ToxSci Advance Access originally published online on February 13, 2006
Toxicological Sciences 2006 91(1):247-254; doi:10.1093/toxsci/kfj128
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A Dose-Response Study Following In Utero and Lactational Exposure to Di(2-ethylhexyl)phthalate: Effects on Female Rat Reproductive Development
Department of Toxicology, Institute of Clinical Pharmacology and Toxicology, Campus Benjamin Franklin, Charité University Medical School Berlin, 14195 Berlin, Germany
1 To whom correspondence should be addressed at Department of Toxicology, Institute of Clinical Pharmacology and Toxicology, Campus Benjamin Franklin, Charité University Medical School Berlin, Garystrasse 5, 14195 Berlin, Germany. Fax: +49 30 8445 1761. E-mail: ibrahim.chahoud{at}charite.de.
Received December 7, 2005; accepted February 1, 2006
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ABSTRACT |
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Phthalates, a class of chemicals used as plasticizers, are economically important due to several industrial applications. Di(2-ethylhexyl)phthalate (DEHP) is the most commonly used phthalate plasticizer, and it has been described as a potent antiandrogen in males. We performed an extensive dose-response study following developmental exposure to DEHP and evaluated the effects on female reproductive development. Two wide ranges of doses that included dose levels relevant for human exposure as well as high doses typically used in toxicological studies were tested. Female Wistar rats were treated daily with DEHP and peanut oil (vehicle control) by gavage from gestation day 6 to lactation day 22. The low doses were 0.015, 0.045, 0.135, 0.405, and 1.215 mg DEHP/kg body weight (bw)/day, and the high doses were 5, 15, 45, 135, and 405 mg DEHP/kg bw/day. At the dose levels tested, no signs of maternal toxicity were observed. A significant delay in the age at vaginal opening (approximately 2 days) at 15 mg DEHP/kg bw/day and above, as well as a trend for a delay in the age at first estrus at 135 and 405 mg DEHP/kg bw/day (approximately 2 days), was observed. Liver enlargement (characteristic of phthalate exposure in rats) was limited to the 135- and 405-mg DEHP/kg bw/day doses. Anogenital distance and nipple development were unaffected. Based on the results of delayed pubertal onset, the no observed adverse effect level for female reproductive development may be set at 5 mg DEHP/kg bw/day.
Key Words: DEHP; female; dose-response; development; endocrine disruptors.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Considerable concern has been growing over the adverse effects that may result from exposure to chemical substances that have the potential to modify the normal endocrine function in wildlife and humans, the so-called endocrine-disrupting chemicals (EDCs). Numerous substances, such as dioxins, polychlorobiphenyls, dichlorodiphenyltrichloroethane, and phthalates, have been suspected of working through endocrine-disruptive mechanisms (Witorsch, 2002
). In recent years, phthalates, a class of chemicals used as plasticizers, have attracted special attention because of their high-volume production, ubiquitous environmental presence, and possible association with adverse reproductive health outcomes (Kavlock et al., 2002; Lovekamp-Swan and Davis, 2003). According to recent biomonitoring studies, exposure of the general population to phthalates is widespread (Barr et al., 2003; Koch et al., 2003; Silva et al., 2004). Di(2-ethylhexyl)phthalate (DEHP) is the most commonly used phthalate plasticizer of polyvinyl chloride (PVC) with a production of approximately one million to four million tons per year (Akingbemi et al., 2001; McKee et al., 2004). It is used in the manufacture of a broad range of consumer goods, including building, car and children's products, clothing, food packaging, and medical devices (Kavlock et al., 2002). DEHP imparts softness and flexibility to PVC, acting as a molecular lubricant. To achieve this purpose, DEHP does not form strong linkages with the polymer at a molecular level. As a result, it can diffuse throughout the matrix and leach into the environment (Bosnir et al., 2003; Petersen and Breindahl, 2000). Most of the observed toxic effects are not caused by DEHP itself but by its monoester metabolite mono(2-ethylhexyl)phthalate (Hoppin, 2003; Latini, 2000), although there is recent evidence that secondary oxidized metabolites may also be responsible for some effects (Regnier et al., 2004; Stroheker et al., 2005).
Several studies have reported widespread human exposure to phthalates. A study by Koch et al. (2003) demonstrated that the general German population is exposed to DEHP at a much higher extent than previously assumed, with an estimated median daily intake of 13.8 µg/kg/day. In addition, because DEHP is able to cross the placenta and pass into breast milk, there is a significant risk of exposure for the developing fetus and newborn (Dostal et al., 1987; Latini et al., 2003). Immature organisms are especially susceptible to endocrine disruption because the reproductive system is under development and relatively small changes in endogenous hormone levels may result in permanent structural and functional changes (WHO, 2002). Recent toxicological studies (Barlow and Foster, 2003; Li et al., 2000; Moore et al., 2001; Mylchreest et al., 2002) have demonstrated that in utero and lactational exposure to individual phthalates (including DEHP) results in a range of reproductive abnormalities in male offspring, consistent with the disruption of androgen-dependent development. Although there is some evidence of female reproductive toxicity in adult animals, little is known about the effects of developmental exposure to DEHP on females. Davis et al. (1994) reported that high-dose exposure (2000 mg DEHP/kg body weight (bw)/day), administered to 6–10 adult female Sprague-Dawley rats per group by gavage for 1–12 days, resulted in prolonged estrous cycles and lowered circulating estradiol levels.
In recent years, there has been growing concern over the possibility of hormonal disruption at low levels of exposure. The traditional risk assessment of environmental chemicals is based on the extrapolation of high-dose data in experimental systems to estimate effects at much lower doses in humans (Faustman and Omenn, 1996; Welshons et al., 2003). However, the validity of such extrapolations is frequently challenged if one considers that low levels of exposure may lead to effects that are qualitatively and quantitatively different from effects seen at high-dose exposures (Kavlock et al., 1996).
The objective of the present study is to evaluate the possible reproductive effects of low (human relevant) and high doses of DEHP on female offspring rats exposed in utero and during lactation. The present results are part of a comprehensive dose-response study designed to evaluate the effects of DEHP on male and female reproductive development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Female Wistar rats, weighing 200 ± 15 g, were purchased from Harlan-Winkelmann (Borchen, Germany) and allowed to acclimatize for 2 weeks. The animals were maintained in the animal facilities of the Institute of Clinical Pharmacology and Toxicology, Charité Medical School Berlin, under controlled temperature (21 ± 1°C), humidity (50 ± 5%), and light (12-h light/dark cycle). Standard pellet food (Altromin 1324, Altromin GmbH, Lage, Germany) and tap water were available ad libitum. Two nongravid female rats were mated with one male for 3 h, and the day of sperm detection in the vaginal smear was considered day 0 of gestation. The gravid females were randomly assigned among the treatment groups and housed individually in type III Macrolon cages with stainless steel covers and softwood granulate as bedding (Altromin GmbH). The experimental protocol was approved by the Office for Work and Health Protection and Technical Safety of the State of Berlin in accordance with the German National Animal Protection Law (Tierschutzgesetz BGBI. I S. 1105, 1998
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Dose selection and treatment.
Rat dams were administered DEHP (lot no. S11126
[GenBank]
-334, Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) or the peanut oil vehicle (Bombastus-Werke AG, Freital, Germany) by daily gavage from day 6 of gestation to day 21 of lactation. Dams were killed by decapitation and pups were weaned on postnatal day (PND) 22. The dosing volume used was 5.0 ml/kg bw. Two wide ranges of doses, low and high, were used in order to provide a clear insight into the shape of the dose-response curve of DEHP. The low-dose levels were 0.015, 0.045, 0.135, 0.405, and 1.215 mg/kg/day, and the high-dose levels were 5, 15, 45, 135, and 405 mg/kg/day. The low-dose range was selected starting from a dose (0.015 mg/kg/day) similar to the estimated median daily intake of the general German population (0.0138 mg/kg/day) reported by Koch et al. (2003). Four additional doses were calculated by applying a space factor of 3 between doses. The high-dose range was chosen starting from 5 mg/kg/day and with a space factor of 3, so that the highest level (405 mg/kg/day) would be a dose known to induce reproductive adverse effects in male offspring rats without causing overt maternal toxicity (Moore et al., 2001). Because data on females are scarce in the literature, the high-dose range was based on previous results obtained in males.
Maternal weight was monitored daily throughout pregnancy and lactation. Variables including litter size, sex ratio, pup weight, postimplantation losses, and the number of viable pups were also assessed. On lactation day 22, the dams were killed by decapitation for collection of organs. The brain, liver, kidney, spleen, thymus, thyroid, and ovaries were removed and weighed. Dams and lactating pups were examined daily for clinical signs of toxicity.
Offspring data.
One or two female pups per litter were randomly selected and necropsied on PND 1. The brain and liver were removed and weighed. Litters were weighed daily during the lactation period. On PND 13, all female pups were examined for the number of nipples/areolas by a single investigator unaware of treatment groups. On PND 22 (weaning), one to three female pups per litter were randomly selected for measurement of the anogenital distance (AGD) and necropsy. The AGD (defined as the distance between the center of the anus and the center of the genital bud) was measured using a manual caliper by a single investigator in a blinded manner. The animals were carefully handled to avoid variation in the measurement due to stretching of the perineal region. Thereafter, pups were killed by decapitation and the brain and liver were removed and weighed.
Beginning on PND 33, all females were evaluated daily for vaginal opening. The day of complete vaginal opening and body weight on that day were recorded. From the day of vaginal opening, daily vaginal smears were collected to detect the day of first estrus, characterized by the predominance of cornified epithelial cells.
Statistical analyses.
Statistical analysis was conducted using either SPSS 12.1 (SPSS Inc., Chicago, IL) or SAS 9.1 (SAS Institute Inc., Cary, NC). Normality and homogeneity of variances were evaluated prior to data analysis. We used a linear mixed model (proc mixed) with treatment as a main effect and litter as a random factor (nested for treatment) to adjust for litter effects. Organ weights and AGD were analyzed with body weight as a covariate. When an overall treatment effect (F-test) was observed, post hoc comparisons were performed using least square means (LSMEANS). The proportion of animals with vaginal opening and first estrus was analyzed by chi-square. Differences were considered to be statistically significant at a probability level of 5% (p < 0.05).
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RESULTS |
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Maternal and Reproductive Outcome Data
At the dose levels tested, DEHP had no statistically significant effect on prenatal and postnatal body weight gain of dams. Litter size, sex ratio, postimplantation losses, and number of viable pups were also unaffected by treatment (Table 1). Pup birth and weaning weight were similar in all groups, and no signs of toxicity were observed in dams and offspring. DEHP had no effect on brain, spleen, thymus, ovary, and thyroid weights of dams. A significant increase in liver and kidney weights was detected at the highest dose level (405 mg/kg/day) (Table 2).
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Offspring Data
In the females selected for necropsy at PND 1, we observed a significant increase in liver weight at 135 and 405 mg/kg/day. Female pups were significantly heavier than controls in the 0.045-, 1.215-, and 5-mg/kg/day groups. Brain weight was similar in all groups. At PND 22, however, no effects were seen in body, liver, and brain weights (Table 3). AGD, with or without covariance adjustment for body weight, was unaffected in all treatment groups (Fig. 1). Likewise, no differences were seen in the number of nipples assessed at PND 13 (Fig. 1).
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Age at Vaginal Opening and First Estrus
Because sexual development landmarks (vaginal opening and first estrus) are categorical variables, we analyzed these end points by chi-square. However, to facilitate comparison to other studies, the mean age at vaginal opening and first estrus were also evaluated. A significant delay in the mean age at vaginal opening (approximately 2 days) was observed in the 15-, 45-, 135-, and 405-mg/kg/day groups (Table 4). This result was also present when the number of animals displaying vaginal opening was analyzed by chi-square (Fig. 2). The body weight at vaginal opening was increased only at 1.215 and 135 mg/kg/day. Although no significant differences were found at the age of first estrus (F-test = 1.85, p = 0.057), there was a trend for a delay (approximately 2 days) at the two highest dose levels (LSMEANS Control vs. 135 mg/kg/day, p = 0.053; Control vs. 405 mg/kg/day, p = 0.008). When analyzed by chi-square, no significant differences were detected at age of first estrus (Fig. 2). The body weight at first estrus was increased at 1.215, 5, 135, and 405 mg/kg/day (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the current study, we evaluated the reproductive effects of DEHP on female offspring rats exposed in utero and during lactation to a wide range of doses. It is well known that exposure to EDCs during critical developmental periods (e.g., in utero, lactation, and puberty) is of particular concern, because small changes in hormone levels may induce long-term deleterious effects on the reproductive tract (Colborn et al., 1993
; U.S. EPA, 1997). Despite some evidence of female reproductive toxicity in adult animals, little is known about the effects of developmental exposure to DEHP on females. There is also a lack of data regarding female exposure to DEHP at environmentally relevant doses.
The major finding of the present study is that exposure to DEHP throughout gestation and lactation causes a significant delay in the age of pubertal onset in female offspring. We detected a significant delay in the age at vaginal opening (approximately 2 days) in the groups exposed to 15, 45, 135, and 405 mg DEHP/kg/day. In addition, a similar delay in the age at first estrus was also observed at 135 and 405 mg DEHP/kg/day, although not significantly so (F-test = 1.85, p = 0.057). Body weight was unaffected or even higher in treated animals when compared to the control group. A previous in utero and lactational exposure study did not detect significant changes at the age at vaginal opening and first estrus (Moore et al., 2001). In the study by Moore et al. (2001), DEHP was given to rat dams from gestation day (GD) 3 to PND 21 at 0, 375, 750, and 1500 mg DEHP/kg bw/day, but adverse effects were only reported in males. Lack of changes in the age at vaginal opening has also been previously described in females exposed in utero and during lactation (Mylchreest et al., 1998) and in utero alone (Mylchreest et al., 2000) to di(n-butyl)phthalate, another commonly used phthalate. However, a recent unpublished multigeneration continuous breeding study (reviewed by NTP-CERHR, 2005) presented results similar to ours. In this study, Sprague-Dawley rats were fed diets containing 1.5 (control), 10, 30, 100, 300, 1000, 7500, or 10,000 ppm DEHP. The age at vaginal opening was significantly delayed by approximately 3 days at 7500 ppm DEHP (estimated intake of 392–592 mg/kg/day) and by 8 days at 10,000 ppm (543–775 mg/kg/day) in F1 animals (NTP-CERHR, 2005). This effect was also detected in F2 and F3 animals at 7500 ppm DEHP (because F1 animals at 10,000 ppm were infertile, no data on subsequent generations were available). No data regarding first estrus were presented. In contrast, Noriega et al. (2004) reported no differences in time to vaginal opening when female Sprague-Dawley rats were treated with DEHP during puberty. The age at vaginal opening and first estrus is mainly dependent on increasing levels of estradiol during puberty, and a delay in these processes may suggest antiestrogenic or androgenic activity of test compounds. However, other factors not specifically controlled by estrogen/androgen status can also influence the time of pubertal onset. Interestingly, in our results, a delay in the age at preputial separation was also observed in males (Andrade et al., in preparation), and the dose levels that affected this end point were the same that resulted in delayed vaginal opening in females. This effect was also observed in the multigeneration study reviewed by NTP-CERHR (2005), suggesting that developmental exposure to DEHP may interfere with a common mechanism controlling the puberty onset in both sexes.
A significant increase in liver weight at PND 1 was detected in female offspring exposed to 135 and 405 mg DEHP/kg/day. We also observed a significant increase in liver and kidney weights of dams in the 405-mg DEHP/kg/day group. Data from previous studies (DeAngelo et al., 1986; Poon et al., 1997) consistently show that the liver and kidney are primary targets for DEHP. It is believed that many of the hepatic effects induced by phthalates (e.g., increase in weight, hepatocellular hypertrophy, hyperplasia) are due to activation of the peroxisome proliferator–activated receptors 
(PPAR). However, because the presence of PPAR has marked species differences (with rodents showing much higher expression than humans), the hepatotoxic effects of DEHP are judged not to be relevant for humans (reviewed by Doull et al., 1999).
No significant changes were detected in nipple development and AGD of female offspring at any dose level tested. In contrast to males, sexual differentiation of females is largely hormone independent, yet still susceptible to hormonal disruption, e.g., masculinization of the female fetus by exposure to androgens (Hughes, 2001; Sharpe, 2001). In a dose-response study conducted by Wolf et al. (2002), in which testosterone propionate was administered to rat dams during late gestation (GDs 14–19), inhibition of nipple development and increased AGD in female offspring were among the most sensitive indicators of masculinization. Previously, changes in nipple development induced by DEHP have been reported in males but no data on females were available. In the study by Moore et al. (2001), male offspring rats exposed in utero and during lactation presented nipple retention at all dose levels tested (375, 750, and 1500 mg DEHP/kg bw/day), and AGD was reduced in males at 750 and 1500 mg DEHP/kg bw/day, but unaffected in females. Our results confirm and extend the observations made by Moore et al. (2001) showing that both end points (nipple development and AGD) are not affected in female offspring exposed to low as well as high doses of DEHP.
In summary, this study demonstrated that in utero and lactational exposure to DEHP at 15 mg/kg/day and above significantly delays the time of pubertal onset in female offspring. Therefore, 5 mg DEHP/kg/day was the highest dose level that did not produce an adverse response and can be considered as the no observed adverse effect level (NOAEL) for this end point. The NOAEL must by definition be one of the experimental doses tested, and the use of a wide range of doses with a short space factor between them (as in the present study) may increase the confidence in this value. In adult females, reproductive toxicity of DEHP has been reported after exposure to very high dose levels (2000 mg/kg/day; Davis et al., 1994), but no comprehensive data on developmental exposure were available. Our results suggest that immature animals may present a greater sensitivity to DEHP. It is important to note that developmental exposure to endocrine disruptors may have either immediate or latent effects. In addition to the delay in age of pubertal onset, it is possible that in utero and lactational exposure to DEHP causes deleterious effects revealed at a later age (sexual maturity).
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ACKNOWLEDGMENTS |
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We thank H. Marburger for exemplary technical assistance and C. Gericke for valuable support on statistical analysis. A. J. M. Andrade is a recipient of a scholarship from CAPES/Brazil.
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REFERENCES |
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