. On PND 16, 1 female per litter was evaluated for vaginal opening, and a second was selected for F1 mating. One male per litter was selected for mating, and 4 or 5 males per litter were evaluated for testicular descent and preputial separation. At weaning on PND 21, pups were given diets containing the same DEHP concentrations as their parents. On PND 81, the F1 rats chosen for mating (17/sex/group) were randomly assigned to breeding pairs (preferably non-sibling) and cohabited for 9 weeks. The study conducted in F0 parents and F1 offspring was repeated in F1 parents and F2 offspring, except that no F3 offspring were mated. Selected F3c males were necropsied on PND 63 – 64 and selected females on PND 60 – 74.
The liver was identified as a target of toxicity with increases in liver weight and hepatocellular hyper-trophy observed at dose levels ≥ 1000 ppm. Changes in organ weights and lesions were also observed in the kidney at ≥ 7500 ppm and the adrenal gland at 10,000 ppm.
Reproductive effects observed in the F0 parents occurred only at 10,000 ppm and included decreases in sperm counts and velocity, reductions in testis and epididymis weights, and increased numbers of rats with small testes. The lowest dose level producing dose-related effects in F1 offspring was 7500 ppm, and those effects included decreases in number of live pups/litter, reduced male anogenital distance, and delays in vaginal opening, preputial separation, and age at testicular descent.
F1 rats from the 10,000 ppm group produced no viable litters. At and above 7500 ppm, rats had reduced sperm counts, seminiferous tubular atrophy, and delayed preputial separation and testicular descent.
In the F3 offspring, reproductive toxicity was noted in numerous endpoints at 7500 ppm but not at lower dose levels. Effects included seminiferous tubule atrophy and decreases in pregnancy index, the number of litters per pair, male reproductive organ weights, sperm counts, and sperm motility.