Inter-laboratory control data for reproductive endpoints required in the OPPTS 870.3800/OECD 416 reproduction and fertility test.

BACKGROUND: The U.S. EPA revised the Reproduction and Fertility Effects Test Guideline (OPPTS 870.3800/OECD 416) in 1998, adding numerous endpoints in an effort to incorporate new methodologies, improve the sensitivity for detecting reproductive toxicants, and more efficiently utilize study animals. Many of these new endpoints have not been used in regulatory reproductive toxicology studies prior to their inclusion in the test guidelines; thus, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute (ILSI) initiated the Reproductive Endpoints Project to examine the utility of these new endpoints. METHODS: This report provides a retrospective analysis of 43 multi-generation studies (16 in Wistar rats, 27 in Sprague-Dawley rats) conducted according to the latest version of the test guidelines. It focuses on vehicle (negative) control values (means and ranges) for the various endpoints to examine inter-laboratory variability. RESULTS: Based on the compiled data, the most variable endpoints across laboratories and their associated coefficients of variation (CV) for each generation were: percent abnormal sperm (166-205%), testicular spermatid concentration (126-147%), postimplantation loss (97-104%), primordial follicle counts (69%, only measured in P2 females), and epididymal sperm concentration (52-57%). Absolute and relative prostate and thymus weights, weanling uterine weights, and anogenital distance had CVs of 25-50%. Sources of variability included procedural differences between laboratories, inherent biological variability, and/or small sample sizes for some endpoints. CONCLUSIONS: These inter-laboratory control data provide a means for laboratories to review their performance on reproductive toxicity measures, and provide perspective for interpreting their own control data and data from treated animals.

Reproductive and developmental toxicity of inhaled 2,3-dichloro-1,3-butadiene in rats.

Inhalation developmental and reproductive toxicity studies were conducted with 2,3-dichloro-1,3-butadiene (DCBD), a monomer used in the production of synthetic rubber. In the reproductive toxicity study, Crl:CD(SD)IGS BR rats (24/sex/group) were exposed whole body by inhalation to 0, 1, 5, or 50 ppm DCBD (6 h/day) for approximately 10-11 weeks total, through premating (8 weeks; 5 days/week), cohabitation of mating pairs (up to 2 weeks, 7 days/week), post-cohabitation for males (approximately 7 days) and from conception to implantation (gestation days 0-7 [GD 0-7]), followed by a recovery period (GD 8-21) for presumed pregnant females. Estrous cyclicity was evaluated during premating (last 3 weeks) and cohabitation. Reproductive organs and potential target organs, sperm parameters, and GD 21 fetuses (viability, weight, external alterations) were evaluated. In the developmental study, pregnant Crl:CD(SD)IGS BR rats (22/group) were exposed whole body by inhalation to 0, 1, 10, or 50 ppm DCBD (6 h/day) on GD 6-20; dams were necropsied on GD 21 (gross post-mortem only) and fetuses were evaluated (viability, weight, and external, visceral and skeletal exams). During the in-life portion of the studies, body weight, food consumption, and clinical observation data were collected. At 50 ppm, gasping and labored breathing occurred in both studies during the first few exposures; body weight and food consumption parameters were affected in parental animals from both studies, but were more severely affected in the developmental study. Fetal weight was decreased in the developmental study at 50 ppm. Degeneration of the nasal olfactory epithelium was observed in the reproduction study at 50 ppm. There were no effects on reproductive function, embryo-fetal viability, or increases in fetal structural alterations in either study. The no-observed-adverse-effect level (NOAEL) for reproductive toxicity was 50 ppm. The NOAEL for systemic toxicity in the reproduction study was 5 ppm based on adverse effects on body weight and food consumption parameters and nasal olfactory epithelial toxicity at 50 ppm in parental rats. The NOAEL for maternal and developmental toxicity was 10 ppm based on reduced maternal weight gain and food consumption and reduced fetal weight at 50 ppm in the developmental toxicity study.

Evaluation of the developmental toxicity of 8-2 telomer B alcohol.

The potential maternal and developmental toxicity of 8-2 Telomer B Alcohol was assessed in rats. Groups of 22 time-mated female Crl:CD (SD)IGS BR rats were administered oral gavage doses as suspensions of 8-2 Telomer B Alcohol in aqueous 0.5% methylcellulose from day 6 through 20 of gestation (G) at daily doses of either 0, 50, 200, or 500 mg/kg. Under the conditions of this study, adverse maternal toxicity was produced at 500 mg kg(- 1) day(- 1) and consisted of maternal mortality, decreased body weights and body weight gains, and increased clinical observations of toxicity. One litter at 500 mg kg(- 1) day(- 1) consisted of one early resorption and was believed to be secondary to overt maternal toxicity, although single conceptus litters occur historically in this strain of rats. Developmental toxicity at 500 mg kg(- 1) day(- 1) consisted of increased fetal skeletal variations (delayed pelvic bone ossification and wavy ribs). At 200 and 500 mg kg(- 1) day(- 1), there were transient reductions in maternal feed consumption. In addition, there were slight increases in the incidence of delayed fetal skull bone ossification at 200 and 500 mg kg(- 1) day(- 1). The no-observed-adverse-effect level (NOAEL), defined as the highest dose at which adverse effects attributable to the test substance were not detected, for both maternal and developmental toxicity, is considered to be 200 mg kg(- 1) day(- 1). Thus, 8-2 Telomer B Alcohol is not considered to be a selective developmental toxicant in rats. The transient and quantitative nature of the observations in the 200 mg/kg group supports the conclusion that these findings were not adverse.

Effects of phthalate esters on the developing reproductive tract of male rats.

Phthalate esters are a large group of chemical agents used predominantly as plasticizers and solvents. Certain members of this chemical class have been shown to cause reproductive and developmental toxicity. Recent attention has focused on the potential of these agents to interfere with male reproductive development through a postulated antiandrogenic mechanism. Observations have focused on di-n-butyl phthalate (DBP), di-(2-ethylhexyl) phthalate (DEHP) and butyl benzylphthalate, with most information relating to dose-response relationships obtained for DBP. Neither DBP, DEHP nor their major metabolites interacted with human or rodent androgen receptors (AR) in transcriptional activation assays. DBP was administered during the critical window of development of the male reproductive system, after which the resulting offspring were examined until adulthood. DBP elicited marked effects on the developing male reproductive tract, including malformations of the epididymis and vas deferens, and hypospadias. Retention of thoracic nipples/areolae and reductions in anogenital distance were also noted. Surprisingly, Leydig cell adenomas were induced in some male offspring at 100 days of age. All these events occurred in the absence of any toxicity in the pregnant dam. Examination of testes from fetal rats indicated markedly reduced testosterone levels and increased Leydig cell numbers after DBP administration to the dams. Leydig cells were positive for AR and 3-betahydroxysteroid dehydrogenase.

Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to Di(n-butyl) phthalate during late gestation.

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 F(1) 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.

Effects of di-n-butyl phthalate (DBP) on male reproductive development in the rat: implications for human risk assessment.

The National Toxicology Program (NTP) conducted a continuous breeding study in SD rats with di-n-butyl phthalate (DBP) given via the diet at dose levels of up to 650 mg/kg/day. In the parental generation effects on reproduction were modest (small decreases in litter size and pup weight following treatment). However, the F(1) male offspring had marked decreases in fertility (at 650 mg/kg/day), with reduced sperm counts and reproductive tract malformations on reaching adulthood. A no-observed-adverse-effect level (NOAEL) was not established for the study [lowest-observed-adverse-effect level (LOAEL) 66 mg/kg/day]. In a study conducted at CIIT, the majority of these adverse changes could be reproduced over a similar dose range, but with a much shorter dosing regimen covering a critical window of development (gestation days 12-20). A default risk assessment for DBP indicates a reference dose (RfD) of 66 microg/kg/day, based on a LOAEL of 66 mg/kg/day and default factors of 10 for inter-species and inter-individual differences and the lack of a NOAEL. Human exposure data would indicate worst-case scenarios to infants (via formula) in the dose range of the RfD. A default risk assessment appears to be inappropriate since rodents, unlike primates, metabolize phthalate diesters (including DBP) to monoesters extensively in the gut following oral administration. It is believed that the monoester is the active principle for induction of reproductive and developmental toxicity of specific phthalate esters. Thus, if humans produce very low levels of the monoester from an environmental exposure to the diester, the likelihood of any reproductive or developmental toxicity via the oral route appears extremely remote.

Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide.

Gestational and lactational exposure to di(n-butyl) phthalate (DBP) at >/=250 mg/kg/day disrupts male rat reproductive development and function. Although this indicates an antiandrogenic mechanism, DBP and its biologically active metabolite do not interact with the androgen receptor (AR) in vitro. In the present study, we compared the effects of DBP and the antiandrogen flutamide using a shorter exposure during the prenatal period of male sexual differentiation in rats. Pregnant CD rats received DBP at 0, 100, 250, or 500 mg/kg/day po (n = 10) or flutamide at 100 mg/kg/day po (n = 5) from Gestation Days 12 to 21. In F1 males, DBP (500 mg/kg/day) and flutamide caused hypospadias; cryptorchidism; agenesis of the prostate, epididymis, and vas deferens; degeneration of the seminiferous epithelium; and interstitial cell hyperplasia of the testis. Flutamide and DBP (250 and 500 mg/kg/day) also produced retained thoracic nipples and decreased anogenital distance. Interstitial cell adenoma occurred at 500 mg DBP/kg/day in two males. The only effect seen at 100 mg DBP/kg/day was delayed preputial separation. In contrast to flutamide, DBP caused a low incidence of prostate agenesis and hypospadias with no vaginal pouch. The low incidence of DBP-induced intraabdominal testes contrasted with the high incidence of inguinal testes seen with flutamide. Thus prenatal male sexual differentiation is a sensitive period for the reproductive toxicity of DBP. A no observed adverse effect level was not established and the lowest observed (adverse) effect level was 100 mg/kg/day. Flutamide and DBP disrupted the androgen signaling necessary for male sexual differentiation but with a different pattern of antiandrogenic effects. DBP is an example of an environmental antiandrogen that disrupts androgen-regulated male sexual differentiation without interacting directly with the AR, as does flutamide.

Male reproductive tract malformations in rats following gestational and lactational exposure to Di(n-butyl) phthalate: an antiandrogenic mechanism?

Di(n-butyl) phthalate (DBP), a widely used plasticizer suspected of having estrogenic properties, was investigated for its effects on the prenatal and early neonatal development of the reproductive tract. Pregnant CD rats (n = 10) were given DBP at 0, 250, 500, or 750 mg/kg/day (p.o.) throughout pregnancy and lactation until their offspring were at postnatal day 20. Maternal body weights throughout the dosing period were comparable in all groups. At 750 mg/kg/day, the number of live pups per litter at birth was decreased and maternal effects on pregnancy and postimplantation loss are likely to have occurred. Anogenital distance was decreased at birth in the male offspring at 500 and 750 mg/kg/day. The epididymis was absent or underdeveloped in 9, 50, and 71% of adult offspring (100 days old) at 250, 500, and 750 mg/kg/day, respectively, and was associated with testicular atrophy and widespread germ cell loss. Hypospadias occurred in 3, 21, and 43% of males and ectopic or absent testes in 3, 6, and 29% of males at 250, 500, and 750 mg/kg/day, respectively. Absence of prostate gland and seminal vesicles as well as small testes and seminal vesicles were noted at 500 and 750 mg/kg/day. Vaginal opening and estrous cyclicity, both estrogen-dependent events, were not affected in the female offspring, although low incidences of reproductive tract malformations were observed at 500 and 750 mg/kg/day. In the male offspring, DBP produced the same spectrum of effects elicited by the antiandrogen flutamide. Thus, DBP specifically impaired the androgen-dependent development of the male reproductive tract, suggesting that DBP is not estrogenic but antiandrogenic in the rat at these high dose levels. For human risk assessment, determining if this toxicity is metabolite-mediated will be critical, since marked species differences in metabolism exist.

Studies on the mechanism of uroporphyrinogen decarboxylase inhibition in hexachlorobenzene-induced porphyria in the female rat.

Hexachlorobenzene (HCB)-induced porphyria occurs in female, but not male, rats after a delay of 35 days following HCB treatment. Uroporphyrinogen decarboxylase (UROD) inhibition has been proposed as a primary causative event. To determine whether there also exists a delay phase and a sexual dimorphism for UROD inhibition, groups of male and female rats were given HCB (100 mg/kg/day) from Days 1 to 5. Hepatic uroporphyrin III was markedly increased only after Day 33. Liver cytosol UROD activity in HCB-treated female rats with porphyria at Days 33, 40, 47, 54, and 100 was decreased by over 70% compared to concurrent control, whereas treated male rats as well as nonporphyric female rats had UROD activity comparable to control levels at Days 6, 12, 19, 26, 33, 40, 47, and 54. Level of immunoreactive UROD in cytosol of porphyric rats was not modified by HCB. No gender-related differences in liver cytosol radiolabel level ([14C]HCB given as the fifth dose) were found at Days 6 and 30. Chromatography of liver cytosol showed nonspecific binding of radiolabel to proteins for males, porphyric and nonporphyric females, and loss of UROD activity did not correlate with the amount of radiolabel in the UROD-containing fractions. Thus, the gender-specific decrease in UROD activity observed when porphyria develops in female rats (delay of about 4 weeks), as well as the persistence of low activity and porphyria for months, suggests that UROD inhibition was causally related to porphyria.

Ultrasound-induced epileptiform activity in rats treated with hexachlorobenzene.

Hexachlorobenzene (HCB), an environmental contaminant, has caused spontaneous convulsions in infants and neonate rats born and breast-fed from exposed mothers as well as in weanling rats exposed for many weeks. This study aimed to determine if HCB causes epileptiform convulsions in adult rats. For this purpose, a controlled stimulus was used. Female Sprague-Dawley rats received HCB (100 mg/kg in corn oil) by daily gavage; on days 7 to 13, rats were exposed to a short ultrasound stimulation 24 hr after each administration. Ultrasound-induced epileptiform activity characterized by a burst of erratic running and leaping and tonic-clonic convulsions was observed in 0, 0, 10, 40, 90, 90 and 100% of rats having received a cumulative dose of 600, 700, 800, 900, 1000, 1100 and 1200 mg/kg, respectively. A similar dose-response pattern was obtained for tremors observed in HCB-treated rats. For a 800 mg/kg group left without further treatment, a three-fold increase in the percentage of rats with epileptiform activity was observed two days after the end of treatment. In summary, ultrasound stimulation has permitted us to demonstrate in a reproducible manner that adult rats treated with HCB display epileptiform activity.