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Bisphenol A (BPA) is an endocrine disrupting chemical with ubiquitous environmental exposure. Animal studies have demonstrated that in utero BPA exposure leads to increased adult body weight. Our aim was to characterize human fetal BPA exposure by measuring BPA concentration in second trimester amniotic fluid (AF) samples and to study its relationship with birth weight (BW) in full term infants. To achieve these goals, we developed a total BPA assay utilizing derivatization with pentafluorobenzyl followed by analysis with LC-ECAPCI-MS/MS with a limit of detection of 0.08 ng/mL and limit of quantification (LOQ) of 0.25 ng/mL. The mean BW of infants with AF BPA 0.40-2.0 ng/mL was 241.8 grams less than infants with AF BPA less than the LOQ after controlling for covariates (p=0.049). No effect was seen outside this range indicating a non-monotonic effect. Our data suggest that low level BPA exposure in utero decreases BW and needs further study.

The objectives of this study were to evaluate the transplacental kinetics and effects on placental function of the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, in the dually perfused human placental lobule system. When placed in the maternal perfusate at a high therapeutic serum concentration (150 ng/ml), enalaprilat was rapidly transferred from the maternal perfusate into placental tissue followed by a gradual release from the placenta into the fetal perfusate. C max and AUC values for enalaprilat in the maternal perfusate were approximately 3 times higher than in the fetal perfusate, and drug levels did not equilibrate between maternal and fetal perfusates during the 4-6 hours of perfusion. The more highly perfused regions of the placental lobule had higher drug levels than non-perfused areas. At the end of the perfusion period, the mean percent of total drug added in the maternal perfusate was 59%, with 23% in the fetal perfusate and the remainder (18%) in placental tissue. Despite the relatively high levels of drug found in placental tissue, there were no alterations in placental function as measured by fetal volume loss, fetal pressure, glucose utilization, lactate production, hCG release, net fetal oxygen transfer, and oxygen consumption. Results from this study clearly document placental transfer of enalaprilat in the perfused human placental lobule system. The lack of effect on placental function suggests that enalaprilat has no direct effect on fetoplacental vascular beds, and that fetal hypotension occurring from ACE inhibitor exposure may be due to direct effects on the fetal kidney and not to decreased perfusion of fetoplacental vascular beds; these findings require further validation in an in vivo model.

Losartan, an AT1-selective angiotensin II receptor antagonist, was evaluated in female rats for effects on fertility, reproduction, and perinatal and postnatal development. In a range-finding study, pregnant rats were treated orally from gestation days 6-17 (GD 6-17) with doses of 25, 75, 150, 225, and 300 mg Losartan/kg/day. There were treatment-related decreases in maternal body weight gain, slight treatment-related decreases in hemoglobin concentration, and slight treatment-related increases in serum urea nitrogen in the 225 and 300 mg/kg/day groups. In a fertility study, female rats were treated for 15 days prior to mating, during mating, and GD 0-19 with doses of 25, 100, and 300 mg Losartan/kg/day. The initial dose of 300 mg/kg/day was lowered to 200 mg/kg/day at the start of mating due to excessive body weight loss during the premating treatment interval. There were no treatment-related effects on reproductive performance, mating, or fertility indices in the F0 generation. There was no evidence of treatment-related or dose-related fetal malformations. However, decreased F1 pup body weights were observed in all drug-treated groups. In the 100 and 300/200 mg/kg/day groups there were treatment-related increases in F1 pup mortality and alterations in the pattern of postweaning body weight gains. There was also a delay in developmental signs in the 100 and 300/200 mg/kg/day groups, which were likely secondary to the decreased weight of the pups in these groups. In a developmental toxicity study, pregnant rats were administered 50, 100, and 200 mg Losartan/kg/day on GD 6-17. There was no evidence of developmental toxicity in any dose group. Maternal toxicity was evident in the 200 mg/kg/day group as a treatment-related decrease in body weight gain during gestation. In a late-gestation/lactation study, pregnant rats were administered 10, 25, and 100 mg Losartan/kg/day on GD 15 through lactation day 20 (LD 20). There were treatment-related decreases in maternal body weight gain during gestation and lactation in the 100 mg/kg/day group. Decreased pup weights were noted in all dose groups, and pre- and postweaning pup deaths were observed in the high dose group which were comparable to those observed in the female fertility study. The lack of fetal body weight effects at 100 mg Losartan/kg/day in the developmental toxicity study, with treatment ending on GD 17, indicates that adverse effects observed in the F1 generation in the fertility and late-gestation/lactation studies were due to exposure during late gestation and/or lactation.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous developmental and reproductive toxicity studies conducted in rats with Losartan, a potent AT1 subtype selective angiotensin II receptor antagonist, noted treatment-related effects on the pups of dams treated beyond the second trimester through lactation, as demonstrated by increases in pre- and postweaning pup deaths and decreased pup body weights [Spence et al. (1995) Teratology 51:000-000]. The studies presented here were designed to define the critical period for the induction of neonatal toxicity and to examine the effects of Losartan on kidney development when the drug is administered to the dam beyond the second trimester through lactation. In a developmental toxicity study with postweaning evaluation, pregnant rats were administered 5, 20, and 100 mg Losartan/kg/day on gestation days 6 through 15 (GD 6-15). There were no adverse effects on the F1 generation as assessed by mortality, clinical signs, weight gain, external examinations, developmental signs, behavioral tests, and gross or microscopic examination of the kidney. In a fostering/cross-fostering study, pregnant rats were administered 100 mg Losartan/kg/day on GD 15 through lactation day 20 (LD 20). Following delivery, pups from dams treated with Losartan were fostered to control dams, pups from control dams were fostered to Losartan-treated dams, and pups were also fostered to dams within the same group. Maternal exposure to Losartan during lactation increased the incidence of pup deaths on postnatal days 1-3 (PND 1-3), caused decreased pup weights on PND 7, and decreased performance in the auditory startle test in females and increased performance on the second swim maze test in males, relative to controls. Maternal exposure to Losartan during gestation was associated with decreased pup weight on PND 21 and effects observed on male performance in the swim maze test. Treatment during gestation was also associated with decreased pup cardiac weight as well as drug-induced histopathological changes of the kidneys from F1 pups, including medial hypertrophy of intracortical arterioles and dilatation of the renal pelvis. While the cardiac and renal vascular effects disappeared with time, significant renal lesions were still evident by PND 90. In a late-gestation/lactation study with renal evaluation, pregnant rats were administered 0.5, 1.0, 5.0, 20, and 100 mg Losartan/kg/day on GD 15-LD 20. Maternal toxicity was evident as decreased body weight gain in the 100 mg Losartan/kg/day group.(ABSTRACT TRUNCATED AT 250 WORDS)

The most unique aspect of the Held of developmental toxicology, which sets it apart from all other areas of toxicologic investigation, is the rapidly changing susceptibility of the conceptus to insult. In this presentation, an overview of the changing susceptibility of the conceptus will be given, with a discussion of the difficulties this presents for risk assessment. Prenatal development of all mammalian species can be divided up into the preim-plantation, embryonic, and fetal periods, with each period possessing its own characteristic susceptibility and unique response to prenatal insult. Most developmental toxicity studies have focused on the embryonic period, with malformations as the outcome of major concern. As understanding of mammalian development has increased, it is now clear that susceptibility to functional impairments, as well as to physiologic alterations, also exist during the embryonic and fetal period, which have major consequences for the offspring. The pattern of outcome can va...

Pregnancy outcomes in women with inadvertent exposure to lovastatin and simvastatin during pregnancy have been examined based on reports submitted to the manufacturer as part of worldwide postmarketing surveillance. There were 134 reports of exposure during pregnancy in which pregnancy outcome was known. Among prospectively followed pregnancies with known outcome, the proportion of normal outcomes was 85%, congenital anomalies 4.0%, spontaneous abortions 8.0%, fetal deaths/stillbirths 1.0%, and miscellaneous adverse outcomes 2.0%. While the number of prospective reports available for evaluation were only sufficient to rule out a three- to fourfold increase in the overall frequency of congenital anomalies, these proportions do not exceed what would be expected in the general population. Based on findings from this interim evaluation, there is no relationship between exposure to therapeutic doses of these agents during pregnancy and the occurrence of adverse pregnancy outcomes.

The purpose of this study was to determine the extent of placental transfer of the angiotensin-converting enzyme inhibitor enalaprilat and the effects on maternal and fetal cardiovascular parameters.Between gestational days 122 and 126 (term 167 days) five rhesus macaques underwent surgery for implantation of maternal and fetal vascular catheters. At least 4 days after surgery maternal and fetal blood pressures and heart rates were recorded for 1 hour. This was followed by a 5-minute maternal venous infusion of saline solution vehicle and recording for an additional hour. Enalaprilat was then infused over 5 minutes through the maternal femoral artery at doses of 0.05, 0.1, or 0.2 mg/kg. Maternal and fetal arterial blood samples were collected for determination of blood gas status and plasma enalaprilat concentrations.Enalaprilat rapidly crossed the placenta, and fetal values for areas under the concentration time curve were 50% to 65% of maternal values across dose groups. Drug was retained in the fetal plasma approximately threefold to fourfold longer than in maternal plasma. Maternal heart rate, blood pressure, arterial Po2 and pH were unchanged after enalaprilat infusion, as were fetal heart rate and blood gases. In contrast, fetal arterial pressure decreased significantly (19% to 23%, p0.01) after maternal treatment with 0.1 and 0.2 mg/kg and remained depressed throughout the 6-hour study interval. At 0.05 mg/kg fetal arterial pressure was decreased by 13% from baseline; differences were not significantly different (p0.05).Results from this study indicate that enalaprilat rapidly crosses the primate placenta with a single intravenous administration to the mother, resulting in significant and prolonged reduction of fetal arterial pressure. Because maternal cardiovascular parameters were unaffected, enalaprilat appears to have a direct effect on fetal arterial pressure.

Effective methods exist for separating epidermis from dermis for many species; however, a simple and effective skin separation method for non-human primates is not available. This investigation describes an easy and reliable method for separating epidermis from dermis in Rhesus monkeys. Skin was shaved and washed prior to necropsy. Skin samples were placed on cardboard and then in Whirl-Pak bags, frozen on dry ice and stored at -70 degrees C. Just prior to the separation procedure, Whirl-Pak bags were returned to dry ice storage. Immediately after removal from dry ice, each closed Whirl-Pak bag was placed into a waterbath maintained between 60 and 67 degrees C. After 2 minutes, the Whirl-Pak bag was removed from the waterbath, opened and the skin surface of the application site was gently scraped with a scalpel blade to remove the epidermis. Effectiveness of removal was verified by histologic examination of the remaining dermal samples.

Females of childbearing potential (FCBP) (between the ages of 15 and 44) and children (under the age of 18) should be considered for inclusion in early clinical trials of medicines if they constitute a substantial portion of the patient population to be treated. The purpose of inclusion in early trials is to determine whether major differences in safety, pharmacokinetics or pharmacodynamics exist relative to adult male volunteers or females of non-childbearing potential (FNCBP). This information is to be used primarily in the design of subsequent efficacy trials in these populations.

As described in previous talks, pharmaceutical products are tested in a lengthy and highly redundant series of tests to identify whether they produce reproductive toxicity in laboratory animals. Considerable progress has been made in improving the design of these studies, and draft guidelines have been promulgated which reduce the redundancy and increase the flexibility of reproductive toxicity tests for drug safety evaluation (Bass et al. 1991; Lumley and Walker, 1991). With improvement in the design of animal studies, it is now possible to turn our attention to the risk assessment process for Pharmaceuticals, the process in which results from animal studies are used to predict whether adverse effects will occur in humans. The perspective I will take is that of a laboratory investigator who condenses the results from animal studies into the narrowly defined categories for use of a drug in pregnancy. This is only the first step in the risk assessment process, and subsequently clinical, legal and regulatory perspectives must be obtained on the potential uses and abuses of the drug during human pregnancy before the final label is produced.

We have examined whether the neonatal mortality accompanied by lung hypoplasia and atelectasis induced by prenatal exposure to the diphenyl ether herbicide 2,4-dichlorophenyl-p-nitrophenyl ether (nitrofen) could be due to alterations in pulmonary surfactant synthesis. The specific hypothesis we tested was whether in utero exposure to nitrofen accelerated the catabolic metabolsim of glucocorticoids by induction of mixed function oxidase enzymes, leading to a depression in glucocorticoid levels and delay in fetal lung surfactant synthesis. Hepatic aminopyrine-n-demethylase and aniline hydroxylase activities in the 9000 g supernatant were not elevated in rat dams exposed to 50 mg/kg/day on days 8–18 of pregnancy. Cortisone and corticosterone levels in fetal plasma were not altered by nitrofen exposure. Fetal lung surfactant synthesis was also not affected, as indicated by measurements of total lung phospholipids, [14C]choline uptake into lung lipids, and cholinephosphotransferase (CPT) activity. Surface tension values from saline extracts of fetal lungs and fetal plasma corticosterone levels were monotored after co-administration of nitrofen and dexamethasone. Nitrofen exposure alone had no influence on these parameters, while dexamethasone, with or without nitrofen, depressed corticosterone levels and minimum surface tension measurements. These results suggest that the level of glucocorticoids and capacity of the fetal lung to respond to glucocorticoids by synthesizing and releasing surfactant are not affected by prenatal exposure to nitrofen.

Female rats were exposed by inhalation to trichloroethylene (TCE) vapors at a concentration of 1800 +/- 200 ppm to determine whether exposure before mating and during pregnancy is more detrimental to reproductive outcome than exposure either before mating alone or during pregnancy alone. Four treatment groups were utilized in a two by two factorial design: exposure to TCE for 2 weeks before mating and during the first 20 days of pregnancy; TCE before mating and filtered air during pregnancy; filtered air before mating and TCE during pregnancy; and filtered air before and during pregnancy. Significant elevations in skeletal and soft tissue anomalies, indicative of developmental delay in maturation rather than teratogenesis, were observed in the group exposed during pregnancy alone. The mixed function oxidase enzymes, ethoxycoumarin and ethoxyresorufin, indicative of cytochrome P-450 and P-448 activities, respectively, were measured in maternal and fetal livers, as well as livers of non-pregnant females, and showed variable levels of activity not uniformly related to treatment or pregnancy. Behavioral evaluation of offspring indicated a lack of treatment effect in tests of general activity levels at 10, 20 and 100 days of age. However, a reduction in postnatal body weights was seen in offspring from mothers with pregestational exposure. No results indicative of treatment-related maternal toxicity, embryotoxicity, severe teratogenicity or significant behavioral deficits were obtained in any of the treatment groups.

Female rats were exposed by inhalation to dichloromethane (DCM) at a concentration of 4500 ppm to determine whether exposure before and during gestation is more detrimental to reproductive outcome than exposure either before or during gestation alone. Four treatment groups were utilized in a two by two factorial design: exposure to DCM for 3 weeks before and during the first 17 days of gestation; DCM before and filtered air during gestation; filtered air before and DCM during gestation; and filtered air before and during gestation. Maternal liver weights were increased and fetal body weights were decreased in the two groups exposed to DCM during gestation compared to those exposed to filtered air during gestation. The incidence of gross external, skeletal, or soft-tissue anomalies was not significantly increased in fetuses in any group. Exposure to DCM both before and during gestation resulted in the same low degree of maternal and embryotoxicity as exposure during gestation alone.

The presence of a drug-metabolizing system in any in vitro bioassay for teratogenicity is necessary because many chemical agents require metabolism before their activity can be expressed, and limb bud cells lack the enzymes necessary to activate these chemicals. We have taken three approaches to add a drug-metabolizing system to limb bud culture; addition of a 9,000 × g supernatant (S9) or a purified microsomal fraction, both from mouse liver, and co-incubation of limb buds with hamster embryo cells (HEC). The liver preparations were able to convert the teratogen cyclophosphamide to five separate metabolites with alkylating activity in limb bud culture but the cytotoxicity of these preparations limited their usefulness as metabolizing systems in limb bud culture. HEC did not exert a toxic effect on limb buds and they were able to continue metabolism of cyclophosphamide for three days. Analysis of the metabolites indicated that HEC did not convert cyclophosphamide to the same products as the liver preparations. The metabolic products were capable of inducing abnormal limb development in vitro. Addition of 14C-cyclophosphamide in the presence of an HEC activating system led to uptake of radioactivity into limb buds. In the absence of HEC, little radioactivity was detected in the target tissue. These results suggest that culturing intact cells capable of drug metabolism with limb buds may be the most likely method to follow in achieving in vitro activation of chemicals.

Nitrofen (2,4-dichloro-4'-nitrodiphenyl ether) is an herbicide with potent teratogenic activity in rats. When administered at doses as low as 0.15 mg/kg/day during organogenesis, abnormal development of the heart, kidneys, diaphragm, and lung occurs. The specific pattern of visceral malformations produced in the absence of overt maternal toxicity or embryolethality/cytotoxicity suggest that the compound perturbs processes unique or highly selective for embryonic differentiation. Despite findings of metabolic activation to mutagenic intermediates and carcinogenic activity in adult rodents, several lines of evidence indicate that teratogenicity is not based on mutagenic insult to the embryo. Rather, evidence is accumulating that nitrofen exerts a teratogenic effect via alterations in thyroid hormone status. The premature and pharmacologic exposure of the embryo to a nitrofen-derived thyromimetic challenge is believed to be the cause of abnormal morphogenesis of the heart, lungs, kidneys, and diaphragm. The parent compound itself could directly bind to embryonic nuclear receptors for T3, leading to altered differentiation of target organs. Alternatively, increased availability and placental transport of free thyroid hormones in the maternal compartment could be the source of thyromimetic challenge to the embryo. Overall, these studies indicate that, in the case of nitrofen, the mode of teratogenic activity is uniquely different from the mode of adult toxicity.

Triamterene (2,4,7-triamino-6-phenylpteridine), a widely used diuretic/antihypertensive agent with weak antifolate activity, has been found to be positive in several in vitro assays for mutagenicity. The present studies were undertaken to characterize the potential mutagenic and antifolate activity of triamterene in the bone marrow and testes of mice with in vivo treatment. Triamterene had no clastogenic effects on the bone marrow at 6, 16, or 24 hr after a single oral dose of 25, 125, or 250 mg/kg. No alterations in hematopoietic cell maturation characteristic of antifolate action were observed in a dose-range study in which triamterene was orally administered to mice at 5–300 mg/kg/day for 5 days. Triamterene had no adverse effects on mating or fertility and did not induce dominant lethal mutations in the germ cells of male mice when given for 5 days at 5–100 mg/kg/day. Oral exposure to mice under identical conditions had no effect on testicular weight, DNA content, or activity of the de novo pathway for thymidine synthesis from deoxy [6-3H]uridine. The present findings are consistent with an absence of mutagenic effect and antifolate action on the bone marrow and testes with in vivo administration.

Many studies have been performed on the in vivo teratogenicity of cyclophosphamide, and there is uncertainty whether the parent compound or P-450 generated alkylating metabolite(s) is the proximal teratogen(s). We have examined the influence of cyclophosphamide and a metabolite, 4-ketocyclophosphamide, on mouse limb development. Pregnant mice were injected with 10, 15 or 20 mg/kg of cyclophosphamide on days 9 to 11 of gestation. Hindlimb buds were maximally sensitive to 20 mg/kg of cyclophosphamide at 9 A. M. on day 11, and the predominant malformations formed were preaxial ectrodactyly and hemimelia. Hindlimb buds of the same gestational age exposed to cyclophosphamide in vitro responded identically to controls in morphology and uptake of 3H-thymidine and 35SO4. Exposure to 4-ketocyclophosphamide in culture, however, resulted in the formation of limbs with a “hemimelic” appearance and distal limb reduction, and with reduced uptake of 3H-thymidine and 35SO4. These findings support the position that the P-450 generated metabolite (s), and not the parent compound, is the proximal teratogen(s).

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces fetal asymmetric limb malformations with exposure of pregnant mice to 50 mg/kg on day 11 of gestation. Hindlimbs were more frequently malformed than forelimbs, and a fourfold greater incidence of postaxial ectrodactyly was found in left forelimbs than in right forelimbs, and a two fold excess in left hindlimbs compared to right hindlimbs. The level of cell death and mitotic index were measured in forelimbs and hindlimbs from treated and control embryos at 1, 4, 18, 24, 48, and 72 hr after exposure to ascertain if these parameters could be correlated with the differential teratogenic susceptibility of the limbs. An increase in necrotic index was first detected in treated limbs at 4 hr, increased at 18 hr, peaked at 24 hr, and began declining at 48 hr to reach the control baseline at 72 hr. At 24 hr, the correlation between the level of cell death and susceptibility of malformation was the strongest, with the left hindlimb having a necrotic index of 58%, the right hindlimb 47%, the left forelimb 30% and the right forelimb 12%. In both forelimbs and hindlimbs, MNNG treatment initially depressed mitotic activity followed by an elevation at 48 hr relative to controls. The magnitude of the depression, extent of the elevation, and overall pattern of mitotic activity could not be uniformly related to limb defects. These results indicate that the amount of cell death in limb buds at 24 hr after MNNG exposure may predict target organ susceptibility. Depressions in mitotic activity and alterations in the pattern of mitosis were also observed which were not as clearly correlated with the incidence of malformations as was the amount of cell death.

The purpose of this study was to determine whether a 30-day administration of SKF 86002-A2, an inhibitor of cyclooxygenase and 5-lipoxygenase pathways of arachidonate metabolism, adversely affected reproductive cycles, ovarian structure, and/or pituitary/ovarian hormone secretion. Cyclooxygenase and 5-lipoxygenase enzymes catalyze the reactions leading to the synthesis of prostaglandins and leukotrienes, respectively, which are physiological regulators of ovarian function. Female rats were dosed once daily by gavage with 0, 1, 5, 10, 30, or 60 mg (base)/kg/day of SKF 86002-A2 for 30 consecutive doses beginning on the day of vaginal proestrus. Vaginal smears were then examined daily until necropsy, when ovaries and uteri were collected for macroscopic and histological examination. In addition, serum concentrations of estradiol, progesterone, luteinizing hormone, follicle stimulating hormone, and prolactin were estimated by radioimmunoassay. Estrous cycle irregularity, resulting from a dose-related lengthening of the interestrous interval, significantly (p less than 0.05) reduced the number of cycles in rats receiving 60 mg/kg/day of SKF 86002-A2 compared to controls. Furthermore, the ovaries from this group of rats weighed significantly more (p less than 0.05) than controls, apparently due to an increased occurrence of enlarged, cystic follicles that occasionally contained blood. Luteinized follicles with entrapped ova were also detected during histological examination. Dilatation of the uterine lumen was observed in some rats receiving doses of SKF 86002-A2 greater than 1 mg/kg/day. Serum progesterone in rats receiving 60 mg/kg/day of SKF 86002-A2 was significantly (p less than 0.05) lower than controls. In contrast, mean levels of serum estradiol were elevated in rats receiving 30 mg/kg/day of SKF 86002-A2. Serum concentrations of FSH, LH, and prolactin were not significantly different in any group. The results of this study suggest that SKF 86002-A2 disrupts cyclic ovarian function by a local, cumulative action that inhibits ovulation and alters steroid secretion.

When pregnant mice were exposed to 40 mg per kg of cytosine arabinoside (ara-C) on days 10 to 12 of gestation, adactylous limbs with large, distally located blisters were found when the fetuses were examined on day 18. Embryonic limbs exposed transplacentally under identical conditions and explanted to culture exhibited the same morphological abnormality as did limbs exposed directly in culture to 0.1 to 1 microgram per ml of ara-C. Two noncytotoxic analogues of ara-C, uridine arabinoside (ara-U) and hypoxanthine arabinose (ara-HX), had no influence on morphological differentiation of limbs in vitro. Ara-C alone caused a dose-related decrease in uptake of 3H-thymidine and 35SO4 in cultured limb buds. Production of this morphologically distinct malformation in vitro will allow detailed biochemical investigations on the effect of ara-C limb ectodermal-mesenchymal interactions.

The human reproductive system in both males and females is highly sensitive to a variety of occupational and environmental hazards. Damage can occur in many target tissues in the fetus and adult, leading to reproductive failure. A more comprehensive awareness of such potential damage is essential to reduce the relatively high rates of reproductive failure in the human population. In this article, human and laboratory animal test systems available for the study of reproductive failure are discussed. Endpoints measured in these systems include adult infertility, spontaneous abortion, growth retardation, stillbirths, birth defects, and functional disease states in the offspring, such as cancer, mental retardation, and sterility. Validation of laboratory animal test systems with known human teratogens is urged. Routine surveillance of the human population via recording of the incidence of spontaneous abortion along with cytogenetic analysis is recommended, as well as semen analysis of occupationally-exposed males. The need for more extensive study of the association between paternal exposure and reproductive outcome is emphasized. Test schemes for the early detection of reproductive failure in the human population and for the identification of agents capable of causing this effect are proposed.

Oral exposure of pregnant Long-Evans rats on day 11 of gestation to the teratogen, nitrofen (2,4-dichloro-4′-nitro diphenyl ether), which was uniformly labeled with 14C in the nitrophenyl ring, resulted in the accumulation of radioactivity in maternal fat with lesser amounts found in liver, kidney, other tissues, and in the embryonic compartment. The peak concentration of radioactivity occured 7–9 h after dosing and the half-life of the label in maternal blood was apporximately 8 days. In the embryonic compartment, radioactivity was first detected at 2 h after dosing, peaked at 4–6 h, and declined to half of that initially seen by 24 h. High performance liquid chromatography of embryo-placental extracts revealed 4 metabolites in addition to the parent compound: 4′-amino and 4′-acetylamine derivatives plus 2 hydroxylated derivatives. A similar metabolic frofile was observed in maternal blood and liver. In another experiment, purified 4′-amino metabolite was found to have no adverse effect on neonatal survival when administered orally on day 11 of gestation at doses up to 215 mg/kg. These results suggest that the teratogenicity of nitrofen cannot be readily explained by preferential distribution of the compound in the embryo or by a unique profile of stable, extractable metabolites in the embyronic compartment.

This study was conducted to determine if the Ah genotype could influence the incidence of tumorigenesis in offspring exposed to 3-methylcholanthrene (3-MC) during the fetal period. Male F1 hybrids (Ahb/Ahd) were backcrossed to Ahd/Ahd females, resulting in pregnant mice containing litters with a 1:1 ratio of Ahb/Ahd (AHH-inducible) and Ahd/Ahd (AHH-noninducible) fetuses. Dams were exposed by gavage to corn oil or to 3-MC at 7, 21, or 63 mg/kg on Days 15, 16, and 17 of gestation, or to the positive control urethane at 1 mg/g ip on Day 17. The phenotype of surviving offspring was determined by hepatic aryl hydrocarbon hydroxylase (AHH) activity. The lung was the major site of neoplastic involvement in adult offspring 6 months after in utero exposure to 3-MC. Dose-related responses in all 3-MC treatment groups were obtained for percent nodule-bearing animals, percentage adenoma-bearing animals, mean number of nodules per animal, mean nodular size per animal, and diffuse bronchiolar hyperplasia. Correlation of Ah phenotype with adult tumorigenesis indicated that genetically nonresponsive (Ahd/Ahd) offspring had a higher incidence of nodules, adenomas, and diffuse bronchiolar hyperplasia than responsive offspring within the same treatment group. Thus, when fetuses are exposed in the same maternal environment to 3-MC, genetic differences in Ah genotype may influence susceptibility to transplacental carcinogenesis.

There are some similarities and differences in the process of sexual differentiation during development in rodents and primates. The most obvious difference is in the timing of the critical periods for morphogenesis of the reproductive tract and sexual differentiation of the central nervous system (CNS). In primates these events occur late in the first trimester while in rodents they occur in the perinatal period. The gonadal hormones involved in morphogenesis of the male reproductive tract are identical for all mammalian species studied. Testosterone is the androgen that induces differentiation of the seminal vesicles, vas deferens, and epididymis, while dihydrotestosterone (DHT) promotes differentiation of the external genitalia, urethra, and prostate. Estrogens, derived from aromatization of testosterone, are the proximal determinants of male sexual differentiation of the CNS in rodents. In nonhuman primates, however, a nonaromatizable androgen, DHT, produces the same effect as testosterone on sexually differentiated behaviors in female offspring. Studies of male patients with 5α-reductase deficiency have suggested that maturation of male gender identity and psychosexual behavior in humans is critically dependent on testosterone but not on normal levels of DHT in prenatal and prepubertal life. Gender identity does not appear to be unalterably fixed in humans until the time of puberty and even at this and later times environmental factors have a strong impact.

This study was conducted to determine if the Ah genotype in mice could influence the incidence of neonatal mortality following maternal exposure to 3-methylcholanthrene (3-MC). Male F1 hybrids ( Ah b Ah d ) produced from a cross of C57BL 6J and DBA 2J were backcrossed to DBA 2J females. This backcross mating resulted in noninducible pregnant mice ( Ah d Ah d ) containing litters with a 1:1 ratio of AHH-inducible ( Ah b Ah d ) and noninducible ( Ah d Ah d ) fetuses. Dams were exposed by the oral route to 3-MC in corn oil at doses of 7, 21, or 63 mg/kg/day on Days 15, 16, and 17 of pregnancy, or to the positive control agent urethane at 1 mg/g ip on Day 17. The phenotype of surviving offspring was determined by zoxazolamine paralysis time and hepatic aryl hydrocarbon hydroxylase (AHH) activity measurements. Dose-related responses in all 3-MC treatment groups were obtained in measures of neonatal toxicity, i.e., number of litters surviving to term, litter size at birth, survival to weaning, and weight gain to 13 weeks of age. Correlation of the Ah phenotype with the neonatal toxicity data indicated that genetically responsive offspring had higher levels of neonatal toxicity than nonresponsive offspring within the same exposure groups. Thus, when fetuses are exposed in the same maternal environment to 3-MC, genetic differences in Ah genotype may influence the susceptibility to neonatal toxicity.

This paper examines the effects of in utero exposure to 2,4-dichlorophenyl- p -nitrophenyl ether (nitrofen) on the viability of neonatal Long-Evans rats. Oral administration of this herbicide on days 8–18 of gestation reduced neonatal survival and birth weight. Day 11 of gestation was the most sensitive day for induction of neonatal mortality; 116 mg/kg to the dam on this day was the LD 50 for the noenate. An increased incidence of hydronephrosis was observed in 35-day survivors. This increase was dose-related in animals exposed on day 11 of gestation. Fetuses exposed on day 11 and examined at term had reduced weights, delayed skeletal ossification, and an increased frequency of hydronephrosis and diaphragmatic hernias. While nitrofen did cause a high incidence of hydronephrosis, BUN or creatinine levels in 4-h neonates were not elevated. Detailed examination of the hearts of term fetuses revealed cardiac malformations classified as ventricular septal defect, double outlet right ventricle, and transposition of the great vessels. We conclude from these studies that the heart and the diaphragm are the target organs in nitrofen-induced neonatal death.

Many teratologic investigations have shown that certain types of chemical insults to the embryo (those altering replication, transcription, and translation) can cause excessive cell death in tissues destined to become malformed. Chemical carcinogens also induce cell death in target tissues, but the critical event is believed to be heritable alteration in the DNA of surviving cells. In the present study, an attempt was made to study the interaction between cell death and DNA damage in the initiation of birth defects. The pattern of DNA damage induced by cyclophosphamide was examined at time intervals before, during, and after the necrotic episode in mouse embryo limb buds. The alkaline elution assay was used to measure alkali-labile sites in single-strand DNA due to its adaptability to small tissue samples. An ip dose of 20 mg/kg of cyclophosphamide induced forelimb malformations in 85% of surviving mouse fetuses and 30% embryolethality when administered at 9 am on Day 11. As early as 5 hr after exposure, a slight excess of necrosis was observed in treated limbs by light microscopy, while at 24 hr, massive necrosis was evident. By 48 and 72 hr, excess necrosis was not observed in treated limbs. When alkaline elution analysis was conducted at prenecrotic (1-, and 5-hr), necrotic (24-hr), and postnecrotic (48-, and 72-hr) intervals, a trend toward increasing DNA damage in treated limbs with time was observed. The greatest differences in elution values occurred during the postnecrotic period. Although mean retention values were not significantly different, significantly increased variance was obtained in retention values of treated limbs at all time intervals other than 1 hr. This may reflect the actual in vivo situation where relatively few cells within a heterogeneous population of cells carry sublethal DNA damage into the postnecrotic period. These results suggest that not all limb bud cells affected by teratogenic exposure to cyclophosphamide die, but that some persist to the postnecrotic period carrying heritable alterations in their DNA.

Nitrofen is a herbicide with potent teratogenic activity in rodent species. Previous studies have indicated that this agent has a stereochemical structure similar to thyroid hormone, and that exposure of adult mice results in depression of thryoxine (T4) levels. The present study was undertaken to determine if teratogenic exposure to nitrofen alters pituitary-thyroid function in nonpregnant, pregnant, and fetal rats, and if these potential alterations could be related to induction of birth defects. In adult thyroparathyroidectomized (TPTX) female rats, nitrofen exposure for 2 weeks resulted in a significant suppression of thyrotropin-stimulating hormone (TSH) levels. When a single dose of nitrofen was administered to euthyroid female rats, a trend toward reduction (p = 0.058) in the release of TSH after a thyrotropin-releasing hormone (TRH) challenge was observed 4 and 5 hr after exposure. Pregnant euthryoid rats given a single dose of nitrofen on Day 11 of gestation had significantly depressed TSH and T4 levels, and fetal T4 levels were markedly depressed at term. Administration of T4 on Day 2 through 22 of pregnancy plus nitrofen on Day 9 through 11 to TPTX dams resulted in a 70% reduction in the frequency of malformed fetuses, especially in regard to the frequency of heart anomalies, compared to nitrofen exposure alone. Competitive displacement studies in radioimmunoassays for T4 and T3 indicated that a nitrofen metabolite (4-hydroxy-2,5-dichloro-4'-aminodiphenyl ether) competed with [125I]T3 for antibody binding, while the parent compound and six isolated metabolites failed to compete with [125I]T4 for antibody binding. These results have been interpreted to indicate that nitrofen teratogenicity is mediated at least in part by alterations in maternal and/or fetal thyroid hormone status, and may be due to a premature and pharmacologic exposure to the embryo to a nitrofen-derived, T3-active metabolite.

In a preliminary 30-day study, oral administration of SK&F 86002-A2, an inhibitor of prostaglandin and leukotriene synthesis, blocked ovulation and altered ovarian structure and hormone production in rats. The purpose of the present study was to determine if the locus of action of SK&F 86002-A2 for these effects was the ovary or some other site in the female reproductive system, using a number of experimental approaches. A single sc or intraovarian injection of SK&F 86002-A2 did not block spontaneous or gonadotropin-induced ovulation in proestrous rats, whereas indomethacin, a positive control, acutely disrupted the ovulatory process. Since neither route of administration blocked ovulation, integrated pituitary and ovarian events were not negatively affected by a single injection of SK&F 86002-A2 at doses which caused ovarian dysfunction when administered repeatedly for 30 days. In contrast to a single dose, oral administration of SK&F 86002-A2 to hypophysectomized rats for 2 weeks suppressed follicular growth and estradiol production in response to sc administration of pregnant mare serum gonadotropin. Although ovarian function was suppressed in hypophysectomized rats, LH surges induced by estradiol in ovariectomized rats were not affected by administration of SK&F 86002-A2 for 2 weeks. Thus, hypothalamic/pituitary dysfunction did not contribute to the ovarian effects of SK&F 86002 that occurred after repeated dosing. In conclusion, these results indicate that disruption of ovarian cycles by SK&F 86002-A2 is related to a direct effect on the ovary, and not to altered hypothalamic/pituitary function and LH release. Specifically, SK&F 86002-A2 may suppress the ovarian response to gonadotrophin, retarding follicular growth and estrogen production. The ovarian effects are consistent with a pharmacological expression of the inhibitory action of SK&F 86002-A2 on prostaglandin and leukotriene synthesis.

Two experiments were undertaken to show that limb regeneration of salamanders could be used as a sensitive index of cadmium toxicity. The first experiment established a range of exposure levels to cadmium that would influence limb regeneration without causing appreciable lethality. Exposure levels of 2 to 4.5 ppM of cadmium appeared suitable, while exposure to 6.75 ppM was associated with high levels of mortality that interfered with assessment of limb regeneration. Consequently, in the second experiment salamanders were exposed to levels of cadmium ranging from 2 to 4.5 ppM, i.e., 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 ppM, for the duration of the regeneration period. Results indicate that: (1) Salamanders are sensitive to cadmium in the aquatic environment; (2) death rate and time of onset of forelimb regeneration are dose related to cadmium exposure; (3) after an initial lag period, cadmium-treated limbs recover from exposure and regenerate to the same length as controls; and (4) cadmium causes an increase in the incidence and severity of abnormal limb regeneration which is related to exposure level. The fact that higher concentrations of cadmium were required to elicit toxic responses from salamanders (2 to 6.75 ppM) than from fish (10 to 100 ppB)more » suggests that the former are less sensitive to cadmium than the latter.« less

Rats divided in four treatment groups were exposed to dichloromethane (DCM) (4500 ppm) or filtered air before and/or during gestation in order to assess the occurrence and extent of toxic effects on developing offspring. The progeny of dams exposed to DCM either prior to and/or during gestation exhibited altered rates of behavioral habituation to novel environments. No simple relationship between exposure period and behavioral outcome was observed. Each of the treatment groups showed effects as a function of age at testing and the behavioral task used. Treatment effects were detectable in offspring as early as 10 days of age and were still demonstrable in 150-day-old male rats. Treatment effects were observed in rats of both sexes in preweaning tests but were not seen in adult female rats. No effects of subacute DCM exposure were evident in growth rate, long-term food and water consumption, wheel running activity, or avoidance learning. This study, which should be viewed as preliminary, is cf interest since altered rates of habituation to novel environments were observed in the absence of overt maternal toxicity, or teratogenicity. The effects cannot be definitely attributed to a direct effect of DCM since elevated maternal carboxyhemoglobin (COHb)- or DCM-induced changes in maternal-litter interactions could have been contributing factors. The findings do suggest that the functional development of progency of DCM-exposed dams should be further investigated.

A workshop was held to discuss potential advancements to improve the precision of risk estimates for developmental toxicity. This paper presents an overview of the discussions at the workshop, focusing on the risk assessment process and science policy considerations important in the use of quantitative models. Some of the pertinent biological considerations are reviewed, particularly those related to the repair capacity of the developing organism and how this affects the concept of a threshold for developmental toxicity effects, as well as the maternal and litter influences on developmental toxicity outcomes. Finally, the current status of use of quantitative approaches is described, possible short-term approaches are discussed, and future research needs in this area are outlined.

Female Long‐Evans rats were exposed by inhalation of 2100 ±. 200 ppm methyl chlorofrom (MC) to determine whether exposure before mating and during pregnancy was more detrimental to the offspring than exposure either before mating or during pregnancy alone. Four groups were exposed for 2 wk before mating and through d 20 of gestation in a 2 X 2 factorial design: (1) MC exposure before and during pregnancy, (2) MC before mating alone, (3) MC during pregnancy alone, and (4) filtered air before and during pregnancy. A t term, half of each group were sacrificed and assessed for maternal toxicity, embryotoxicity, and teratogenicity; the other half delivered their young for later behavioral evaluation and examination for gross lesions. No significant differences were found in measurements of maternal toxicity or embryotoxicity except for a decrease in fetal body weight when dams were exposed during pregnancy alone. Significantly increased incidences of skeletal and soft tissue variations were seen in fetuses fro...