Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the developing male Wistar(Han) rat. I: No decrease in epididymal sperm count after a single acute dose.

It has been reported that fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat. We set out to replicate and extend these effects using a robust experimental design. Groups of 75 (control vehicle) or 55 (50, 200, or 1000 ng of TCDD/kg bodyweight) female Wistar(Han) rats were exposed to TCDD on gestational day (GD)15, then allowed to litter. The high-dose group dams showed no sustained weight loss compared to control, but four animals had total litter loss. Pups in the high-dose group showed reduced body weight up till day 21, and pups in the medium dose group showed reduced body weight in the first week postpartum. Balano-preputial separation was significantly delayed in the high-dose group male offspring. There were no significant effects of treatment when the offspring were subjected to a functional observational battery or mated with females to assess reproductive capability. Twenty-five males per group were killed on postnatal day (PND) 70, and approximately 60 animals per group (approximately 30 for the high-dose group) on PND120 to assess seminology and other end points. At PND120, the two highest dose groups showed a statistically significant elevation of sperm counts, compared to control; however, this effect was small (approximately 30%), within the normal range of sperm counts for this strain of rat, was not reflected in testicular spermatid counts nor PND70 data, and is therefore postulated to have no biological significance. Although there was an increase in the proportion of abnormal sperm at PND70, seminology parameters were otherwise unremarkable. Testis weights in the high-dose group were slightly decreased at PND70 and 120, and at PND120, brain weights were decreased in the high-dose group, liver to body weight ratios were increased for all three dose groups, with an increase in inflammatory cell foci in the epididymis in the high-dose group. These data show that TCDD is a potent developmental toxin after exposure of the developing fetus but that acute developmental exposure to TCDD on GD15 caused no decrease in sperm counts.     

Relationships between tissue levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mRNAs, and toxicity in the developing male Wistar(Han) rat.

We compared the effects of a single acute dose, or chronic fetal exposure, to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the male reproductive system of the Wistar(Han) rat. Tissue samples were taken from dams on gestation day (GD)16 and GD21, and from offspring on postnatal days (PND)70 and 120. Steady-state concentration of TCDD was demonstrated in the chronic study: body burdens were comparable in both studies. Fetal TCDD concentrations were comparable after acute and chronic exposure, and demonstrate more potent toxicity after chronic versus acute dosing. In maternal liver, cytochrome P450 (CYP)1A1 and CYP1A2 RNA were induced. In fetus, there was induction of both CYP1A1 and CYP1A2 RNA at medium and high doses, but inadequate evidence for induction at low dose in either study. The low level induction of CYP1A1 RNA at low dose in fetus argues against AhR activation in fetus as a mechanism of toxicity of TCDD in causing delay in balanopreputial separation (BPS), and the greater induction of CYP1A1 RNA in PND70 offspring liver from chronically-dosed dams suggests that lactational transfer of TCDD is crucial to this toxicity. These data characterize the maternal and fetal disposition of TCDD, induction of CYP1A1 RNA as a measure of AhR activation, and suggest that lactational transfer of TCDD determines the difference in delay in BPS between the two studies.     

Adult 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and effects on male reproductive organs in three differentially TCDD-susceptible rat lines.

The contribution of genetic factors to adult male reproductive system toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was analyzed in three rat lines differentially resistant to TCDD acute lethality: line A, B, and C rats (selectively bred from TCDD-resistant Han/Wistar [Kuopio; H/W] and TCDD-sensitive Long-Evans [Turku/AB; L-E] rats). The resistance is linked to a mutated H/W-type aryl hydrocarbon receptor allele in line A and to an H/W-type unknown "B" allele in line B. Line C rats do not have resistance alleles. Mature male line A, B and C rats were given single oral doses up to 1000, 300, and 30 micrograms/kg TCDD, respectively. The dose-responses of TCDD effects on male reproductive organ weights, sperm numbers, and serum testosterone concentrations were analyzed 17 days after exposure. Serum testosterone concentrations were decreased by the highest doses of TCDD, and there were no major sensitivity differences among the rat lines. Correspondingly, the decrease in relative weight of ventral prostate and seminal vesicles was seen only after a dose of >/=100 micrograms/kg TCDD. Thus the effect was observed only in resistant lines A and B. The relative weights of testes and epididymides were not affected. Significant decrease in spermatogenesis was observed in each rat line, but the amount of decrease was reduced by resistance alleles. The highest TCDD dose decreased the daily sperm production by 37, 38, and 60% in line A, B, and C rats, respectively. Therefore, the resistance alleles appear to selectively modify the TCDD effects on the adult male reproductive system. The fact that the influence of resistance alleles on spermatogenesis is different from that on androgenic status indicates that the effect of TCDD on sperm numbers is not fully related to decreased serum testosterone.     

Early developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure decreases chick embryo heart chronotropic response to isoproterenol but not to agents affecting signals downstream of the beta-adrenergic receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes cardiovascular toxicity in laboratory animals, including alteration in several processes in which beta-adrenergic receptor (beta-AR) signaling plays important roles. Thus, our laboratory investigated the effects of TCDD on beta-AR expression and signal transduction. Fertile chicken eggs were injected with vehicle (corn oil), 0.24 or 0.3 pmol TCDD/g egg on incubation day 0 (D0) or D5. On D10, heart function was assessed by ECG in ovo. Exposure to TCDD increased the incidence of arrhythmias and decreased the positive chronotropic responsiveness of the heart to isoproterenol. The reduced beta-AR responsiveness was, in part, independent of any overt morphological changes in the heart as chick embryos exposed to TCDD on D5 displayed an intermediate responsiveness to beta-AR agonist in the absence of the dilated cardiomyopathy observed in chick embryos exposed to TCDD on D0. TCDD did not decrease the chronotropic response of the heart to agents that stimulate signals downstream of the beta-AR. In fact, TCDD-exposed embryos were more sensitive than controls to forskolin, increasing heart rates (HR) 21.8 +/- 3.5 beats per min (bpm) above baseline versus control values at 6.3 +/- 2.7 bpm above baseline. TCDD exposure also augmented the negative chronotropic response of the heart to verapamil, decreasing HR -23.2 +/- 7.4 bpm relative to baseline versus control embryos at -12.7 +/- 5.9 bpm below baseline. Finally, the mean cardiac beta1-AR mRNA expression in D10 embryos was not significantly altered by exposure to TCDD on D0. These findings establish that a functional end point of the developing chick heart is sensitive to TCDD exposure and that the TCDD-induced reduction in beta-AR responsiveness may result from alterations in signal transduction upstream of adenylyl cyclase.     

Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental defects in the rat vagina.

At puberty, female rats exposed in utero to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exhibit a persistent thread of mesenchymal tissue surrounded by keratinized epithelium that partially occludes the vaginal opening. Our objective was to determine the earliest time during fetal development that morphological signs of this vaginal canal malformation could be detected and to obtain greater insight into mechanisms involved in this effect. Pregnant rats were administered a single dose of vehicle (control) or TCDD (1.0 microg/kg, po) on gestation day (GD) 15 and were sacrificed on GD 18, 19, 20, and 21 for histological evaluation of female. Gestational exposure to TCDD affected vaginal morphogenesis as early as GD 19, 4 days after exposure of pregnant dams. In exposed fetuses, the thickness of mesenchymal tissue between the caudal Mullerian ducts was increased, which resulted in a failure of the Mullerian ducts to fuse, a process normally completed prior to parturition. In addition, TCDD exposure appeared to inhibit the regression of Wolffian ducts. Thus, TCDD interferes with vaginal development by impairing regression of the Wolffian ducts, by increasing the size of interductal mesenchyme, and by preventing fusion of the Mullerian ducts. Taken together, these effects appear to cause the persistent vaginal thread defect observed in rats following in utero and lactational TCDD exposure.     

Acute neurobehavioural effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Han/Wistar rats.

The neurobehavioural effects of a single non-lethal dose (1000 micrograms/kg intraperitoneally) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were assessed in young male Han/Wistar rats, highly resistant to acute lethality of TCDD. TCDD decreased body weight significantly compared with ad libitum fed controls. TCDD did not change the behaviour or the motility of rats in the open field test 8 days after the treatment nor did it affect the spontaneous motor activity up to 27 days after the exposure. In the elevated plus-maze test for anxiety, TCDD-treated rats did not differ from either ad libitum fed controls or pair-fed controls. In the 24-hr passive avoidance test, the learning of TCDD-treated rats did not differ significantly from that of ad libitum fed controls or pair-fed controls from 8 hr to 16 days after the treatment. TCDD did not affect the motor coordination or the maintenance of balance on the rotating rod but it impaired them slightly in the elevated horizontal bridge test 16 hr after exposure. It did not affect nociception in the hot plate test 16 hr or 8 days after the injection. The results suggest that a single sublethal dose of TCDD does not alter markedly the general behaviour of Han/Wistar rats, in contrast to its striking effect on feeding behaviour which results in a marked decrease in body weight gain.     

Promotion of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin and 17beta-estradiol in male Sprague-Dawley rats.

The determination of differences in hormonal regulation of tumor promotion-related response to 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) between males and females may identify factors contributing to the female-specific hepatocarcinogenicity of TCDD in rats. In the current study, diethylnitrosamine-initiated male Sprague-Dawley rats were exposed to TCDD or corn oil vehicle in the presence and absence of 17beta-estradiol (E2), and cell proliferation and development of preneoplastic altered hepatic foci (AHF) were determined. After 30 weeks of exposure, gamma-glutamyltranspeptidase (GGT)-positive AHF and the number of placental glutathione-s-transferase (PGST)-positive AHF were significantly higher in TCDD-treated rats than in control rats. Both the number and volume fraction of GGT-positive AHF were significantly lower in rats cotreated with E2 regardless of TCDD exposure compared with corresponding non-E2-treated groups and were unaffected by TCDD. In contrast, the number of PGST-positive AHF was significantly higher in E2-treated rats in the absence of TCDD treatment. In addition, whereas E2 had no effect on the volume fraction of PGST-positive foci, the levels in rats cotreated with both E2 and TCDD were significantly higher than in controls. No differences were observed in cell proliferation between TCDD-treated and control rats, although cell proliferation was lower in rats exposed to E2 compared with placebo controls. The weaker potency of tumor promotion and lack of induction of cell replication and DNA damage in male rats likely explain the female-specific hepatocarcinogenicity of TCDD in chronic bioassays.     

Physiologically based pharmacokinetic model for developmental exposures to TCDD in the rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent developmental toxicant in rodents, and these effects occur at exposures similar to background human body burdens. A physiologically based pharmacokinetic (PBPK) model can aid in quantitatively describing the relationship between exposure, dose, and response. The aim of this work was the development a PBPK model to describe the relationship between maternal TCDD exposure and fetal TCDD concentrations during critical windows of susceptibility in the rat. This PBPK model is a modification of an eight-compartment model that describes the adult female rat. The modified model reduces the compartments from eight to four maternal compartments (liver, fat, placenta and rest of the body). Activation of the placental compartment and a separate fetal compartment occurs during gestation. The systemic circulation connects the maternal compartments. The physiological and biochemical parameters were obtained from the literature. The model validation used experimental data from acute and subchronic exposures prior to and during gestation. The simulations predict the TCDD tissue concentrations of the maternal compartments within the standard deviation of the experimental data. The model overestimates the fetal concentrations by approximately a factor of two at low subchronic exposures, but does predict the fetal tissue concentrations within the range of the experimental data at the higher exposures. This model may provide a framework for the development of a human PBPK model to estimate fetal TCDD concentrations in human health risk assessments.     

Characterization of the enhanced responsiveness to postingestive satiety signals in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated Han/Wistar rats.

Previous studies have shown that under free-feeding conditions, TCDD-treated Han/Wistar (H/W) rats consume less sucrose solution but ingest more saccharin solution than their controls thus implying hyperresponsiveness to postingestive satiety signals. In this study, nutrient preloads were employed to further elucidate this phenomenon. Male H/W rats were given a single high but usually non-lethal intraperitoneal dose (1000 micrograms/kg) of TCDD. Feed intake was stimulated by 24 hr feed deprivation at various time points after TCDD exposure. When TCDD-dosed rats were allowed to drink either a 20% sucrose or a 0.25% saccharin solution and then given access to feed, those that had had sucrose ate only about 50% of the amount consumed by the saccharin group. Although the preloads were similar in control rats, no such difference in subsequent feeding occurred. The sucrose solution also produced a longer-lasting suppression of feed intake in TCDD-treated compared with control rats when infused directly into the stomach. By contrast, TCDD-treated H/W rats failed to exhibit an augmented satiety response to parenterally applied glucose independent of testing time. Oral corn oil reduced feed intake in both control and TCDD-exposed rats, but the inhibition was slightly larger in TCDD-treated animals. TCDD did not markedly affect the responsiveness of H/W rats to the suppression of feeding by CCK-8 or bombesin. It is concluded that gastrointestinal factors appear critical to the exaggerated response of TCDD-treated H/W rats to nutrient energy.     

The in vivo biotransformation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rat

This study presents evidence for the in vivo biotransformation of TCDD in the rat. Three male rats with indwelling bile loop cannulas were given repeated daily po doses of 15 μg [¹⁴C]TCDD/kg body weight. After either two, four, or six doses, the total output of bile from one rat was collected for 24 hr following the last dose. Biliary ¹⁴C activity was excreted at a rate similar to the excretion of ¹⁴C activity in the feces of normal (noncannulated) rats given po doses of [¹⁴C]TCDD. Therefore it is not likely that enterohepatic recycling plays a significant role in the retention of ¹⁴C activity following a dose of [¹⁴C]TCDD. High-pressure liquid chromatography of the bile from these rats showed the presence of at least eight radioactive peaks and very little, if any, unchanged TCDD. These metabolites were all more polar than TCDD, and the chromatographic profile was altered following incubation of the bile with β-glucuronidase. These data, in conjunction with previous studies, indicate that the metabolic transformation of TCDD in the liver may be the rate-limiting step in the elimination of TCDD from the body.     

Hepatic metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rat and guinea pig

Marked interspecies variability exists in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), with the rat having an LD50 about 25-fold greater than the guinea pig. The metabolism of TCDD was examined by incubating hepatocytes isolated from these animals with purified [14C]TCDD (2.2 microM) for 8 hr. Over the 8-hr incubation, cytochrome P-450 content and ethoxyresorufin O-deethylase and benzphetamine N-demethylase activities were well maintained, indicating the functional viability of the hepatocytes. Quantitative differences were observed in the rate of [14C]TCDD metabolism, with hepatocytes from control rats metabolizing TCDD at a rate 2.8-fold greater than hepatocytes from control guinea pigs. The role of the hepatic cytochrome P-450-448-dependent monooxygenase system in the metabolism of TCDD was examined through the use of hepatocytes isolated from animals pretreated with either TCDD (5 micrograms/kg, ip; 72 hr prior to hepatocyte isolation) or phenobarbital (80 mg/kg, ip X 3 days; 24 hr prior to isolation). The rate of [14C]TCDD metabolite formation in hepatocytes from TCDD pretreated guinea pigs (0.26 +/- 0.14 pmol mg cell protein-1 hr-1) was unchanged from the control rate (0.25 +/- 0.07), while the rate in hepatocytes from TCDD pretreated rats (2.26 +/- 0.43 pmol mg-1 hr-1) was 3.2-fold greater than control (0.70 +/- 0.10) and nine times greater than in hepatocytes from TCDD-pretreated guinea pigs. In addition, significant differences were observed in the profiles of the metabolites formed by hepatocytes from TCDD-pretreated rats and guinea pigs. On the other hand, phenobarbital pretreatment produced little change in the rate of [14C]TCDD metabolism in rat hepatocytes (0.98 +/- 0.13 pmol mg-1 hr-1). These results suggest that TCDD may be metabolized by a TCDD inducible form of cytochrome P-448 which is expressed in the rat but not in the guinea pig. Furthermore, the differences in the hepatic metabolism of TCDD in the rat and guinea pig and in the ability of TCDD to induce its own rate of metabolism may play a major role in explaining the varying susceptibility of these species to the acute toxicity of TCDD.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits regression of the common cardinal vein in developing zebrafish.

A role for the aryl hydrocarbon receptor (AHR) pathway in vascular maturation has been implicated by studies in Ahr-null mice. In this study the hypothesis that activation of AHR signaling by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters common cardinal vein (CCV) development in the zebrafish embryo was investigated. The CCV is a paired vessel that grows across the yolk, connecting to the heart. It is extensively remodeled and regresses as the heart migrates dorsally within the pericardium. TCDD significantly reduced CCV growth as early as 44 h post fertilization (hpf), and CCV area was reduced to 63% of control at 62 hpf. This vascular response to TCDD was at least as sensitive as previously defined endpoints of TCDD developmental toxicity in zebrafish. TCDD also blocked regression of the CCV (by 80 hpf), possibly contributing to the "string-like" heart phenotype seen in TCDD-exposed zebrafish larvae. Dependence of the block in CCV regression on zebrafish (zf) AHR2 was investigated using a zfahr2 specific morpholino to knock down expression of AHR2. The zfahr2 morpholino had no effect on CCV regression in the absence of TCDD, but did protect against the TCDD-induced block of CCV regression. This demonstrates that the TCDD-induced block in CCV regression is AHR2 dependent. It is significant that decreased CCV growth occurs before and inhibition of CCV regression occurs concurrent with overt signs of TCDD developmental toxicity. This suggests that alterations of vascular growth and remodeling may play a role in TCDD developmental toxicity in zebrafish.     

Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure during gestation has revealed reproductive anomalies in rat offspring, including inconclusive reports of stunted mammary development in females (Brown et al., 1998, Carcinogenesis 19, 1623-1629; Lewis et al., 2001, TOXICOL: Sci. 62, 46-53). The current studies were designed to examine mammary-gland development in female offspring exposed in utero and lactationally to TCDD, and to determine a critical exposure period and cellular source of these effects. Long-Evans rats were exposed to 1 microg TCDD/kg body weight (bw) or vehicle on gestation day (GD) 15. TCDD-exposed females sacrificed on postnatal days (PND) 4, 25, 33, 37, 45, and 68 weighed significantly less than control litter mates, and peripubertal animals exhibited delayed vaginal opening and persistent vaginal threads, yet did not display altered estrous cyclicity. Mammary glands taken from TCDD-exposed animals on PND 4 demonstrated reduced primary branches, decreased epithelial elongation, and significantly fewer alveolar buds and lateral branches. This phenomenon persisted through PND 68 when, unlike fully developed glands of controls, TCDD-exposed rats retained undifferentiated terminal structures. Glands of offspring exposed to TCDD or oil on gestation days 15 and 20 or lactation days 1, 3, 5, and 10 were examined on PND 4 or 25 to discern that GD 15 was a critical period for consistent inhibition of epithelial development. Experiments using mammary epithelial transplantation between control and TCDD-exposed females suggested that the stroma plays a major role in the retarded development of the mammary gland following TCDD exposure. Our data suggest that exposure to TCDD prior to migration of the mammary bud into the fat pad permanently alters mammary epithelial development in female rat offspring.     

Evaluation of the developmental toxicity of formamide in Sprague-Dawley (CD) rats.

Timed-pregnant CD(R) outbred albino Sprague-Dawley rats received formamide (50, 100, or 200 mg/kg/day) or vehicle (5 ml/kg deionized/distilled water, po) on gestational days (gd) 6 through 19. Maternal food and water consumption (absolute and relative), body weight, and clinical signs were monitored at regular intervals throughout gestation. At termination (gd 20), confirmed-pregnant females (21-23 per group) were evaluated for clinical status and gestational outcome; live fetuses were examined for external, visceral, and skeletal malformations and variations. There were no maternal deaths and no dose-related clinical signs. At 200 mg/kg/day, maternal body weight on gd 20, weight gain, and gravid uterine weight were significantly decreased. Maternal weight gain, corrected for gravid uterine weight, liver weight (absolute or relative), and food and water consumption (absolute or relative), were not affected. Formamide did not affect prenatal viability or incidences of fetal malformations or variations. Average fetal body weight/litter was decreased at 100 and 200 mg/kg/day. Fetal body weight was affected at lower daily doses than in previously published studies, possibly due to the longer total exposure period and/or lack of a recovery period between cessation of exposure and termination. In summary, the maternal toxicity no-observed-adverse-effect level (NOAEL) was 100 mg/kg/day and the low observed adverse effect level (LOAEL) was 200 mg/kg/day under the conditions of this study. Similarly, the developmental toxicity NOAEL was 50 mg/kg/day and the LOAEL was 100 mg/kg/day.     

Evaluation of the developmental toxicity of isoeugenol in Sprague-Dawley (CD) rats.

Isoeugenol, used as a perfumery and flavoring agent, was evaluated for developmental toxicity. Timed-pregnant CD((R)) outbred albino Sprague-Dawley rats received isoeugenol (250, 500, or 1000 mg/kg/day) or vehicle (5 ml/kg corn oil) by gavage on gestational days (gd) 6 through 19. Maternal food and water consumption, body weight, and clinical signs were monitored at regular intervals throughout gestation. At termination (gd 20), confirmed-pregnant females (23-25 per group) were evaluated for gestational outcome. All live fetuses were weighed and examined for external malformations, and approximately 50% were evaluated for visceral or skeletal malformations. There were no treatment-related maternal deaths. Clinical signs associated with isoeugenol exposure included dose-related evidence of sedation and aversion to treatment (rooting behavior) in all isoeugenol groups, as well as an increased incidence of piloerection at >/= 500 mg/kg/day. Maternal body weight, weight gain, and gestational weight gain (corrected for gravid uterine weight) were reduced at all doses in a dose-related manner. Gravid uterine weight was significantly decreased at the mid and high doses, whereas maternal relative liver weight was increased at all three dose levels. During treatment (gd 6 to 20), maternal relative food consumption was significantly decreased at the high dose, and maternal relative water consumption was elevated in the mid- and high-dose groups. Prenatal mortality (resorption or late fetal death) was unaffected. At 1000 mg/kg/day, average fetal body weight/litter was decreased by 7% (male) or 9% (female). Incidences of fetal morphological anomalies were statistically equivalent among groups, except for an increase in the incidence of unossified sternebra(e), a skeletal variation, at the high dose. In summary, the maternal toxicity lowest observed adverse effect level (LOAEL) was 250 mg/kg/day based primarily on reduced body weight and gestational weight gain (corrected for gravid uterine weight), and the maternal toxicity no observed adverse effect level (NOAEL) was not determined in this study. The developmental toxicity LOAEL was 1000 mg/kg/day based on intrauterine growth retardation and mildly delayed skeletal ossification. The developmental toxicity NOAEL was 500 mg/kg/day.     

The reproductive and developmental toxicity of the antifungal drug Nyotran (liposomal nystatin) in rats and rabbits.

Nyotran is a liposomally encapsulated i.v. formulation of the antifungal polyene nystatin. This drug was evaluated in a series of reproductive toxicity studies, according to the guidelines outlined by the International Conference on Harmonization (ICH). A fertility and early embryonic development study (SEG I) and a prenatal and postnatal development (SEG III) study were conducted in rats, and embryo-fetal development (SEG II) studies were conducted in rats and rabbits. Nyotran was administered iv in all studies. In SEG I and SEG III, rats were administered daily doses of 0.5, 1.5, or 3.0 mg/kg Nyotran. In both studies, parental mortality and toxicity in the 3.0 mg/kg dose group necessitated the lowering of the high dose to 2.0 mg/kg/day. Parental toxicity, in the form of decreased body weights, decreased food consumption, and piloerection were also observed at the 1.5 mg/kg/day dose level in the SEG I and SEG III studies. Despite the parentally toxic doses in the SEG I study, there was no effect of Nyotran on F0 male or female fertility or early embryonic development of F1 offspring. In the SEG III study, lactational body weights of the F1 generation were decreased at all Nyotran dose levels. There was no effect on pre-wean developmental landmarks, but post-wean development was affected by Nyotran administration at all dosage levels. Preputional separation was delayed in the 1.5 and 3.0/2.0 mg/kg/day F1 offspring, auditory startle function was decreased in F1 females at all dose levels, and motor activity was decreased in male F1 offspring at all dose levels. However, there were no treatment-related effects on the subsequent mating of the F1 generation and resulting F2 offspring. In SEG II studies, rats and rabbits were also administered 0.5, 1.5, or 3.0 mg/kg/day of Nyotran during gestation. The high dose in these SEG II studies was not lowered, as the maternal animals were able to tolerate the shorter duration of dosing. Maternal effects in rabbits were observed only in the high-dose group and were limited to decreased food consumption and decreased absolute and relative liver weight. Decreased food consumption in high-dose dams and clinical weight loss in some animals at the mid- and high-dose levels evidenced maternal toxicity in rats. Nyotran did not have any effect on Caesarian section parameters in either rats or rabbits and no effect on the incidence of fetal malformations in rabbits. A statistically significant increase in mild hydrocephaly, observed in 4 rat fetuses, was seen at the highest dose level of 3.0 mg/kg/day. The biological significance and relationship to Nyotran treatment of this finding is not clear. This finding may represent a change in the background incidence or a change in the pattern of responsiveness of this strain of rat fetus to the test chemical. Toxicokinetic data were also collected in the SEG II rabbit and rat studies for comparison to human exposures. In both species, systemic exposure to the nystatin at effective antifungal concentrations was demonstrated. The systemic exposures in rats and rabbits were, however, considerably less than have been reported in humans administered clinical doses of 2 or 4 mg/kg/day Nyotran. Thus, humans tolerate higher dosages and systemic exposures of Nyotran relative to rats and rabbits and there is no margin of safety in either dosage level or systemic exposure to drug. Given this lack of a margin of safety and the effects on postnatal development in F1 rats, caution should be exercised when using this drug in females of childbearing potential.     

Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in four (sub)strains of adult male rats

Four (sub)strains of adult male rats were given single oral doses of various concentrations of TCDD to establish and compare the oral 30-day LD50 values. The strains of rats were Fischer (F/334N) supplied by Harlan Industries, Frederick Cancer Research Center, and Charles River Breeding Laboratories; and CD supplied by Charles River Breeding Laboratories. The Charles River/Fischer rats were most sensitive to TCDD (LD50 = 164, 95% confidence limits 104-217 micrograms/kg), the Frederick/Fischer and Charles River/CD rats were moderately sensitive to TCDD (LD50 = 303, 250-360; and 297, 240-360 micrograms/kg, respectively), and the Harlan/Fischer rats were most resistant to TCDD (LD50 = 340, 281-409 micrograms/kg). The mean times of death were from 24.5 +/- 1.0 to 28.3 +/- 0.5 days and the percentage body weight loss at death was 37.4 +/- 1.2 to 42.7 +/- 1.3%. One week after exposure of the Charles River/Fischer animal to 45 micrograms TCDD/kg (1/4 the established 30-day LD50 dose), the same serum profile was induced as previously observed in the Harlan/Fischer rat, which includes hypoglycemia, hypertriglyceridemia, and hypercholesterolemia. These results emphasize the importance of indicating the precise dose, strain of rat, and time after dosing before termination in reporting the effects of TCDD on a particular biological response.     

Developmental stage-specific effects of perinatal 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on reproductive organs of male rat offspring.

Exposure to a relatively low dose of 2,3,7,8-tetrachlorodebenzo-p-dioxin (TCDD) during mid-gestation induces a reduction of ventral prostate weight in rat offspring. Recently we reported that a single administration of TCDD (12.5-800 ng/kg body weight) to pregnant Holtzman rats on gestational day (GD) 15 caused a decrease in androgen receptor (AR) mRNA level in the ventral prostate during the prepubertal period, and we proposed that this reduction of AR mRNA is one of the most sensitive adverse endpoints due to perinatal exposure to TCDD (S. Ohsako et al., 2001, TOXICOL: Sci. 60, 132-143). In the present study, to investigate the mechanism of a decrease in AR mRNA level, we administered TCDD to rats at other developmental stages and compared possible alterations of the male reproductive system. Pregnant Sprague-Dawley rats were given a single oral dose of 1 microg TCDD/kg body weight on GD 15 or GD 18, or male pups born from untreated dams were subcutaneously given a single dose of 1 microg TCDD/kg body weight on postnatal day 2 (PND 2). Offspring exposed on GD 15, GD 18, and PND 2 were sacrificed on PND 70. TCDD exposure on GD 15 resulted in significant decreases in the urogenital complex and ventral prostate weights and urogenital-glans penis length of male rat offspring, but not on GD 18 and PND 2. Testicular and epididymal weights were also lower than control group only in the TCDD-exposed GD 15 group. Anogenital distance was significantly reduced in the TCDD-exposed GD 15 and GD 18 groups, but not in the TCDD-exposed PND 2 group. Semiquantitative RT-PCR analysis showed that AR mRNA levels were decreased in the TCDD-exposed GD 15 group only, and that the constitutive level of cytochrome P450 1A1 (CYP1A1) mRNA in the ventral prostate was not changed by TCDD in any of the exposed groups. No changes in AR mRNA level were detected in the testis or brain in any of the TCDD-exposed groups. These results suggest the presence of a critical window during development with regard to impairments of male reproductive organs by in utero and lactational exposure to a low dose of TCDD.