Dose-Dependent Alterations in Androgen-Regulated Male Reproductive Development in Rats Exposed to Di(n-butyl) Phthalate during Late Gestation

Eve Mylchreest2, Duncan G. Wallace, Russell C. Cattley3 and Paul M. D. Foster1

Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709

Received October 18, 1999; accepted January 4, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Di(n-butyl) phthalate (DBP) is a commercially important plasticizer and ubiquitous environmental contaminant. Since previous, limited dose-response studies with DBP that reported alterations in male reproductive development and function failed to establish a NOAEL (no-observed-adverse-effect level), an extensive dose-response study was conducted. Pregnant CD rats were given DBP by gavage at 0, 0.5, 5, 50, or 100 mg/kg/day (n = 19–20) or 500 mg/kg/day (n = 11) from gestation day 12 to 21. In male offspring, anogenital distance was decreased at 500 mg DBP/kg/day. Retained areolas or nipples were present in 31 and 90% of male pups at 100 and 500 mg/kg/day, respectively. Preputial separation was not delayed by DBP treatment in males with normal external genitalia, but cleft penis (hypospadias) was observed in 5/58 rats (4/11 litters) at 500 mg/kg/day. Absent or partially developed epididymis (23/58 rats in 9/11 litters), vas deferens (16/58 animals in 9/11 litters), seminal vesicles (4/58 rats in 4/11 litters), and ventral prostate (1/58 animals) occurred at 500 mg/kg/day. In 110-day-old F1 males, the weights of the testis, epididymis, dorsolateral and ventral prostates, seminal vesicles, and levator ani-bulbocavernosus muscle were decreased at 500 mg/kg/day. At 500 mg/kg/day, widespread seminiferous tubule degeneration was seen in 25/58 rats (in 9/11 litters), focal interstitial cell hyperplasia in 14/58 rats (in 5/11 litters), and interstitial cell adenoma in 1/58 rats (in 1/11 litters). For this 10-day prenatal (embryonic and fetal) exposure to DBP, the NOAEL and LOAEL (lowest-observed-adverse-effect level) were 50 and 100 mg/kg/day, respectively. This is currently the lowest NOAEL described for the toxicity of DBP.

Key Words: phthalate, di(n-butyl); male reproductive development; in utero exposure; reproductive tract malformations; testicular toxicity; androgen; Leydig cell hyperplasia.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Di(n-butyl) phthalate (DBP) is a constituent of a large group of chemicals called phthalate esters that are mainly used as plasticizers to impart flexibility to nitrocellulose, polyvinyl acetate, and products containing polyvinyl chloride. DBP is also a component of adhesives, coatings for paper and other materials, printing inks, aerosols, nail polish, and hair spray. Other prominent uses of DBP are as a lubricating oil, an antifoaming agent, a skin emollient and a solvent in cosmetics. In western industrialized countries, annual production of DBP is 10–50,000 tons, and 1–4 million tons for di(ethylhexyl) phthalate (DEHP), the most commercially important phthalate ester plasticizer. DBP is ubiquitous but not very persistent in the environment, probably because it is rapidly metabolized in the environment and in exposed animals. The monoester metabolite mono (n-butyl) phthalate (MBP), not the parent compound, is believed to be the toxic species, but little is known about environmental levels. Human exposure occurs primarily through contaminated food, particularly foods with high-fat content, that may have been in contact with plastic, inks, adhesives, and other packaging materials that contain DBP.

In laboratory animals, DBP and some other phthalate esters elicit liver, testicular, and developmental toxicity at relatively high doses. Subchronic oral exposure to DBP causes peroxisome proliferation and hepatomegaly in rats; the lowest reported NOAEL (no-observed-adverse-effect level) and LOAEL (lowest-observed-adverse-effect level) for liver effects are 138 and 279 mg/kg/day, respectively (NTP, 1995Go). Unlike DEHP, which is a more potent peroxisome proliferator, DBP does not induce liver tumors in rats. Following exposure during major organogenesis, DBP is embryotoxic at 630 mg/kg/day and is teratogenic at even higher and frankly maternally toxic doses in rats (Ema et al., 1993Go). DEHP, DBP and some other phthalate esters produced extensive testicular toxicity in rats exposed to extremely high subacute oral doses (1–2 g/kg/day) (Cater et al., 1977Go, Gray et al., 1977Go) and somewhat lower subchronic doses (571 mg/kg/day) (NTP, 1995Go). The widespread germ-cell loss in the seminiferous epithelium of the testis appears to result from Sertoli cell dysfunction (Foster et al., 1982Go, Gray and Beamand, 1984Go). The embryo and fetus appear to be more sensitive to the testicular toxicity of phthalate esters (Mylchreest et al., 1999Go) than neonates and pubertal rats, with adults being the least sensitive (Dostal et al., 1988Go, Foster et al., 1980Go).

Exposure to chemicals with hormone- or antihormone-like activity is one possible cause for the alleged decline in male reproductive health in humans (Toppari et al., 1996Go) and the increases in reproductive deficits in wildlife (Kendall et al., 1998Go). These so-called endocrine disruptors are chemicals that interfere with normal male reproductive development in laboratory animals by acting like estrogen, antiandrogens, and potentially by other mechanisms. There is growing concern that phthalate esters may cause endocrine disruption, more so recently since DBP has been shown to produce severe alterations in male reproductive development. DBP caused abnormal development of the epididymis and testis in rats exposed at lower doses in utero, neonatally, or as adults in a long-term breeding study (66, 320, 651 mg/kg/day) (NTP, 1991Go), when compared with doses given to adult animals that produce liver toxicity or other adverse effects. We have shown that a much shorter exposure throughout gestation and lactation at 250–750 mg DBP/kg/day also produced malformations of reproductive organs, namely of the epididymis, as well as seminiferous tubule degeneration reported in the NTP study (Mylchreest et al., 1998Go). The major period of male sexual differentiation in the rat (days 12–21 of gestation) was a sensitive developmental window for the reproductive tract malformations, degenerative and proliferative lesions in the testis, feminized anogenital distance, and nipple development induced by DBP. We previously proposed that the embryonic/fetal testis is the primary target and that DBP produces antiandrogenic effects by altering androgen-signaling pathways during prenatal male sexual differentiation. DBP disrupts the development of tissues that require androgens for their differentiation, but it does not do so by direct interaction with steroid receptors (Mylchreest et al., 1999Go).

A NOAEL for male reproductive and developmental toxicity has not been identified for DBP. The current LOAEL is 66 mg/kg/day for absent testis and epididymis, and reduced F2 pup weight in the NTP continuous-breeding study (NTP, 1991Go). The purpose of this study was to determine the NOAEL for the effects of DBP on male reproductive development in rats exposed during the major period of prenatal male sexual differentiation (gestation days [GD] 12–21). The dose levels selected were 0, 0.5, 5, 50, 100, and 500 mg DBP/kg/day. The highest dose was chosen because it was previously shown to produce reproductive tract malformations and testicular toxicity in the absence of maternal toxicity and fetal death (Mylchreest et al., 1998Go). The mid-dose levels encompass the LOAELs of 66 and 100 mg DBP/kg/day established in previous studies (Mylchreest et al., 1999Go, NTP, 1991Go), while the lower dose levels were selected to achieve a NOAEL.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
This study was approved by the CIIT Institutional Animal Care and Use Committee and followed Federal guidelines for the care and use of laboratory animals (NRC, 1996Go). Animals were housed in a HEPA-filtered, mass air-displacement room that was maintained on a 12–h light–dark cycle at approximately 18–26°C with a relative humidity of 30–70%.

Female Sprague-Dawley CD rats, approximately 8 weeks-old, were mated overnight at Charles River Laboratories, Inc. (Raleigh, NC), and sperm-positive females were received at the CIIT animal facility the following morning. The day sperm was found in the vagina of the mated female was considered gestation day 0 (GD 0). Upon arrival, animals were allocated to dose groups, using randomization of body weights to ensure equal weight distribution among groups. Timed pregnant rats were housed individually and postweaning offspring, in groups of up to 4 by gender and dose group, in polycarbonate cages with cellulose fiber chips (ALPHA-dri, Shepherd Specialty Papers, Kalamazoo, MI) as bedding. Rodent feed (NIH–07, Zeigler Bros., Gardner, PA) and deionized water were provided ad libitum. We used glass, rather than plastic, water bottles with Teflon-lined caps.

Treatment.
From GD 12 to 21, timed pregnant female rats were administered DBP (99.8%, Aldrich Chemical Co., Milwaukee, WI) or the corn-oil vehicle by daily gavage at 1 ml/kg/day between 0830 and 1130. The dose levels were 0, 0.5, 5, 50, 100, and 500 mg/kg/day. The stability of DBP in corn oil at the low and high concentrations (0.5 and 500 mg/kg/day) over a 21–day period was verified by gas chromatography. DBP was stable in corn oil at these concentrations, which fell within 4–7% of expected values at the end of the study.

The study was conducted in 2 blocks, each comprising half the total number of animals per dose level. The targeted number of pregnant dams delivering litters was 10 in the 500-mg/kg/day group and 20 at the other dose levels. Two additional dams (one per block) were included and designated as replacements a priori in each dose group, should the targeted number of litters not be achieved; these potential replacements were retained only to substitute for animals that were removed from the study (e.g., nonpregnant or dead animals). Dams were examined for clinical signs of toxicity and their body weights recorded daily immediately prior to dosing and weekly during lactation. Food consumption of dams was monitored twice weekly throughout the dosing period and during lactation. Dams were killed by carbon dioxide asphyxiation when their pups were weaned at postnatal day (PND) 21. Tissues were examined grossly, and weights of liver, kidneys, adrenals, uterus, and ovaries and numbers of implantation sites were recorded.

Litters.
On the day of delivery, which was considered to be post-natal day 1 (PND 1), the following end points were recorded: numbers of live and dead pups, clinical signs of toxicity, pup weight (by sex and litter), and anogenital distance (Mylchreest et al., 1999Go). Litters were weighed weekly by gender during the lactation period. The location and number of nipples and areolas were recorded for each F1 male pup on PND 14 by examining the epidermis in the region of the nipples. Observations were scored based on the presence or absence of a nipple bud or a discoloration of the skin surrounding the nipple (i.e., areola). At weaning (PND 21), pups were identified individually by ear tag and housed by gender, in groups of 3–4 animals, according to treatment. Pups were examined daily for vaginal opening or preputial separation (separation of the prepuce from the glans penis) from PND 30 and 38, respectively, until acquisition.

Necropsy and pathology of F1 animals.
F1 pups were killed at sexual maturity (postnatal day 110 ± 10 for males and 80 ± 5 days for female) and body weights recorded. The internal and external genitalia were examined for malformations and undescended testes. The following organ weights were recorded: liver, kidneys, adrenals, uterus, ovaries, testes, seminal vesicles with coagulating glands and seminal fluid, epididymides, vas deferens, ventral prostate, dorsolateral prostate, and the levator ani-bulbocavernosus muscle complex. The right testis and epididymis were fixed in Bouin's and embedded in paraffin; 5-µm sections were stained with hematoxylin and eosin. Two transverse sections of the testis (from median and cranial regions) and one longitudinal section of the epididymis were evaluated for the presence of lesions. Glycol methacrylate/toluidine blue was used instead of paraffin/hematoxylin and eosin in the preparation of the cranial testis section of two animals per litter. Focal proliferative lesions of the interstitial cells were classified using current established criteria: lesions having a diameter greater than that of one seminiferous tubule were identified as interstitial cell adenoma, while those of lesser diameter were considered interstitial cell hyperplasia (Boorman et al., 1987Go). Degeneration of the seminiferous tubules was graded by subjective evaluation of the percentage of tubules affected in the two sections examined microscopically.

Statistical analysis.
The litter was used as the experimental unit unless stated otherwise. Statistical analyses were conducted using JMP (SAS Institute Inc., Cary, NC). Because this study was conducted in 2 blocks (of equal number of pregnant dams), differences between least-squares estimates of mean values were evaluated (F-test). Treatment and block were fixed effects in the model, while litter (nested for treatment and block) was a random effect. Body weight was used as a covariate for the analysis of anogenital distance, nipple retention, preputial separation, and organ weight data from the F1 animals. Post hoc contrasts of least squares means of each DBP level and of the control group were analyzed by an F-test. A logistic regression analysis was performed on preputial separation data (Fig. 4Go) and differences between control and treated groups were evaluated by comparing the slopes of the regression curves using a 95% confidence interval. The level of significance for all tests was p < 0.05.



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FIG. 4. Effect of exposure to di(n-butyl) phthalate (DBP) during late gestation (GD 12–21) on the onset of preputial separation in F1 male rats. Data are expressed as the percentage of males with preputial separation as a function of age. This landmark of pubertal development was evaluated in males with gross morphologically normal external genitalia, not in animals with hypospadias. Slopes of all DBP-exposed groups were not significantly different from control (p < 0.05).

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reproductive Performance
At the dose levels tested, DBP had no statistically significant effect on maternal body weight or body weight gain, food consumption, number of implantation sites, live pups per litter and pup weight at birth, or survival to weaning (Table 1Go). No treatment-related clinical signs of toxicity were observed. DBP had no effect on liver, kidney, adrenal, uterus, or ovary weight of dams (data not shown). Female pups were significantly (p < 0.05) heavier than controls in the 5-mg/kg/day group at PND 7 and in the 5-, 50-, 100-, and 500-mg/kg/day groups at PND 14 (results not shown) and at weaning on PND 21 (Table 1Go). Male pup weights were increased in the 5-mg/kg/day group at PND 7, 14 and 21 (Table 1Go). After weaning, no differences in pup body weights were seen between treatment and control groups (results not shown).


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TABLE 1 Reproductive Parameters in Rats Treated with Di(n-Butyl) Phthalate during Late Gestation (GD 12–21)
 
Developmental Landmarks
Exposure to DBP resulted in a statistically significant decrease (12% lower than controls) in anogenital distance in F1 males at 500 mg/kg/day only (Fig. 1Go) in the absence of changes in male pup weight (Table 1Go, Fig. 1Go). Anogenital distance was also significantly reduced when pup weight was used as a covariate. A dose-dependent increase in the incidence of thoracic areola and nipple development was also observed in F1 males on PND 14 (Fig. 2Go) and was highly correlated with anogenital distance, but not with pup weight, on PND 1. At 500 mg/kg/day, male pups had nipple buds similar to those of females. The areolas and nipples in the thoracic area were the most prominent. In contrast to the marked nipple bud development at 500 mg/kg/day, nipple development was more rudimentary at 100 mg DBP/kg/day but was still clearly different from control males. The nipple bud was rarely visible, but a dark spot on the skin was apparent at the position of the nipple, also mainly in the thoracic area. In the control group, only one male pup had nipple buds; others had a discoloration of the skin in the nipple region, but it was faint and did not show a predilection for the thoracic area. Typical nipple development in females, and in male pups at 100 and 500 DBP/kg/day compared to a control male, is shown in Figure 3Go.



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FIG. 1. Anogenital distance at birth (PND 1) in F1 male rats exposed to di(n-butyl) phthalate (DBP) daily from GD 12 to 21. Values are litter means ± SE for 19–20 litters per group except at 500 mg/kg/day (n = 11). Asterisk represents significant difference from control group (p < 0.05).

 


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FIG. 2. Effect of di(n-butyl) phthalate (DBP) treatment during late gestation (GD 12–21) on areola and nipple development in F1 male rats on postnatal day 14. The ratio of affected to total number of rats or litters is indicated above each bar. Asterisk represents significant difference from control group (p < 0.05).

 


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FIG. 3. Comparison of nipple development in control female (A), male (B), and male pups exposed to DBP from GD 12–21 at 100 (C) and 500 mg/kg/day (D). Note the prominent thoracic nipple buds at 500 mg DBP/kg/day (D) and the areolas at 100 mg DBP/kg/day (C) compared to the control male, which has no visible areolas in the thoracic area (B).

 
No significant change in the age at preputial separation was seen at any dose level (Fig. 4Go). Least squares means, tested for dose, block, litter, and body weight effects, were 45.2 ± 0.3, 44.9 ± 0.4, 44.1 ± 0.4, 44.3 ± 0.4, 45.0 ± 0.3,and 44.6 ± 0.6 at 0, 0.5, 5, 50, 100, and 500 mg DBP/kg/day, respectively. Preputial separation could not be evaluated in the 5 animals with malformed penis (hypospadia) at 500 DBP/kg/day. The age of onset of vaginal opening was similar in all F1 females (data not shown).

Reproductive Organ Weights in F1 Males
At sexual maturity, F1 males in the 500-mg/kg/day group had significantly smaller epididymides, dorsolateral prostate, and levator ani-bulbocavernosus muscle (Table 2Go). Body, kidney, adrenal, ventral prostate, vas deferens, seminal vesicle, and testis weights were only marginally decreased at 500 mg/kg/day, and no changes in organ weights were seen at lower dose levels (Table 2Go). Enlarged testes (2.7–3.9 g) were seen in 1, 2, and 8 animals at 0.5, 100, and 500 mg/kg/day, respectively. Testis weight was significantly decreased (1.589 ± 0.053; p < 0.0003) when enlarged testes were excluded. Liver weight was not significantly affected at any dose level.


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TABLE 2 Effect of Prenatal (GD12-21) Exposure to Di(n-Butyl) Phthalate on Body and Organ Weights in F1 Rats
 
Reproductive Organ Weights in F1 Females
At sexual maturity, F1 females showed no treatment-related changes in body, liver, kidney, adrenal, ovary, or uterus weight or gross morphology of the reproductive organs (data not shown).

Reproductive Tract Malformations
Malformations of the male reproductive tract were observed only at the highest dose level (Fig. 5Go). Absent or malformed epididymis was observed in 40% of the animals (82% of litters). The malformed epididymis consisted of missing body or head, or tail and head not attached to the rete testis. Twenty-eight percent of males (82% of litters) had an absent or malformed vas deferens and was generally associated with a malformed epididymis. Hypospadia was present in 9% of animals at 500 mg/kg/day. This malformation of the external genitalia consisted of small penis with a cleft on the ventral aspect near the tip and a cleft prepuce in some instances. Two of the 5 malformed animals had more severe hypospadias in which the os penis was exposed; these animals also had a cleft prepuce. Four animals had unilaterally absent seminal vesicle. One animal had no ventral prostate and 4 animals (in 3 litters) had a small intraabdominal testis. Malformations of the external genitalia, prostate, or seminal vesicles occurred in animals that had testicular lesions or malformed epididymis, and affected animals usually had multiple malformations.



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FIG. 5. Reproductive tract malformations induced by di(n-butyl) phthalate (DBP) in F1 male rats following exposure during late gestation (GD 12–21). Malformations occurred only at 500 mg DBP/kg/day. The number of affected animals or litters is indicated above each bar.

 
Histopathology
The testicular lesions induced by DBP consisted of seminiferous tubule degeneration, focal interstitial hyperplasia, and adenoma at 500 mg/kg/day (Table 3Go) and have been previously described morphologically in more detail (Mylchreest et al., 1998Go, 1999Go). Decreased tubule size and partial-to-complete depletion of the germinal epithelium, usually with retention of Sertoli cells, characterized the lesion of the seminiferous tubule. At 500 mg/kg/day, 43% of animals (82% of litters) had degeneration of the seminiferous epithelium in more than 50% of tubules (grade 4) in the 2 cross-sections of testis that were examined for each animal. A significant number of these animals (24% of animals in 45% of litters) also had focal interstitial-cell hyperplasia. In one animal, the focal hyperplasia was of a diameter sufficiently large to be considered an interstitial cell adenoma. The significance of a grade-4 testicular lesion in one animal at 100-mg/kg/day group is equivocal (see Discussion).


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TABLE 3 Histopathologic Lesions in the Testis of Adult F1 MaleRats Exposed Prenatally (GD 12–21) to Di(n-Butyl)Phthalate
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Androgen-dependent nipple regression was the most sensitive marker of altered male sexual differentiation by DBP. Following 10 consecutive days of oral exposure during the major period of prenatal male sexual differentiation, a dose-dependent increase in retained nipples occurred in male pups at 100 and 500 mg DBP/kg/day. Previously, nipple development was seen at 250 and 500 mg DBP/kg/day but not at 100 mg/kg/day (Mylchreest et al., 1999Go), possibly because the less noticeable nipple development at 100 mg/kg/day was previously overlooked. Development of the nipple anlagen is dependent on androgen produced by the fetal testis (Imperato-McGinley et al., 1986Go, Neumann et al., 1970Go, Neumann and Goldman, 1970Go). Furthermore, nipple retention appears to be a very sensitive marker of prenatal exposure to the environmental antiandrogen vinclozolin (Gray et al., 1999aGo), p, p–DDE (You et al., 1998Go) and procymidone (Ostby et al., 1999Go). Thus the development of nipples in male pups exposed to 100 mg DBP/kg/day can be considered indicative of altered androgen status during the critical window for imprinting of nipple sexual dimorphism in utero. Whether the nipples found in neonatal males were permanent as they are following antiandrogen exposure was not evaluated in the present study.

Aside from nipple retention at 100 mg/kg/day, other effects induced by DBP were restricted to the high-dose group. We observed decreased anogenital distance, reproductive tract malformations, seminiferous tubule degeneration, interstitial cell hyperplasia and adenoma in the testis, and small reproductive organs in F1 males at 500 mg/kg/day. Overall, these observations are in good agreement with our previous studies showing these effects on male sexual differentiation at 250, 500, and 750 mg/kg/day. The single incidence of severe seminiferous tubule degeneration at 100 mg/kg/day may not be due to DBP treatment, since there is a low spontaneous occurrence of severe testicular lesions in the Sprague-Dawley rat.

Preputial separation, a noninvasive marker of androgen-dependent postnatal pubertal development (Korenbrot et al., 1977Go), was not affected by DBP exposure from GD 12 to 21. This finding contrasts with previous observations of a delay at 500 mg DBP/kg/day and an equivocal change at 100 mg/kg/day (Mylchreest et al., 1999Go) but confirms earlier data revealing no statistically significant change in preputial separation at 250–750 mg/kg/day (Mylchreest et al., 1998Go). Nipple retention and anogenital distance also appear to be the most useful noninvasive markers of altered male sexual differentiation upon in utero exposure to other chemicals (Gray et al., 1999aGo, You et al., 1998Go).

The incidence of malformed epididymis and seminiferous tubule degeneration (generally occurring in the same animal) was highly consistent across studies with DBP, which is in contrast with the low and variable occurrences of other malformations (Mylchreest et al., 1998Go, 1999Go). Our present observations thus support those made in the NTP study (NTP, 1991Go). This outcome suggests that the fetal testis and upper Wolffian duct (the anlage of the epididymis) are more sensitive to DBP toxicity compared with the urogenital sinus and genital tubercle (the anlagen of the prostate and external genitalia). Since testosterone produced by the fetal testis is essential for Wolffian duct differentiation, potential effects of DBP on fetal testis androgen could lead to impairment of normal Wolffian duct differentiation. We have preliminary evidence of Leydig cell proliferation, germ cell degeneration, and reduced testosterone production in the fetal testis exposed to DBP. The Wolffian duct may be more sensitive to decreases in testosterone than the urogenital sinus and genital tubercle, which are dihydrotestosterone-dependent. High levels of testosterone may need to be reached and maintained between GD 17 and 18, the critical period of Wolffian duct stabilization (Jost et al., 1973Go). Also, the timing of testosterone production may be critical for Wolffian-duct differentiation, which begins at GD 16.5, concurrent with the rise in fetal testosterone in the rat (Warren et al., 1984Go). DBP could possibly interfere with the timing or the level of androgen produced by the fetal testis. In contrast, differentiation of the urogenital sinus and external genitalia in the rat begins just prior to birth along with local expression of 5{alpha}-reductase, the enzyme that produces dihydrotestosterone from testicular testosterone. Thus the greater sensitivity of the Wolffian duct to DBP may be due to its unique requirements for testicular testosterone. In addition to this early developmental toxicity, the absence of a functional epididymis may contribute to the severe seminiferous tubule degeneration observed in the adult rats exposed in utero to DBP. Lack of fluid reabsorption by the initial segment of the epididymis causes reflux of luminal fluids and increased pressure in the testis that can result in severe tubule damage and testicular atrophy (Hess et al., 1997Go).

Evaluation of risk to humans from exposure to DBP should consider its possible toxicity to the developing male reproductive system, since this is the most sensitive toxicity for DBP. Although the effects on male reproductive development described in our study were seen at relatively high doses, they occurred at lower dose levels and with a shorter exposure period than any other known toxicity elicited by DBP. For instance, testicular toxicity occurs after subacute high doses (1 g DBP/kg/day and higher) to neonates, and pubertal or adult rats (Dostal et al., 1988Go, Foster et al., 1980Go, IPCS, 1997Go). Hepatic peroxisome proliferation, which is believed to induce liver tumors in rodents, requires chronic high-dose exposure to DBP. Unlike rats, humans do not appear to respond to peroxisome-proliferating compounds (Cattley et al., 1998Go). Thus toxicity to male reproductive development may be more relevant to humans than liver toxicity of DBP.

MBP is probably the metabolite responsible for abnormal male sexual differentiation in rats exposed to DBP. DBP is cleared rapidly from the body; both DBP and MBP cross the placental barrier but MBP does not accumulate in the rat fetus (Saillenfait et al., 1998Go). It has been proposed that species differences in pharmacokinetics of phthalate esters may protect humans against the toxic effects seen in rats. Indeed, at high-dose levels, hydrolysis of DEHP to the monoester metabolite mono(ethylhexyl) phthalate (MEHP) is much lower in nonhuman primates than in rodents (Rhodes et al., 1986Go). At low levels, however, pharmacokinetic differences do not appear to be protective of humans for testicular toxicity of DEHP (Keys et al., 1999Go). Whether this is also true for DBP remains to be determined.

Currently, the lowest dose level of DBP producing reproductive effects originated from a continuous breeding study in CD rats in which no NOAEL was found and the LOAEL was 66 mg/kg body weight/day (NTP, 1991Go). Using the default risk assessment approach, which estimates a worst-case scenario for human risk, a reference dose of 66 µg/kg body weight/day has been proposed for DBP based on the NTP reproductive toxicity data (IPCS, 1997Go). This guidance value was derived by assuming that there is a threshold for a reproductive and developmental effect of DBP (in adherence to the default risk assessment process) and by applying default uncertainty factors of 10 for interspecies differences, 10 for interindividual differences and 10 for the lack of a NOAEL.

Our present evaluation has tested dose levels below those used in the NTP study and identified 100 and 50 mg/kg/day as the LOAEL and NOAEL for the effects of DBP on male reproductive development. Based on the findings described here and considering that nipple development, although probably a reversible change, is an indicator of altered androgen status during the critical developmental period, we consider the NOAEL to be 50 mg/kg/day. From this NOAEL and by applying default uncertainty factors of 10 for interspecies differences and 10 for interindividual differences, the reference dose would be 500 µg/kg/day. Because the extrapolation is based on a NOAEL and not a LOAEL, this value appears more reliable than the reference dose inferred from the NTP data. However, both experimental outcomes were remarkably consistent since effects of DBP were seen at similar dose levels (100 and 66 mg/kg/day).

The reference dose of 500 µg/kg/day, calculated using default assumptions, is well above the dietary intake of 0.5–30 µg DBP/kg/day, a range that encompasses exposure estimates for adults (IPCS, 1997Go, UK MAFF, 1996aGo, UK MAFF, 1990Go) and babies feeding on milk formula (IPCS, 1997Go, UK MAFF, 1998Go, UK MAFF, 1996bGo). Thus the risk to the human fetus from exposure to DBP is extremely low. Although the human male embryo/fetus is at low risk from exposure to DBP alone, the combined risk associated with exposure to other phthalates (e.g., DEHP) that perturb male sexual differentiation through the same basic mechanism should be considered. In fact, the developing male reproductive system of rats appears to be more sensitive to DEHP than to DBP (Arcadi et al., 1998Go, Gray et al., 1999bGo, Poon et al., 1997Go). Consequently, total exposure to those phthalate esters found to be toxic should be considered when establishing acceptable exposure levels for humans. However, dose-response and NOAELs for the effects on male reproductive development, using the appropriate study design, are not presently available for phthalate esters other than DBP, including DEHP.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Derek Janszen for guidance on statistical analyses and for performing the logistic regression analysis, Drs. Frank Welsh and David Dorman for reviewing the manuscript, and Dr. Barbara Kuyper for language editing. We are grateful to Max Turner for conducting the DBP stability analysis.


    NOTES
 
1 To whom correspondence should be addressed at Chemical Industry Institute of Toxicology, 6 Davis Drive, Research Triangle Park, NC 27709. Fax: (919) 558-1300. E-mail: foster{at}ciit.org. Back

2 Present address: DuPont Haskell Laboratory, P.O. Box 50, Newark, DE 19714. Back

3 Present address: Amgen Inc., 1840 DeHavilland Drive, Thousand Oaks, CA 91320. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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