We are developing a data base that will allow us to select endpoints that would be useful in the detection of reproductive toxicity in a multigenerational test. In this effort, carbendazim (MBC), a known reproductive toxicant, was administered to male and female rats from weaning, through puberty, gestation, and lactation. A similar study was conducted with hamsters. In rats, MBC was administered at 0, 50, 100, 200, or 400 mg/kg/day. Hamsters were dosed at 0 or 400 mg/kg/day. In the parent (P0) generation, landmarks of puberty were measured. In females, estrous cyclicity, litter size, the number of implants, organ weights, and histology were assessed. Our assessment of the male rat included organ weights, testicular and epididymal sperm counts, a quantitative measure of sperm motility, sperm morphology, testicular histology, and endocrine measures. The growth, viability, and reproductive function of the offspring (F1) were observed during a 4-month period of continuous breeding. In the P0 of both species, MBC did not alter pubertal development, growth, or viability. The reproductive potential of the rats treated with MBC at 200 and 400 mg/kg/day was reduced due to effects on sperm production and fetal viability. In the male rat, MBC treatment markedly altered sperm morphology, testicular and epididymal weights, and sperm numbers and testicular histology. Fertility, sperm motility, and hormonal levels altered, primarily in the males with very low sperm counts. The ability to conceive did not appear to involve a female factor. In P0 female rats, MBC administration caused postimplantation losses in the high-dosage groups and a few malformed rat pups were found in the litters from the 100 and 200 treatment groups. MBC was less toxic to the hamster than the rat. The only reproductive effects induced by MBC treatment were on sperm measures. Fertility of the P0 generation and fetal and neonatal (F1) viability were not decreased by MBC administration. In the male rat, testis weight, sperm numbers in the cauda epididymis and testis and sperm morphology were sensitive to the effects of MBC. In females, counting implantation scars at necropsy was useful, as this information allowed us to confirm pregnancy and identify postimplantation losses induced by MBC administration.

Reproductive Toxicity of a Single Dose of 1,3-Dinitrobenzene in Two Ages of Young Adult Male Rats. LINDER, R. E., STRADER, L. F., BARBEE, R. R., REHNBERG, G. L., AND PERREAULT, S. D. (1990). Fundam. Appl. Toxicol14, 284–298. These studies evaluated the reproductive response and the possible influence of testicular maturation on the reproductive parameters, in male rats treated with 1,3-dinitrobenzene (m-DNB). Young adult male rats (75 or 105 days of age) were given a single oral dose of 0, 8, 16, 24, 32, or 48 mg/kg of m-DNB and killed at 14 days post-treatment. Mortality and neurotoxicity were observed at 48 mg/kg, but only in the older animals. Epididymis weight, testicular sperm head counts, cauda sperm reserves, and sperm morphology were affected at 16 and 24 mg/kg and higher in the older and younger animals, respectively. Testis weight and sperm motility were affected at 24 mg/kg and higher in both age groups. Histologic changes included maturation depletion of mid and late spermatids at 16 mg/kg and higher, atrophy of a few to many seminiferous tubules at 24 mg/kg and higher, and immature germ cells in the epididymis. The movement and/or mixing of luminal elements in the epididymis appeared to be influenced by severe testicular effects. In separate groups given only the 48 mg/kg dosage, fertilizing ability was lost by 5–6 weeks post-treatment and several animals failed to recover in 5 months. In the breeder males, minimal to extensive degrees of seminiferous tubule atrophy and sloughed germ cells in the epididymis were still present after 175 days. The studies indicated that the lowest dosage to produce reproductive changes was 16 mg/kg with a no-effect level of 8 mg/kg. A few animals suffered protracted or permanent reproductive damage. Since the older animals were more susceptible to both the general and the reproductive toxicity of m-DNB, the less severe reproductive changes in the younger animals cannot be attributed solely to maturational differences in the testis.

This paper compares the statistical precision and biological sensitivity of multiple indices of reproductive function to infertility in the male rodent. The studies discussed include those that examined reproductive function in the male following perinatal exposure to reproductive toxicants and others in which the compounds were administered to young-adult males, often with very diverse results. For example, some chemicals that alter sex differentiation reduce fertility by affecting breeding performance alone (polychlorinated biphenyls (PCBs), fenarimol, or losulazine), without altering sperm and testicular measures. Others also markedly alter sex differentiation of the genitalia, the accessory glands and the testis in addition to their effects on central nervous system (CNS) sex differentiation and mating behavior (testosterone, flutamide, cyproterone acetate, tamoxifen, estradiol and diethylstilbestrol (DES)). In contrast, prenatal exposure to compounds that alter primary germ cell survival (busulphan, congo red) induce partial gonadal/germ cell agenesis without altering sex differentiation. These chemicals dramatically reduce testicular sperm production in the male offspring, and the most severely affected males are infertile. In a series of studies conducted in our laboratory, young male rats were exposed to known reproductive toxicants in a dose related manner from puberty, through young adulthood and breeding. We have found that the profile of effects varies considerably depending upon the chemical's mechanism of toxicity. When a compound produced infertility through direct effects of testicular function (Carbendazim (MBC) and dibutyl phthalate (DBP)), then testis weight, testicular histology, and testicular sperm head counts provided sensitive indicators of toxicity. In general, dramatic reductions in sperm production are required to induce infertility and these changes were accompanied by elevated serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and changes in human chorionic gonadotropin (hCG)-stimulated testosterone synthesis. Chemicals that have hormonal activity, alter the internal endocrine environment, or directly effect CNS function induce a completely different profile of effects. For example, estrogen administration alters the function of the seminal vesicle and the endocrine system, and reduces epididymal sperm reserves; while testicular measures are relatively unaffected. Since very different spectrums of effects are produced by different compounds, no single endpoint will consistently be the most sensitive indicator of reproductive toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

To explore the relationship between sperm measures and reproductive success in the laboratory rat, a review has been conducted of results of several male reproductive toxicology studies containing both fertility and multiple endpoint data. Comparisons are made between subchronic and acute exposure studies, emphasizing for each approach the value of multiple endpoint data. Further, the choice of endpoints in male reproductive toxicology is discussed with select examples of endpoints for which an understanding is evolving. We conclude that sperm measures are far more sensitive indicators of reproductive organ impairment than is the measurement of fertility, and that evaluation of multiple endpoints in acute studies is a key step in determining mechanisms of toxicant action that are critical for accurate interspecies risk extrapolation.

In the present study rats were dosed from weaning, through puberty and gestation, to Day 15 of lactation with methoxychlor at 25, 50, 100, or 200 mg/kg/day. Morphological landmarks of puberty were measured, including the ages at vaginal opening, first estrus, and first estrous cycle in females and at preputial separation in males. In the female, estrous cyclicity, fertility, litter size, number of implantation sites, organ weights, and ovarian and uterine histology were also measured. The viability of the offspring (F1) and their fertility were evaluated using a continuous breeding protocol. Males were necropsied after breeding, the reproductive organs were weighed, and the cauda epididymal sperm counts were determined. One testis was used for histopathology, while the other was used to quantify interstitial fluid (IF) content, IF testosterone concentration, and testicular sperm production. Testosterone and androgen-binding protein were measured in the caput epididymis, and sperm motility and morphology were evaluated from a caudal sample. The serum and pituitary were saved for hormonal determinations. Methoxychlor accelerated the age at vaginal opening and first estrus, and the vaginal smears were cornified. Growth was retarded at 100 and 200 mg/kg/day and fertility was reduced when the females were bred with untreated or similarly treated males. In the highest-dose group, the mated females went from constant estrus into pseudopregnancy following mating, but they had no implants. In males, methoxychlor treatment markedly reduced growth, seminal vesicle weight, cauda epididymal weight, caudal sperm content, and pituitary weight. Puberty was delayed in the two highest-dosage groups. Testicular sperm measures were much less affected than caudal measures. Testis weight and histology were slightly affected, and testicular sperm production, sperm morphology, and motility were unaffected. Endocrine function of the testes and pituitary was altered by methoxychlor administration. Leydig cell testosterone production, in response to human chorionic gonadotropin challenge, was reduced and pituitary levels of prolactin, thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH) were altered. In contrast, serum levels of prolactin, FSH, and luteinizing hormone were unaffected. Serum TSH was reduced by 50% of control at 100 and 200 mg/kg/day, while pituitary levels were increased. Gonadotropin-releasing hormone concentration in the mediobasal hypothalamus was also elevated. In spite of the many reproductive alterations, the fertility of treated males was not reduced when they were mated with untreated females.(ABSTRACT TRUNCATED AT 400 WORDS)

The determination that a chemical poses a reproductive risk to man typically relies upon fertility studies using rodents. However, fertility in rodents is often difficult to disrupt and more sensitive indicators of reproductive function should be included in the risk assessment process. The present discussion compares the sensitivity of fertility to other endpoints following exposure to known reproductive toxicants. In our studies rats were dosed from weaning through puberty , gestation, and lactation. The reproductive function of the male, the female, and the offspring was assessed. The effects of methoxychlor, carbendazim (MBC), dibutyl phthalate (DBP), and lindane are discussed and compared to fertility. For each compound a ratio (SR = sensitivity ratio) of the lowest effect level (LEL) for infertility or reduced fecundity to the LEL for the most sensitive physiologic endpoint was calculated. The SR should be large when a compound produces effects over a wide range of doses, but should equal unity when the dose-response curve is very steep. For methoxychlor, which blocked implantation, pubertal landmarks and estrous cyclicity provided rapid and sensitive indicators of the subsequent reproductive failure. The SR = 8 (100/12) for methoxychlor using data from females. In contrast, DBP and MBC directly altered testicular function, and for these compounds, sperm and testicular measures provided sensitive indicators of toxicity. The SR for MBC was 2 (100/50), while DBP had a SR of 1 (500/500). In the lindane study, fertility was not reduced but most of the pups (F1) died shortly after birth. The SR for lindane is equal to 0.5 (10/20). At 20 mg/kg the treated females were larger and their estrous cycles were erratic.(ABSTRACT TRUNCATED AT 250 WORDS)

This study determined the quantitative and qualitative histopathologic effects of a single oral dose of 1,3-dinitrobenzene (48 mg/kg) on the rat testis from 1 to 175 days postexposure. The testis was damaged severely by hour 24, as evidenced by increased numbers of regressive seminiferous tubules that exhibited degenerating pachytene spermatocytes, chromatin margination in spermatids, giant cells, deformed spermatid heads, retained spermatids, and reduced numbers of meiotic figures. The major effects during the first 48 hours posttreatment were degeneration or exfoliation of pachytene spermatocytes and round spermatids and the retention of step 19 spermatids. These regressive effects continued until 24 days, after which the tubules either recovered or became atrophic. At the end of the study (175 days), three males were normal, one had regressed testicles, and three males had atrophic tubules (15 to 45%). Several cellular abnormalities were common throughout the period. In addition, the frequency of the stages of spermatogenesis was altered, an indication of a disturbance in the kinetics of spermatogenesis. 1,3-Dinitrobenzene produced profound and specific lesions in the seminiferous tubules, and recovery was slow and incomplete. Atrophic tubules seemed to form if the normal cellular associations were not reestablished within 24 days, possibly due to the inability of Sertoli cells to reorganize the synchrony of germ cell development.

Tri-o-cresyl phosphate (TOCP) and O-ethyl O-(4-cyanophenyl) phenylphosphonothioate (cyanofenphos, Surecide) were found to be delayed neurotoxicants. They were administered to chickens by gavage at 100 and 30 mg/kg . d for 15 d, respectively. In CD-1 mice neither TOCP nor cyanophenphos induced any of the usually recognized clinical symptoms of neuropathy when administered daily by gavage at 262 or 31.25 mg/kg . d for 30 d, respectively. In the chickens, TOCP and cyanofenphos produced about 98 and 90% in vivo inhibition of brain neurotoxic esterase (NTE) activity. In the mice, 24 h after the last daily dose, TOCP and cyanofenphos produced only about 50 and 40% in vivo inhibition of the brain NTE activity. Parathion [O,O-diethyl O-(4-nitrophenyl) phosphorothioate], at 2 or 6.75 mg/kg . d for 15 or 30 d, did not induce neuropathy in either chicken or mice and produced no significant in vivo inhibition of brain NTE activity at the end of the dosing regimen. The specific activity of NTE in control chicken brain crude homogenate was much higher than that in mouse brain homogenate. These results suggest that the differences between chickens and mice in susceptibility to neurotoxic organophosphates may be attributed to (1) inhibitor specificity of NTE forms in the brain in these two different animal species and/or (2) inability of the active metabolites of these neurotoxic compounds to reach the site of action.

Diets containing the herbicide dinoseb (2-sec- butyl 4,6-dinitrophenol) were fed to adult male Sherman rats for 11 weeks. One-half the survivors were killed for terminal studies during the eleventh week and the remainder bred to untreated females during posttreatment, and then killed for terminal studies. Interim sacrifices were made in groups fed 0 and 300 ppm. In rats fed 300 ppm, differential classification of spermatozoa from the cauda epididymidis indicated 90% of the cells were atypical by 20 days of treatment. By 30 days, bizarre and amorphous forms were observed and epididymal sperm counts were decreased. Histologic changes in the testes included abnormal spermatozoa and spermatids and multinucleated spermatogenic cells at 20 and 30 days and severe damage to spermatogenic cells by 50 days. Dietary levels of 225 and 300 ppm produced marked oligospermia and extensive loss of spermatogenic cells in rats fed dinoseb for 11 weeks. Evidence of necrotic spermatogenic cells was seen in some tubules, and in many tubules, only Sertoli cells remained. Reproductive failure occurred at 225 and 300 ppm, although mating behavior and libido were unaffected. There was little or no remission of these effects during the 16-week post-treatment period. Decreased epididymal sperm counts, atypical epididymal spermatozoa, and minimal testicular changes were seen in rats fed 150 ppm, but reproduction was unaffected and the anomalies were reversible. No effects were detected in animals fed 75 ppm.

Oral LD50 values for pentachlorobenzene (QCB) in rats were 1125, 1080, and 940 mg/kg for adult males, adult females, and weanling females, respectively. The oral LD50 values in mice were 1175 mg/kg for males and 1370 mg/kg for females. Clinical signs of toxicity included tremors and narcosis. Dermal application of 2500 mg/kg did not produce clinical signs in rats. In subchronic studies weanling male rats were fed 0, 125, or 1000 ppm QCB for 100 days and weanling females fed 0, 125, 250, 500, or 1000 ppm for 180 days. No clinical signs of toxicity or effects on growth were observed in these rats throughout the exposures. QCB accumulated in adipose tissues at approximately 1.5-2.2 times the dietary concentrations. Porphyrin measurements were made only in females. Terminal values for urinary uro-and coproporphyrin and accumulation of liver porphyrins were not remarkably different in control and QCB-treated groups. In groups fed 1000 ppm, the WBC was increased and red blood cell indices were generally decreased compared to controls. The rats were pair-bred with untreated partners after 67 days of treatment. Fertility and fecundity were unaffected in either sex; however, suckling pups of QCB-treated mothers fed 250 ppm or more developed tremors and at 1000 ppm most died before weaning. Adrenal weights in males and kidney weights in both sexes were increased in adults fed 1000 ppm. In groups fed 250 ppm or more liver/body weight ratios were increased in both adults and in weanling offspring of QCB-treated dams. Hepatocellular enlargement was particularly evident in the 500 and 1000 ppm groups. In the kidneys of adult males, more numerous and larger foci of tubular atrophy and lymphocytic infiltration were seen at 1000 ppm than were seen in controls and dose-related increases in hyaline droplet formation occurred at 125 and 1000 ppm.