Butylparaben multigenerational reproductive assessment by continuous breeding in Hsd:Sprague Dawley SD rats following dietary exposure.

Butylparaben (BP) is an antimicrobial agent utilized for decades as a preservative in numerous consumer products. The safety of parabens has recently come under scrutiny based on reports of estrogenic activity and suggested adverse effects upon the reproductive system. Due to the limited availability of studies that address the potential for BP exposure to induce reproductive toxicity, and clear evidence of human exposure, the National Toxicology Program conducted a multigenerational continuous breeding study to evaluate the impact of dietary BP-exposure at 0, 5000, 15,000, or 40,000 ppm on reproductive and developmental parameters in Hsd:Sprague Dawley SD rats. BP-exposure was not associated with adverse alterations of fertility, fecundity, pubertal attainment, or reproductive parameters in F0, F1, or F2 generations. Exposure-dependent increases in liver weights, and incidences of non-neoplastic liver lesions suggest the liver is a target organ of BP toxicity. No findings were observed that would support the purported mechanism of BP-induced endocrine disruption in perinatally-exposed rodents.

Toxicologic Pathology Forum*: Opinion on Sexual Maturity and Fertility Assessment in Long-tailed Macaques ( Macaca fascicularis) in Nonclinical Safety Studies.

If nonhuman primates represent the only relevant species for nonclinical safety evaluation of biotechnology-derived products, male and female fertility effects can be assessed in repeat dose toxicity studies given that sexually mature monkeys are used. This opinion piece provides recommendations for determining sexual maturity and when/how fertility assessments should be conducted in the cynomolgus monkey. Male sexual maturity should be proven by presence of sperm in a semen sample, female sexual maturity by at least two consecutive menstrual bleedings. As per regulatory guidance, default parameters for an indirect assessment of fertility in both sexes are reproductive organ weight and histopathology. Beyond default parameters, daily vaginal swabs are recommended for females, and for males, it is recommended to include blood collections (for potential analysis of reproductive hormones), testis volume sonography, and collection of frozen testis samples at necropsy. Only if there is a cause for concern, blood collection for potential reproductive hormone analysis should be conducted in females and semen analysis in males. In principle, adverse reproductive effects can be detected within 4 weeks of test article administration, depending on study design and reproductive end point chosen. Therefore, there are options for addressing reproductive toxicity aspects with studies of less than 3 months dosing duration. *This is an opinion article submitted to the Toxicologic Pathology Forum. It represents the views of the authors. It does not constitute an official position of the Society of Toxicologic Pathology, British Society of Toxicological Pathology, or European Society of Toxicologic Pathology, and the views expressed might not reflect the best practices recommended by these Societies. This article should not be construed to represent the policies, positions, or opinions of their respective organizations, employers, or regulatory agencies.

Environmental lead exposure and pubertal trajectory classes in South African adolescent males and females.

The effects of environmental lead exposure in the neuro-endocrine system have been shown to impact the maturation and tempo of puberty development in adolescents. In low and middle income countries very little is known regarding the detrimental health effects of childhood lead exposure with regard to the tempo of puberty development. To help address this gap in data, we examined the association between lead exposure and puberty progression in males and females. Study participants from the urban Birth to Twenty Plus (BT20+) birth cohort in Soweto-Johannesburg, South Africa with data for blood lead levels at age 13years, cord blood lead levels, pubic hair development and breast development in females, and pubic hair development and genital development in males, were included in this study. The sample comprised 1416 study participants (n=684 females). Pubertal development trajectory classes were defined using Latent Class Growth Analysis. Data were examined for (i) an association between cord blood lead levels and pubertal trajectory classes; and (ii) an association between blood lead levels at age 13years and pubertal trajectory classes. In females, there was an association between adolescent elevated blood lead levels (≥5µg/dL) and lower level of maturation at age 9years and slower progression of pubic hair and breast development (relative risk ratio (RRR)=0.45, p<0.0001; 95% CI (0.29-0.68)) and (RRR=0.46, p<0.01; 95% CI (0.27-0.77)), respectively. In males, elevated blood lead levels at birth were associated with slower tempo of pubic hair development (RRR=0.20, p<0.05). Findings from this study suggest a possible role for environmental lead in altering pubertal development in South African adolescents as shown by slower tempo of progression through the Tanner stages pubertal development in females and males. There were also gender-differences between the effects of prenatal and postnatal lead exposure during pubertal development.

Safety assessment of propylparaben in juvenile rats.

There are conflicting literature reports that parabens, useful antimicrobial additives in pharmaceuticals, may have estrogenic activity. We conducted a comprehensive study to determine whether propylparaben (PP) administration to juvenile rats is associated with adverse effects on reproductive development and function. PP was administered orally once daily to groups of Crl:CD(SD) rats at doses of 0 (vehicle), 10, 100, or 1,000 mg/kg on Postnatal Days (PNDs) 4-90. In-life observations, clinical pathology, reproductive organ weights and histopathology, landmarks of sexual maturation, estrous cyclicity and functional reproductive competence were assessed. A conventional uterotrophic assay was conducted separately using the same doses. Systemic exposures to PP and 3 metabolites were evaluated on PND 7, 21 and 83. These studies demonstrated that PP was well tolerated when administered from PND 4-90 at all doses (AUC[0-T] on PND 83 = 69.9 ng•h/mL). Para-hydroxybenzoic acid, a non-estrogenic compound, was the predominant metabolite contributing to 95% of the total exposure at 1,000 mg/kg/day on PND 7. There was no evidence of estrogenic activity at any dose, and no effects on reproductive organs or function. The No-Observed-Adverse-Effect-Level (NOAEL) was 1,000 mg/kg/day.

Method to assess component contribution to toxicity of complex mixtures: Assessment of puberty acquisition in rats exposed to disinfection byproducts.

A method based on regression modeling was developed to discern the contribution of component chemicals to the toxicity of highly complex, environmentally realistic mixtures of disinfection byproducts (DBPs). Chemical disinfection of drinking water forms DBP mixtures. Because of concerns about possible reproductive and developmental toxicity, a whole mixture (WM) of DBPs produced by chlorination of a water concentrate was administered as drinking water to Sprague-Dawley (S-D) rats in a multigenerational study. Age of puberty acquisition, i.e., preputial separation (PPS) and vaginal opening (VO), was examined in male and female offspring, respectively. When compared to controls, a slight, but statistically significant delay in puberty acquisition was observed in females but not in males. WM-induced differences in the age at puberty acquisition were compared to those reported in S-D rats administered either a defined mixture (DM) of nine regulated DBPs or individual DBPs. Regression models were developed using individual animal data on age at PPS or VO from the DM study. Puberty acquisition data reported in the WM and individual DBP studies were then compared with the DM models. The delay in puberty acquisition observed in the WM-treated female rats could not be distinguished from delays predicted by the DM regression model, suggesting that the nine regulated DBPs in the DM might account for much of the delay observed in the WM. This method is applicable to mixtures of other types of chemicals and other endpoints.

Organochlorine pesticides and female puberty in South Kazakhstan.

The purpose of the study was to assess the puberty of females living in cotton-growing regions of South Kazakhstan, where organochlorine pesticides are widely used. The physical growth and sexual development were assessed; organochlorine pesticides, gonadotropic and steroid hormones, as well as insulin-like growth factor 1 in the peripheral blood were determined. 524 females (adolescents aged 10-17) were examined. They were divided into 2 groups, depending on their place of residence. The clinical research included the assessment of physical and sexual development. All examined females lived in rural areas, i.e. they were comparable in terms of social, welfare, and climatographic factors. A high concentration of organochlorine pesticides (lindane-18.51±0.16mg/l, dieldrin-169.16±3.13mg/l, DDT-177.78±2.71mg/l, endrin-37.57±0.9mg/l) in the blood of females living in regions exposed to pesticides compared to their peers (4.05±0.41mg/l, 30.8±3.7mg/l, 109.7±2.58mg/l, 4.85±0.69mg/l respectively) was found (p<0.001). The physical and sexual development of such females was delayed. The research established a correlation between the concentration of pesticides and the endocrine status, as well as with the insulin-like growth factor 1. This shows the adverse effect of organochlorine pesticides on the development of the female reproductive system during puberty.

Safety assessment of Hibiscus sabdariffa after maternal exposure on male reproductive parameters in rats.

CONTEXT: Hibiscus sabdariffa L. (Malvaceae) is a species widely used in folk medicine for the treatment of some disorders. OBJECTIVE: This study evaluated the effects of H. sabdariffa (HS) on the development of the male reproductive tract in rats following in utero exposure. MATERIALS AND METHODS: Pregnant rats received 250 or 500 mg/kg of HS extract or vehicle from gestational day 12 until day 21 of lactation. RESULTS AND DISCUSSION: Both doses of HS increased the body weight of male offspring at weaning, without compromising the puberty onset parameters. At puberty, there was a significant increase in the vas deferens absolute weight and a significant reduction in the relative weight of kidney at higher dose. These animals also presented a significant reduction in the sperm number in the caput/corpus of epididymis after exposure to both doses and a reduction in the sperm number in the cauda epididymis for the lower dose. At adulthood, the highest dose significantly reduced the sperm production in relation to controls and both doses provoked a reduction in the relative sperm number in the epididymis without affecting the sperm morphology. CONCLUSION: These findings demonstrated that maternal exposure to H. sabdariffa can adversely influence the male reproductive system in rats.

Assessment of estrogenic potential of di-n-butyl phthalate and butyl benzyl phthalate in vivo.

Phthalate compounds are widely used industrial chemicals; when incorporated into polyvinyl chloride, they are not covalently bound and released into the surrounding media. Some of them have estrogenic potential in vitro but data on in vivo studies are scanty. For the 3-day uterotrophic assay, di-n-butyl phthalate (DBP;10 and 100 mg/kg), butyl benzyl phthalate (BBP; 20 and 200 mg/kg), and diethylstilbestrol (DES, 40 µg/kg, positive control) were administered orally to immature female rats for three consecutive days from postnatal day (PND) 21. For the 20-day pubertal onset assay, DBP (10 and 20 mg/kg), BBP (20 and 200 mg/kg), and DES (6 µg/kg) were administered orally from PND 21 daily for 20 days. In the uterotrophic assay, in groups treated with higher dose of DBP and BBP, the uterine wet weight significantly decreased in the higher dose, and there were minor variations in the ovary wet weight, while the wet weight of these organs increased significantly in DES-treated group. In the 20-day pubertal assay, the weight of uterus and ovary declined significantly and changes in vaginal weight were nonsignificant in DBP- and BBP-treated groups. However, in DES-treated group nonsignificant elevation in vagina weight was observed. All the DES-treated animals showed the vaginal opening (VO) on day 26.17 ± 0.16. However, VO was not observed in any of the animals in control, vehicle control, BBP-, and DBP-treated groups up to PND 42, except in one animal each in vehicle control and DBP (100 mg/kg)-treated groups. The data indicated that both DBP and BBP were unable to induce elevation in the uterine and ovarian weight. While DES treatment can accelerate the growth of uterus and ovary and alter the onset of puberty and estrous cyclicity in prepubertal rats. These suggest that these compounds may not have estrogenic potential in vivo.

Comprehensive assessment of a chlorinated drinking water concentrate in a rat multigenerational reproductive toxicity study.

Some epidemiological studies report associations between drinking water disinfection byproducts (DBPs) and adverse reproductive/developmental effects, e.g., low birth weight, spontaneous abortion, stillbirth, and birth defects. Using a multigenerational rat bioassay, we evaluated an environmentally relevant "whole" mixture of DBPs representative of chlorinated drinking water, including unidentified DBPs as well as realistic proportions of known DBPs at low-toxicity concentrations. Source water from a water utility was concentrated 136-fold, chlorinated, and provided as drinking water to Sprague-Dawley rats. Timed-pregnant females (P0 generation) were exposed during gestation and lactation. Weanlings (F1 generation) continued exposures and were bred to produce an F2 generation. Large sample sizes enhanced statistical power, particularly for pup weight and prenatal loss. No adverse effects were observed for pup weight, prenatal loss, pregnancy rate, gestation length, puberty onset in males, growth, estrous cycles, hormone levels, immunological end points, and most neurobehavioral end points. Significant, albeit slight, effects included delayed puberty for F1 females, reduced caput epidydimal sperm counts in F1 adult males, and increased incidences of thyroid follicular cell hypertrophy in adult females. These results highlight areas for future research, while the largely negative findings, particularly for pup weight and prenatal loss, are notable.

Selecting surrogate endpoints for estimating pesticide effects on avian reproductive success.

A Markov chain nest productivity model (MCnest) has been developed for projecting the effects of a specific pesticide-use scenario on the annual reproductive success of avian species of concern. A critical element in MCnest is the use of surrogate endpoints, defined as measured endpoints from avian toxicity tests that represent specific types of effects possible in field populations at specific phases of a nesting attempt. In this article, we discuss the attributes of surrogate endpoints and provide guidance for selecting surrogates from existing avian laboratory tests as well as other possible sources. We also discuss some of the assumptions and uncertainties related to using surrogate endpoints to represent field effects. The process of explicitly considering how toxicity test results can be used to assess effects in the field helps identify uncertainties and data gaps that could be targeted in higher-tier risk assessments.

Toxicological assessment of drugs that affect the endocrine system in puberty-related disorders.

INTRODUCTION: Toxicologists must ensure that clinical risk-to-benefit analysis should be made both for genders and age groups, with any treatment. Puberty concerns physiological changes leading to organism's maturation. Pubertal growth disorders are increasing in last decades: besides causing physical and psychological distress, they may signal underlying endocrine-metabolic abnormalities with serious health consequences later on. Therapeutic approaches for some health conditions in childhood and adolescence are considered. AREAS COVERED: The authors discuss how some diseases and treatments can impact pubertal growth. The authors look at particular immunological disorders such as asthma and how both the disease and treatment affects pubertal growth. They also discuss how the provision of available data can help to assess the dose-response of the drug, in these cases, and minimize the chance of side effects. The authors also discuss pediatric inflammatory bowel disease and how both the disease and treatment can mitigate the growth delay. Last, but not least, the authors discuss how the effects of the drugs used in the treatment of psychiatric disorders may accentuate endocrine issues in juvenile patients. Hyperprolactinemia induction by some antipsychotics is highlighted as an example. EXPERT OPINION: Appropriate risk-benefit analysis of drugs prescribed during childhood and adolescence and intended to be used in the long term is required. Furthermore, future treatment strategies and safer compounds development should be supported by the knowledge of mechanisms underlying adverse side effects in pubertal growth and development.

Precocious sexual signalling and mating in Anastrepha fraterculus (Diptera: Tephritidae) sterile males achieved through juvenile hormone treatment and protein supplements.

Sexual maturation of Anastrepha fraterculus is a long process. Methoprene (a mimic of juvenile hormone) considerably reduces the time for sexual maturation in males. However, in other Anastrepha species, this effect depends on protein intake at the adult stage. Here, we evaluated the mating competitiveness of sterile laboratory males and females that were treated with methoprene (either the pupal or adult stage) and were kept under different regimes of adult food, which varied in the protein source and the sugar:protein ratio. Experiments were carried out under semi-natural conditions, where laboratory flies competed over copulations with sexually mature wild flies. Sterile, methoprene-treated males that reached sexual maturity earlier (six days old), displayed the same lekking behaviour, attractiveness to females and mating competitiveness as mature wild males. This effect depended on protein intake. Diets containing sugar and hydrolyzed yeast allowed sterile males to compete with wild males (even at a low concentration of protein), while brewer´s yeast failed to do so even at a higher concentration. Sugar only fed males were unable to achieve significant numbers of copulations. Methoprene did not increase the readiness to mate of six-day-old sterile females. Long pre-copulatory periods create an additional cost to the management of fruit fly pests through the sterile insect technique (SIT). Our findings suggest that methoprene treatment will increase SIT effectiveness against A. fraterculus when coupled with a diet fortified with protein. Additionally, methoprene acts as a physiological sexing method, allowing the release of mature males and immature females and hence increasing SIT efficiency.

Venom alkaloid and cuticular hydrocarbon profiles are associated with social organization, queen fertility status, and queen genotype in the fire ant Solenopsis invicta.

Queens in social insect colonies advertise their presence in the colony to: a) attract workers' attention and care; b) gain acceptance by workers as replacement or supplemental reproductives; c) prevent reproductive development in nestmates. We analyzed the chemical content of whole body surface extracts of adult queens of different developmental and reproductive stages, and of adult workers from monogyne (single colony queen) and polygyne (multiple colony queens) forms of the fire ant Solenopsis invicta. We found that the composition of the most abundant components, venom alkaloids, differed between queens and workers, as well as between reproductive and non-reproductive queens. Additionally, workers of the two forms could be distinguished by alkaloid composition. Finally, sexually mature, non-reproductive queens from polygyne colonies differed in their proportions of cis-piperidine alkaloids, depending on their Gp-9 genotype, although the difference disappeared once they became functional reproductives. Among the unsaturated cuticular hydrocarbons characteristic of queens, there were differences in amounts of alkenes/alkadienes between non-reproductive polygyne queens of different Gp-9 genotypes, between non-reproductive and reproductive queens, and between polygyne and monogyne reproductive queens, with the amounts increasing at a relatively higher rate through reproductive ontogeny in queens bearing the Gp-9 b allele. Given that the genotype-specific piperidine differences reflect differences in rates of reproductive maturation between queens, we speculate that these abundant and unique compounds have been co-opted to serve in fertility signaling, while the cuticular hydrocarbons now play a complementary role in regulation of social organization by signaling queen Gp-9 genotype.

Morphometric assessment of the impact of serum thymulin immunoneutralization on pituitary cell populations in peripubertal mice.

Thymulin is a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Thymulin also possesses hypophysiotropic activity which suggests that this metallopeptide may play an important role in thymus-pituitary communication, particularly during early life. The aim of the present study was to evaluate the impact of serum thymulin suppression from birth to peripuberty on the morphology of different pituitary cell populations in prepubertal C57Bl/6 mice. Animals were submitted to immunoneutralization of circulating thymulin from postnatal day 1 to the end of the study (age 32 days). From their 1st day of life, the animals were submitted to a protocol of intraperitoneal injections of rabbit anti-thymulin serum (alpha-FTS) and normal rabbit serum (NRS) in the controls. On their 33rd day of life, the animals were killed and their pituitaries were immediately dissected, fixed and immunostained using the EnVision system with primary antibodies against growth hormone, thyrotropin, corticotropin, gonadotropins and prolactin. Morphometry was performed by means of an image analysis system. The following parameters were calculated: volume density = Sigma cell area/reference area (RA); cell density (CD) = number of cells/RA, and cell size (expressed in microm2). Serum thymulin was measured by a rosette bioassay while pituitary hormones were assayed by radioimmunoassay. Serum prolactin, luteinizing hormone, follicle-stimulating hormone, growth hormone and thyroid-stimulating hormone were significantly lower in the alpha-FTS animals of either sex compared with the corresponding NRS counterparts. The somatotrope, lactotrope and corticotrope populations showed a significant decrease in CD, while cell hypertrophy was observed in some of the pituitary cell populations of the alpha-FTS group compared to the NRS group. In the alpha-FTS group, there were sex differences in the morphometric changes observed. Our results suggest that serum thymulin plays a significant role during early life in the postnatal maturation of endocrine cells of the mouse anterior pituitary gland.

Assessment of DE-71, a commercial polybrominated diphenyl ether (PBDE) mixture, in the EDSP male and female pubertal protocols.

DE-71, a commercial mixture, was used to test the sensitivity of the female and male pubertal protocol to detect thyroid active chemicals. These protocols are being evaluated for the U.S. EPA's Endocrine Disruptor Screening Program as part of a Tier I Screening Battery. To examine the ability of these protocols to screen for chemicals that induce the clearance of thyroid hormone, we examined male and female Wistar rats following DE-71 exposure. Rats were gavaged daily with 0, 3, 30, or 60 mg/kg DE in corn oil from postnatal day (PND) 23-53 in the male or PND 22-41 in the female. The temporal effects of DE-71 on liver enzymes and thyroid hormones were measured in another group of males and females following only 5 days of dosing (PND 21 to 26 in females and PND 23 to 28 in males). Serum T4 was significantly decreased at 30 and 60 mg/kg following the 5-day exposures and in the 21-day exposed females. Doses of 3, 30, and 60 mg/kg decreased T4 in 31-day exposed males. Serum T3 was decreased and TSH elevated by 30 and 60 mg/kg in the 31-day exposed males only. Decreased colloid area and increased follicular cell heights (indicative of the hypothyroid state) were observed in thyroids of the 60 mg/kg groups of 20- and 31-day exposed female and males. Increased liver-to-body weight ratios coincided with a significant induction of uridinediphosphate-glucuronosyltransferase (UDGPT; two to four-fold), and ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD) at the two highest doses in all exposures. Of the androgen dependent tissues in the 31-day exposed males, seminal vesicle (SV) and ventral prostate (VP) weights were reduced at 60 mg/kg, while testes and epididymal weights were not affected. Preputial separation (PPS) was also significantly delayed by doses of 30 and 60 mg/kg. In the female, the 60 mg/kg dose also caused a significant delay in the age of vaginal opening. Based upon the thyroid hormone response data, this study provides evidence that the 31-day alternative Tier 1 male protocol is a more sensitive test protocol than the 5-day or female pubertal protocol for thyrotoxic agents that act via up-regulation of hepatic metabolism. This apparent greater sensitivity may be due a greater body burden attained following the longer dosing regimen as compared with that of the female protocol, or to gender specific differences in thyroid hormone metabolism. Also, the delay in PPS and reduction in SV and VP weights may indicate a modification or inhibition of endogenous androgenic stimulation directly by DE-71 or a secondary effect that occurs in response to a DE-induced change in thyroid hormones.

Low level lead (Pb) exposure during gestation and lactation: assessment of effects on pubertal development in Fisher 344 and Sprague-Dawley female rats.

Studies using both Fisher 344 and Sprague-Dawley (SD) rat lines have shown that gestational and/or lactational maternal lead (Pb) exposure causes delayed reproductive maturation in their respective female offspring. Because these studies utilized different experimental regimens for dosing and for monitoring Pb levels, it has not been possible to determine which rat line provides the best model for low level Pb toxicity studies. This study was designed to address this issue. Adult Fisher and SD female rats were dosed with either a solution of PbAc containing 12 mg of Pb/ml or sodium acetate (NaAc) for controls. Dosing began 30 days prior to breeding and continued until their pups were weaned at 21 days of age. At the time of breeding and through weaning the blood lead (BPb) levels in the Fisher dams averaged 37.3 microg/dl and the SD dams averaged 29.9 microg/dl. Pb delayed the timing of puberty (p < 0.01) in Fisher offspring, and suppressed serum levels of luteinizing hormone (LH, p < 0.001) and estradiol (E2, p < 0.01). These effects did not occur in the SD offspring. Doubling the dose given to the SD rats increased their BPb levels to 62.6 microg/dl, yet there were still no effects noted. These results indicate that Fisher offspring are more sensitive to maternal Pb exposure with regard to puberty related insults than are SD rats, suggesting that the Fisher line may be a more reliable rodent model to study the effects of low level Pb toxicity.

Assessment of pubertal development in juvenile male rats after sub-acute exposure to bisphenol A and nonylphenol.

The effects of bisphenol A and nonylphenol on pubertal development in the intact juvenile/peripubertal male Sprague-Dawley rats was observed in this study from PND23-52/53. Two groups of rats were administered orally with either 100 mg/kg body weight of nonylphenol or bisphenol A. Another group of rats were administered orally with a mixture of 100 mg/kg body weight of nonylphenol and bisphenol A. Control group was administered with the vehicle of Tween-80 with corn oil (1:9 v/v). Observations made in this study included growth, age at preputial separation, thyroid, liver, testis and kidney weight and histology, epididymal and seminal vesicle plus coagulation gland weight. Nonylphenol and bisphenol A have been observed to cause delay in puberty onset as well as testicular damage in the treatment groups when compared to the control; spermatogenesis was affected in most treated rats. Bisphenol A also caused the enlargement of the kidney and hydronephrosis. Administration of nonylphenol and bisphenol A as a mixture has caused less than additive effects.

The effect of atrazine on puberty in male wistar rats: an evaluation in the protocol for the assessment of pubertal development and thyroid function.

Since atrazine (ATR), a chlorotriazine herbicide, has been shown previously to alter the secretion of luteinizing hormone (LH) and prolactin (PRL) through a direct effect on the central nervous system (CNS), we hypothesized that exposure to ATR in the EDSTAC male pubertal protocol (juvenile to peripubertal) would alter the development of the male rat reproductive system. We dosed intact male Wistar rats from postnatal day (PND) 23 to 53 and examined several reproductive endpoints. ATR (0, 12.5, 25, 50, 100, 150, or 200 mg/kg) was administered by gavage and an additional pair-fed group was added to compare the effects of any decreased food consumption in the high dose group. Preputial separation (PPS) was significantly delayed in the 12.5, 50, 100, 150, and 200 mg/kg ATR dose groups. PPS was also delayed in the pair-fed group, although significantly less than in the high dose-ATR group. The males were killed on PND 53 or 54, and pituitary, thyroid, testes, epididymides, seminal vesicles, and ventral and lateral prostates were removed. ATR (50 to 200 mg/kg) treatment resulted in a significant reduction in ventral prostate weights, as did the reduced food consumption of the pair-fed group. Testes weights were unaffected by atrazine treatment. Seminal vesicle and epididymal weights were decreased in the high dose-ATR group and the control pair-fed group. However, the difference in epididymal weights was no longer significantly different when body weight was entered as a covariable. Intratesticular testosterone was significantly decreased in the high dose-ATR group on PND 45, but apparent decreases in serum testosterone were not statistically significantly on PND 53. There was a trend for a decrease in luteinizing hormone (LH) as the dose of ATR increased; however, dose group mean LH was not different from controls. Due to the variability of serum prolactin concentrations on PND 53, no significant difference was identified. Although prolactin is involved in the maintenance of LH receptors prior to puberty, we observed no difference in LH receptor number at PND 45 or 53. Serum estrone and estradiol showed dose-related increases that were significant only in the 200 mg/kg-ATR group. No differences were observed in thyroid stimulating hormone (TSH) and thyroxine (T4) between the ATR groups and the control; however triiodothyronine (T3) was elevated in the high dose-ATR group. No differences in hormone levels were observed in the pair-fed animals. These results indicate that ATR delays puberty in the male rat and its mode of action appears to be altering the secretion of steroids and having subsequent effects on the development of the reproductive tract, which appear to be due to ATR's effects on the CNS. Thus, ATR tested positive in the pubertal male screen that the Endocrine-Disrupter Screening and Testing Advisory Committee (EDSTAC) is considering as an optional screen for endocrine disrupters.