Sex ratio of the offspring of Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally in a three-generation study.

Reports of a decreased male/female sex ratio in children born to males exposed to TCDD in Seveso, Italy, at a young age have sparked examinations of this endpoint in other populations exposed to TCDD or related compounds. Overall, the male/female sex ratio results reported in these studies, with slightly different age-exposed male populations, have shown mixed results. Experimental studies of the effects of in utero exposure to TCDD in laboratory animals have reported no effect on the f(1) sex ratio and mixed results for the sex ratio of the f(2) generation. In order to better understand the potential effects of TCDD on second generation sex ratio, we retrieved archived data from a comprehensive three-generation feeding study of TCDD in rats that was conducted and published in the 1970s, but which did not publish data on sex ratio of the offspring [Murray, F.J., Smith, F.A., Nitschke, K.D., Humiston, C.G., Kociba, R.J., Schwetz, B.A., 1979. Three-generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet. Toxicol. Appl. Pharmacol. 50, 241-252]. A re-examination of the original Murray et al. data found no statistically significant treatment-related changes in postnatal day 1 sex ratio in any generation of treated animals, consistent with one other relatively large study reporting on this endpoint. We discuss mechanistic data underlying a potential effect of TCDD on this endpoint. We conclude that the inconsistency in findings on sex ratio of the offspring of male rats exposed to TCDD in utero is likely due to random variation associated with a relatively small sample size, although differences between studies in strain of rat, dose regimen, and day of ascertainment of sex ratio cannot be ruled out.

In utero and lactation exposure of rats to 1R4F reference cigarette mainstream smoke: effect on prenatal and postnatal development.

Childhood cognitive and behavioral deficits have been reported in children born to mothers who smoked during pregnancy (Institute of Medicine, 2001). To investigate these potential responses in an animal model, reproductive and neurotoxicity evaluations based on the U.S. FDA guidelines were used to examine the offspring of male and female Sprague-Dawley rats exposed 2 h/day, 7 days/week by nose-only inhalation to whole mainstream smoke total particulate matter (TPM). Concentrations of 150, 300, or 600 mg/m(3) were used (males: 4 weeks prior to and during mating; and females: 2 weeks prior to mating, during mating, and through weaning at postnatal day 21). Sham air controls receiving filtered air and cage controls were also maintained. F(1) rats were weighed, identified by gender, examined for clinical signs of toxicity, and evaluated for neurobehavioral effects through postnatal day 65. Parental exposure was evidenced by smoke concentration-related increases in blood carboxyhemoglobin, nicotine, and cotinine and by characteristic cigarette smoke-related rodent respiratory tract histopathology. Also, nicotine and cotinine were found in F(1) blood through the lactation period. Maternal toxicity occurred at concentrations of 300 and 600 mg TPM/m(3), where total body weight gain during gestation was significantly (p < or = 0.05) decreased compared to sham controls. While smoke concentration-related decreases in F(1) birth weight and growth were evident (600 mg TPM/m(3), significantly different from sham at all time points), no adverse effects on developmental landmarks, including age at vaginal patency or preputial separation, motor activity, acoustic startle response or learning, and memory, were observed in the F(1) generation. This study confirmed that maternal exposure to high levels of mainstream cigarette smoke during gestation and lactation reduces birth weight and retards growth in the rat neonate; however, the developmental and neurobehavioral testing methodologies employed did not appear to be sensitive for an evaluation of neonatal behavioral effects following parental smoke exposure.

In utero exposure to 1R4F reference cigarette smoke: evaluation of developmental toxicity.

The potential developmental effects of 1R4F reference cigarette smoke were examined using Sprague-Dawley rats exposed for 2 h/day, 7 days/week, by nose-only inhalation at target mainstream smoke concentrations of 150, 300, and 600 mg/m3 total particulate matter (TPM). Males were exposed 4 weeks prior to and during mating, with females exposed 2 weeks prior to mating and during mating, and through gestation day (GD) 20. Sham controls received filtered air to simulate nose-only exposure, while cage controls were maintained untreated. Smoke exposure was confirmed through biomarker evaluation (parental: carboxyhemoglobin, nicotine, and cotinine; fetal: nicotine and cotinine). Characteristic cigarette smoke-related histopathologic changes including nasal epithelial hyperplasia and squamous metaplasia and pigmented macrophages in the lung were observed in all exposed parental groups. Maternal toxicity during gestation was indicated at smoke concentrations of 300 and 600 mg TPM/m3, where corrected total body weight gain was significantly (p

Developmental toxicity of an octyltin stabilizer in NMRI mice.

The octyltin stabilizer ZK 30.434 is a mixture of 80% dioctyltin diisooctylthioglycolate (DOTTG) and 20% of monooctyltin triisooctylthioglycolate (MOTTG) and is used as stabilizer for rigid polyvinylchloride (PVC) materials. One of the applications of such stabilized films is the packaging of foodstuffs. Exposure to humans occurs via migration of DOTTG/MOTTG from PVC materials. In the present study the developmental toxicity of DOTTG/MOTTG in NMRI mice was investigated. Dams were treated orally with doses of 20, 30, 45, 67, or 100 mg/kg/day DOTTG/MOTTG from gestation day 6 through 17 (plug = day 1). Resorption rates were significantly increased and fetal weights significantly reduced in the study group at the 2 highest doses. External anomalies, such as bent forelimbs, cleft palate, and exencephaly were reported in the group treated with 100 mg/kg/day DOTTG/MOTTG, with the 67-mg/kd dose also exhibiting a significant increase in cleft palate. Moreover, an increase in skeletal anomalies was reported in fetuses exposed to 100 mg/kg/day. The doses of 20, 30, and 45 mg/kg/day elicited a significant increase in supernumerary lumbar ribs. It can be concluded that DOTTG/MOTTG is embryo-fetotoxic and induces developmental effects. The study revealed the need for the establishment of different No-Observed Adverse Effect Levels (NOAEL) for the endpoints investigated.

The calciuric response to dietary salt of Dahl salt-sensitive and salt-resistant female rats.

BACKGROUND: We have shown previously that the calciuric response to salt does not differ in Dahl salt-sensitive (S) and salt-resistant (R) male rats. Clinical studies with women, however, suggest an effect of salt sensitivity on the calciuric response to salt. The objective of this study was to determine whether there is an effect of salt sensitivity on the calciuric response to salt of female S and R rats. METHOD: Dahl S and R female rats were fed high- (8%) or low- (0.3%) salt diets for 3 weeks. The rats were placed in metabolic cages for 24-hour urine collection at baseline and weekly (for sodium and calcium determination). RESULTS: Blood pressure of female S rats was 177+/-3.0 mm Hg at week 3 of high salt intake compared with 96+/-1 mm Hg for female R rats. Female S rats excreted significantly more calcium than female R rats at baseline (P < 0.001), when fed a nonpurified diet, and during high salt intake (P = 0.004). Salt sensitivity significantly increased calcium excretion, water intake, and urine output when rats were fed a high-salt diet. Calcium excretion, water intake, and urine output of female S rats were time-dependent during high salt intake. Plasma 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D concentrations were markedly lower in female S rats fed a high-salt diet, but not in female R rats. Plasma parathyroid hormone and 1,25-dihydroxyvitamin D concentrations did not significantly differ between female S and R rats, but plasma concentrations of these two hormones at week 3 were significantly higher in S rats fed a high-salt diet compared with S rats fed a low-salt diet. CONCLUSIONS: Our data indicate that the calciuric response to salt is greater in female S compared with female R rats, thus supporting findings on the effect of salt sensitivity reported in several clinical studies with women. The greater calciuric response to salt of female S rats compared with female R rats, which was not seen in a previous study when male S rats were compared to male R rats, suggest a gender difference in the calciuric response to salt.

Male reproductive toxicity and beta-luteinizing hormone gene expression in sexually mature and immature rats exposed to 2-bromopropane.

1. The reproductive effects of 2-bromopropane (2-BP) in sexually mature and immature male Sprague-Dawley rats were investigated. The animals were randomly divided into three treatment groups and one control group each of which comprised six mature and six immature rats. The treated groups were injected s.c. 200, 600 and 1800 mg/kg of 2-BP on 5 days a week for 5 - 7 weeks and the control group received the vehicle. 2. The absolute and relative testis weights were significantly reduced in 600 and 1800 mg/kg b.w. dose groups in both mature and immature rats. The absolute epididymis, prostate, seminal vesicle, and pituitary weights and the relative epididymis weights, however, were significant only at the highest dose level used in both mature and immature rats. 3. The sperm concentration and sperm viability in epididymal duct decreased and the percentage of abnormal sperm increased in a dose-dependent manner in both mature and immature rats. Additionally, serum testosterone level was significantly decreased in all dose groups in mature rats, and was significantly reduced only in the group treated with the middle and highest dose in immature rats. 4. In both mature and immature rats treated with 200 and 600 mg/kg, the seminiferous tubules were atrophied and all types of germ cells were decreased in number. At the highest dose level, the effect was more marked showing severely atrophied seminiferous tubules and a complete loss of all types of germ cells. 5. The mating, pregnancy and fertility indices were significantly reduced in the 600 and 1800 mg/kg groups. Additionally, at the highest dose group the number of implantations and viable fetuses per litter were reduced and the resorption rate was increased significantly. 6. In the mature rats, the beta-LH gene expression increased significantly in the 1800 mg/kg group when compared to the control group. 7. It can be concluded that 2-BP induces alterations in both neuro-endocrine axis and the reproductive tract under the present experimental conditions. The no observed adverse effect level (NOAEL) in this study could be estimated to be lower than 200 mg/kg/b.w. based on the alteration in testicular morphology as well as on sperm parameters observed at the dose level of 200 mg/kg.

Correlation between maternal toxicity and embryo/fetal effects.

It has been widely debated whether embryo/fetal toxicity is secondary to maternal toxicity. This argument has led to great difficulties for administrative decision makers involved in public health evaluation of drugs or chemicals. The present study sought to characterize whether there is a correlation between maternal toxicity and embryo/fetal toxicity. Developmental data from control and treated animals in our laboratory were collected and evaluated. Maternal toxicity, defined here as maternal body weight change, was statistically correlated with embryo/fetal parameters. The result showed that embryo/fetal parameters did not correlate with the body weight change. It can be concluded that maternal toxicity does not always lead to embryo/fetal toxicity; therefore, findings should be handled on a case by case basis and causal relationships should be established.

Reproductive effects of endosulfan on male offspring of rats exposed during pregnancy and lactation.

1. The reproductive effects of endosulfan on the male offspring of rats were examined. Dams were treated orally with 0, 1.5 or 3.0 mg endosulfan/kg from day 15 of pregnancy to postnatal day (PND) 21 of lactation. The male offspring rats were investigated at PND 65 or 140, corresponding to the pubertal and adulthood stage of development. 2. The dose of 3.0 mg endosulfan/kg induced a decrease in maternal body weight during pregnancy, but litter size and mean birth weight were not affected. Similarly, the age at testis descent and preputial separation was not affected on the male offspring. 3. The daily sperm production (x10(6)) was permanently decreased in the highest dose group when investigated at puberty and at adulthood. At the lowest dose, however, the daily sperm production was significantly reduced only at puberty. 4. Histologically, the percentage of seminiferous tubules showing complete spermatogenesis was significantly decreased at puberty. This finding may explain the decrease in daily sperm production observed in the endosulfan-exposed male rats. 5. The results of this study show that low doses of endosulfan have no apparent effect on developmental landmarks or on the weight of reproductive and accessory sex organ. Daily sperm production was the most susceptible endpoint in the male offspring exposed to endosulfan during pregnancy and lactation. To further understand the reproductive effects of endosulfan on male rat offspring, additional reproductive and toxicokinetic studies should be carried out to determine the extent of endosulfan exposure in male rat offspring in utero and during lactation.

Effects on developmental landmarks and reproductive capability of 3,3',4,4'-tetrachlorobiphenyl and 3,3',4,4',5-pentachlorobiphenyl in offspring of rats exposed during pregnancy.

1. Pregnant Wistar rats were treated orally with a single dose of 100 microg 3,3',4,4'-tetrachlorobiphenyl (PCB 77)/kg b.w. or 10 microg 3,3',4,4',5 pentachlorobiphenyl (PCB 126)/kg b.w. on day 15 of pregnancy. The control rats received peanut oil at the same day. Developmental landmarks were assessed in all offspring rats and reproductive effects of PCB 77 and PCB 126 on male offspring were studied on postnatal day 65 (at puberty) and on postnatal day 140 (at adulthood). 2. The ano-genital distance as well as the ratio ano-genital distance to body length was reduced in male pups of the PCB 126 group and the age at vaginal opening was significantly delayed in the female pups. 3. Testis, brain weights and daily sperm production were permanently increased and seminal vesicle weights were decreased in male offspring of the PCB 77 group. In male rats of PCB 126 group, the brain weights were permanently increased and ventral prostate weights permanently reduced. In both PCB groups, however, serum testosterone concentration was reduced only at adulthood. Additionally, the male rats of the PCB 126 group showed alterations in sexual behavior. In these rats the number of mounts with intromissions was significantly increased. 4. The results of this study show that PCB 126 elicits some TCDD-like reproductive effects after in utero exposure, while the reproductive effects of in utero exposure to PCB 77 on male offspring may be attributed to the neonatal hypothyroidism induced by the substance during early fetal development. Further studies using multiple doses and providing thyroid hormone data will be necessary to support this hypothesis.

Reproductive toxicity and tissue concentrations of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male offspring rats exposed throughout pregnancy and lactation.

The effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the reproductive system of male offspring rats were examined. The dams were treated subcutaneously 2 weeks prior to mating and throughout mating, pregnancy, and lactation. They received an initial loading dose of 25, 60, or 300 ng TCDD/kg body wt, followed by a weekly maintenance dose of 5, 12, or 60 ng TCCD/kg body wt (TCDD 25/5, TCDD 60/12, and TCDD 300/60). Three dams per group were killed on Gestation Day 21 and the fetuses were removed. The concentration of TCDD in the maternal liver and fat was measured. After birth, developmental landmarks in male rats were monitored. At weaning, the concentration of TCDD in the offspring liver and testis was determined. Effects on male reproduction were studied on Postnatal Days (PND) 70 and 170. At weaning, the concentration of TCDD in the offspring liver was 0.24, 0.39, and 1.78 ng/g in the TCDD 25/5, TCDD 60/12, and TCDD 300/60 groups, respectively. In the testes, the concentration of TCDD was 0.25 ng/g in the TCDD 25/5 and TCDD 60/12 groups and 0.28 ng/g in the TCDD 300/60 group. The number of sperm per cauda epididymis was reduced in TCDD groups at puberty and at adulthood. Daily sperm production was permanently decreased as was the sperm transit rate in the TCDD-exposed male rats, thus increasing the time required by the sperm to pass through the cauda epididymis. Moreover, the male rats of the TCDD groups showed an increased number of abnormal sperm when investigated at adulthood. Similarly, mounting and intromission latencies were significantly increased in the TCDD 25/5 and TCDD 300/60 groups. In the highest dose group, serum testosterone concentration was decreased at adulthood. Likewise, in this dose group permanent changes including pyknotic nuclei and the occurrence of cell debris in the lumen were revealed. The lowest adverse effect level and the no observed effect level can be estimated to be substantially lower than the estimated daily dose of the lowest dose which is 0.8 ng/kg body wt/day. Sperm parameters were more susceptible than the other end points investigated. However, the question as to whether such doses exposed throughout gestation and lactation induce subtle changes in humans remains to be determined.

Reproductive toxicity and tissue concentrations of 3,3',4,4'-tetrachlorobiphenyl (PCB 77) in male adult rats.

1 The aim of this study was to ascertain the reproductive effects of PCB 77 on adult male rats and to determine its concentration in the liver and testis. Adult male rats (n = 15/group) were treated subcutaneously with a single dose of 18 mg/kg bw (PC18) or with 60 mg/kg bw (PC60). The substance was dissolved in a 10 ml volume of peanut oil/kg. Control rats received the same volume of the vehicle. The reproductive effects as well as the concentration of PCB 77 in the liver and testis were investigated 1, 4 and 8 weeks after treatment. 2 In both groups, the daily sperm production (DSP; x10(6)) remained permanently reduced in the PC18 as well as in the PC60 groups throughout the entire investigation period (DSP week 8: control: 31 +/- 7; PC18: 22 +/- 5; PC60: 20 +/- 7). The sperm number (x10(6)) per cauda epididymis was affected only at the 1st and 4th week after treatment (control week 1: 211 +/- 67; PC18 week 1: 135 +/- 62; PC60 week 1: 142 +/- 49). Moreover, a significant increase in the percentage of abnormal sperm was observed 4 weeks following treatment in the PC18 and PC60 groups and 8 weeks after treatment in the PC60 group. Abnormal tails were the most frequent changes observed. 3 The relative testicular and prostata weights (g) were slightly increased in the PC60 group at the 1st and 4th week following treatment (testis weight: control/I: 0.46 +/- 0.02; PC60/I: 0.51 +/- 0.03). 4 The serum testosterone concentrations and effects on testis morphology were not reported. 5 The maximum concentration of PCB 77 was detected in the liver and testis 1 week after treatment. The concentration declined 4 weeks after treatment in both organs, but still a significant amount of PCB 77 was detectable in the liver as well as in the testis 8 weeks after treatment. 6 The results demonstrate that PCB 77 affects sperm variables when applied to adult rats and that the elimination of PCB 77 in the testis parallels that of the liver.

An optimized approach for the assessment of sexual behavior in male rats.

The improvement and optimization of methods used to detect reproductive disorders in experimental animals are among the main challenges facing researchers in this field. The conventional method for testing male sexual behavior uses ovariectomized females rendered sexually receptive by injection of estradiol benzoate and progesterone prior to testing. The receptive females are then mated with exposed male rats during the dark phase of the cycle under dim red light followed by direct visual and momentary observation of the mating activity. The ovariectomized females may respond differently to hormonal injection (individual differences), leading to variations in the intensity of lordosis. Additionally, the data obtained by the direct visual and momentary evaluation of copulatory activity are very subjective and may produce inaccurate results. In the optimized method, the sexual cycles of female rats are determined, and only those in estrus are selected and mated with sexually experienced male rats. The mating activity is videotaped, enabling the correct observation and evaluation of the different components of mating behavior. This method fosters animal welfare by avoiding surgical intervention and enables the videotape to be kept as permanent documentation.

Ganciclovir induces reproductive hazards in male rats after short-term exposure.

1. The effect of short-term treatment of ganciclovir on male reproduction in adult rats was studied. The animals were treated subcutaneously with either a single dose of 60 mg/kg daily for 5 days (Gan5day) or with 100 mg/kg administered three times at 4 h-intervals (Gan1day). The effects were investigated every 2 weeks up to 8 weeks, followed by investigations 16 and 24 weeks after treatment to detect the potential of recovery. 2. Time to mating was significantly increased in Gan1day group. The pregnancy index and outcome were only decreased 8 weeks (Gan5day and Gan1day) or 16 weeks (Gan1day) after treatment. 3. The lowest values of sperm variables studied were registered 8 weeks after treatment: The number of spermatid was reduced up to 4% (Gan5day) or 2% (Gan1day) of control; the sperm number was 5% and 8% of control in Gan5day and Gan1day, respectively. Over 80% of sperm were abnormal in Gan5day group, and only few normal sperm was detected in Gan1day group. 4. Morphological investigation of testes revealed a clearcut time-dependency effect. Four weeks after treatment distinct alterations were located exclusively in the peripheral part of the tubuli which included fat inclusions, cell and pyknotic nuclear debris and swellings of Sertoli cells. The effect was reversible 24 weeks after treatment. 5. Ganciclovir induces testicular damage and affects sperm variables after short-term exposure. The intensity and degree of the hazards varied in between the time of investigation after treatment.

Standard values for reproductive and clinical chemistry parameters of Japanese quail.

Historical control data of Japanese quail collected from reproduction studies conducted at the Federal Institute for Health Protection of Consumers and Veterinary Medicine between 1988 and 1994 are presented in this paper. Reproductive and clinical chemistry data from control animals of 10 ecotoxicological studies are summarized and discussed to develop a normal data base of this species. The data obtained were compared to the control reproductive parameters of bob-white quail and mallard ducks available in the literature. For a long time these two species have been most used in avian reproductive toxicology studies. In summary, the data obtained indicate that Japanese quail appears to be more appropriate to be used for the determination of reproductive effects of pesticides on birds.

Reproductive toxicity and toxicokinetics of lindane in the male offspring of rats exposed during lactation.

1. Reproductive toxicity and toxicokinetics of lindane during lactation were studied. For the reproductive toxicity study the dams were treated with a single dose of 6 mg/kg on day 9 or 14 of lactation, or with 1 mg/kg on days 9 to 14 of lactation. The male offspring were investigated at puberty and adulthood. For the toxicokinetic study, two groups of dams were treated with 6 mg/kg on day 9 or 14 of lactation. The concentration of lindane was measured in maternal plasma and milk, as well as in male offspring organs. 2. At adulthood, testicular weight and the number of sperm and spermatids were significantly reduced in all treated groups. 3. The testosterone level of the groups treated with 6 mg/ kg was significantly reduced to approximately 50% at puberty as well as in adulthood. In the group treated with 1 mg/kg, the testosterone level was in both age periods reduced, however, only at puberty was the reduction statistically significant. 4. The concentration of lindane in the testis was similar to that found in brain and was half the concentration found in the liver. 5. Histologically some areas of the testis exhibited distinct alterations ranging from small changes to a pronounced effect. 6. Exposure to lindane during lactation induces reproductive hazards to male offspring rats which are detectable at adulthood.

Reproductive toxicity and tissue concentrations of lindane in adult male rats.

1. The effects of lindane on the reproductive system and its concentration in different tissues were investigated. Groups were dosed orally with 6 mg lindane/kg for 5 days or with a single dose of 30 mg/kg body weight. 2. The results indicate that the number (mio) of spermatids counted in the testes of both treated groups 2 weeks after treatment were significantly reduced compared to the control rats. Effects on the number of sperms were observed in both treated groups, but significant differences were only revealed on those with 30 mg/kg. 3. The concentration (microgram/g tissue) of lindane in adipose tissue, liver, brain and testis was determined 24 h and 2 weeks after the last treatment. Lindane was detected in the testis of both groups 24 h and 2 weeks after the last treatment. 4. Histological investigation by electron microscopy revealed a pronounced ballooning of Sertoli cells accompanied by fragmentation or complete loss of organelles. 5. According to the result obtained, it is evident that lindane passes through the testis and exerts its toxic effect.