Retinoic-acid-induced limb-reduction defects: perturbation of zones of programmed cell death as a pathogenetic mechanism

Pregnant C57Bl/6J mice were treated with 100 mg/kg body weight of all-trans retinoic acid in sesame oil on day 11.0 of gestation. Among the live fetuses harvested on day 18 of gestation, 100% had mesomelic defects of the limbs as determined by gross examination and skeletal staining. Control fetuses treated with sesame oil had no observable limb malformations. Some treated and control embryos were harvested 12 hr after treatment and examined for patterns of cell death by using the supravital stain Nile blue sulphate and methylene-blue- and acid-fuchsin-stained histological sections. Retinoic-acid-induced cell death in the core of the limb was always associated with the zones of programmed cell death as seen in control embryos of comparable stages. This, in concert with previous studies demonstrating excessive cell death in regions of programmed cell death that correlated with subsequent malformations, leads us to conclude that the pathogenesis of mesomelic malformations has a primary association with the phenomenon of programmed cell death.     

Developmental toxicity of dichloroacetate in the rat.

Dichloroacetic acid (DCA) is a principal by-product of the chlorine disinfection of water containing humic and fulvic acids, and is also a drug of interest in the therapeutic management of metabolic disorders. The developmental effects of DCA were evaluated in the pregnant Long-Evans rat. In two separate studies, animals were dosed by oral intubation on gestation days 6-15 (plug = 0) with 0, 900, 1,400, 1,900 or 2,400 mg/kg/day and 0, 14, 140, or 400 mg/kg/day. The vehicle control was distilled water. Maternal observations included clinical signs, weight change, and gross evaluation of organ weights and uterine contents at necropsy (day 20). Corpora lutea were counted and uteri stained for implantation sites. Live fetuses were examined for external, skeletal, and soft tissue malformations. Seven dams died during treatment (1,400 mg 1/19, 1,900 mg 2/19, 2,400 mg 4/21), and maternal weight gain was reduced at all except the lowest treatment levels. Liver, spleen, and kidney weights increased in a dose-related manner. The mean percentage of resorbed implants per litter was significantly elevated at greater than or equal to 900 mg/kg/day. Live fetuses showed dose-dependent reductions in weight and length at doses above 140 mg/kg. Statistically significant frequencies of soft tissue malformations ranged from 2.6% (140 mg/kg) to 73% (2,400 mg/kg). These were principally in the cardiovascular system and predominantly comprised defects between the ascending aorta and the right ventricle. Skeletal malformations were not observed in significant numbers in any dose group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Malformations in rat fetuses induced by trypan blue

Malformations of fetuses obtained from Wistar rat dams treated with trypan blue during gestation were studied. Fetuses were examined on day 20 of gestation. One hundred and twenty-seven fetuses showed abnormalities of the external features, skeleton and internal organs, separately or in combination. External malformations were found in 108 fetuses. The most frequent external malformation was anomaly of tail. Spina bifida, club foot, exencephaly and anal atresia were also observed frequently. Skeletal malformations were detected in 48 fetuses. Deformity of vertebrae in the lumbar, sacral and/or caudal regions was found in 46 fetuses. Internal malformations were observed in 27 fetuses. Anomaly of heart and/or great vessels, hydrocephaly and micro- or anophthalmia were observed frequently. About 90% of the fetuses with skeletal malformations also showed some external malformations. In contrast, about 48% of the fetuses with internal malformations also had some external malformations. These results suggest that, for teratological study, internal examination is more important in detecting malformations of fetuses than skeletal examination.     

Evaluation of heart malformations in B6C3F1 mouse fetuses induced by in utero exposure to methyl chloride

C57BL/6 female mice impregnated by C3H males mice to produce B6C3F1 fetuses were exposed daily for six hr to atmospheres containing 0, 250, 500, or 750 ppm methyl chloride, from gestation day 6 to gestation day 18. There were 74 to 77 females with copulation plugs per exposure concentration. Females exposed to 750 ppm ethyl chloride exhibited ataxia commencing on the seventh day of exposure (gestation day 12). They also showed hypersensitivity to touch or sound, tremors and convulsions. Six females in the 750 ppm group died and one was euthanized in extremis prior to scheduled sacrifice. On gestation day 18, all other females were euthanized for evaluation. Only dams exposed to 750 ppm exhibited significant decrease in body weight by gestation day 18, weight gain during the gestation period, and absolute weight gain (weight gain minus gravid uterine weight) versus controls. There were no treatment related-effects on these parameters in the other exposure groups. None of the groups exhibited exposure-related differences in pregnancy rate, gravid uterine weight, or maternal liver weight. There were no differences in the numbers of implantations, resorption, dead fetuses, nonlive (dead plus resorbed) fetuses, live fetuses, sex-ratio, or mean fetal body weight per litter. There was a significant exposure-related increase in the number and percentage of affected (nonlive plus malformed) fetuses per litter with the incidence of affected fetuses in the 750 ppm group significantly higher than controls. There was a statistically significant increase in the incidence of heart defects in the 500 and 750 ppm group relative to controls. Of the 37 fetuses in the study with heart defects, 23 were females, 14 were males. The heart defects observed included: absent or abnormal tricuspid valve, reduced number of papillary muscles and/or chordae tendineae on the right side, small right ventricle, globular heart, and white spots in the left ventricular wall. Multiple malformations were observed in one fetus from the 500 ppm group and in three fetuses in the 750 ppm group. It is concluded that methyl chloride inhalation exposure to pregnant C57BL/6 mice from gestation day 6 through gestation day 17 resulted in maternal toxicity only at the 750 ppm exposure concentration and was teratogenic to B6C3F1 conceptuses at exposure concentrations of 750 and 500 ppm, leading to fetal heart malformations. No evidence of embryo or fetotoxicity other than teratogenicity was seen at any of the exposure concentrations employed. No maternal, embryo or fetotoxicity or teratogenicity was associated with exposure of mice, during critical periods of embryo and fetal development, to 250 ppm of methyl chloride.     

Methanol exposure during gastrulation causes holoprosencephaly, facial dysgenesis, and cervical vertebral malformations in C57BL/6J mice

BACKGROUND: Exposure of pregnant outbred CD-1 mice to methanol during the period of gastrulation results in exencephaly, cleft palate, and cervical vertebra malformations [Rogers and Mole, Teratology 55: 364, 1997], while inbred C57BL/6J mice are sensitive to the teratogenicity of ethanol. C57BL/6J fetuses exhibit the holoprosencephaly spectrum of malformations after maternal exposure to ethanol during gastrulation, but the sensitivity of C57BL/6J mice to methanol-induced teratogenesis has not been previously described. METHODS: Pregnant C57BL/6J mice were administered two i.p. injections totaling 3.4 or 4.9 g/kg methanol or distilled water four hrs apart on gestation day 'GD' 7. On GD 17, litters were examined for numbers of live, dead and resorbed conceptuses, fetuses were weighed as a litter and examined externally, and all fetuses were double stained for skeletal analysis. RESULTS: No maternal intoxication was apparent, but the high dosage level caused a transient deficit in maternal weight gain. The number of live fetuses per litter was reduced at both dosages of methanol, and fetal weight was lower in the high dosage group. Craniofacial defects were observed in 55.8% of fetuses in the low dosage group and 91.0% of fetuses in the high dosage group, including micro/anophthalmia, holoprosencephaly, facial clefts and gross facial angenesis. Skeletal malformations, particularly of the cervical vertebrae, were observed at both dosages of methanol, and were similar to those previously reported in the CD-1 mouse following methanol exposure. CONCLUSIONS: The types of craniofacial malformations induced in the C57BL/6J mouse by methanol indicate that methanol and ethanol have common targets and may have common modes of action.     

The teratogenic effects in rabbits of doxycycline, dissolved in polyvinylpyrrolidone, injected into the yolk sac.

A technique was developed for injecting agents into the yolk sac of rabbits to study their teratogenic effects. On the 9th day of gestation the rabbits were laparotomized during ether-oxygen narcosis. The test substance was injected into the yolk sacs in one horn of the uterus, and as control the corresponding solvent was injected into the other horn. On the 28th day of gestation the rabbits were killed, resorptions counted, and fetuses examined for malformations. Three series of studies were made: physiological saline; polyvinylpyrrolidone; and doxycycline dissolved in polyvinylpyrrolidone (DIP). The rate of resorptions and malformations significantly increased after injection of Vibravenos. As polyvinylpyrrolidone was not teratogenic it is concluded that the teratogenic effects of DIP were probably caused by doxycycline.     

Developmental toxicity of 1,6-hexamethylene diisocyanate (HDI) in the Sprague-Dawley rat

BACKGROUND: 1,6-Hexamethylene diisocyanate (HDI), a widely used chemical in commercial polyurethane manufacture, has been shown to affect the respiratory tract of experimental animals. However, its potential to affect neonatal development, particularly after inhalation exposure, is less well described. The present study was conducted to assess the developmental toxicity of HDI. METHODS: Gravid Sprague-Dawley rats were exposed to concentrations of 0, 0. 005, 0.050, or 0.300 ppm HDI via inhalation (whole-body exposure) on days 0-19 of gestation. Maternal toxicity, as demonstrated by clinical signs and changes in body weight gain during gestation, was characterized. Dams were sacrificed on gestation day 20, at which time fetuses were removed by cesarean section, the uterus was examined, and a gross maternal necropsy was performed. Maternal evaluation also included lung weight and a detailed histopathologic assessment of the nasal turbinates, larynx, trachea, and lungs. All fetuses were evaluated for external anomalies. Approximately one-half of each litter was examined for visceral effects, the other half underwent a skeletal (bone and cartilage) examination. RESULTS: Maternal toxicity was demonstrated in the 0.300- and, to a lesser extent, in the 0.050-ppm exposure groups. No maternal effects were noted in the 0.005-ppm group. Test compound-related maternal effects were restricted to histopathological findings and included acanthosis, hyperkeratosis, inflammation of the nasal turbinates, and, more seriously, degeneration of the olfactory epithelium. No pathological alterations were noted in the larynx, trachea, or lungs in any dose group. No test compound-related effects were observed on any reproductive parameters, or any embryonic endpoints, including pre/postimplantation loss and resorption. There were no effects on litter size or the number of fetuses per implantation site and no effects on fetal or placental weights were observed. No test compound-related fetal external, visceral, or skeletal findings were observed. No effect on the fetal or litter incidence of total malformations or variations was observed, and there was no difference in the incidence of malformations between males and females. CONCLUSIONS: Administered as described in this study, 1, 6-HDI produced maternal effects (nasal turbinate histopathology) at concentrations of 0.050 and 0.300 ppm with no developmental toxicity observed at any concentration.     

Developmental toxicity evaluation of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in Wistar rats

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a genotoxic chlorination by-product in drinking water. There is some evidence that it has developmental toxic effects in vitro but its potential to cause developmental effects in vivo is not known. The developmental effects were evaluated in Wistar rats. Rats (22-26 dams per dose group) were administered MX by gavage at the dose levels of 3, 30, or 60 mg/kg in water on gestation days 6-19. Control animals received plain water. Clinical signs, body weight, and food and water consumption were recorded for the dams. On gestation day 20, a cesarean section was performed and the ovaries anduterine contents of the dams were examined and the liver, kidneys, spleen, and thyroid glands weighed. The fetuses of all dose groups were weighed, sexed, and observed for external and skeletal malformations and the fetuses of the two highest dose groups were evaluated for visceral malformations. The highest dose, 60 mg/kg of MX, was slightly toxic to the dams. It decreased the corrected body weight gain of dams by 32% and the water consumption by 16-17%. Kidney and liver weights were slightly increased. MX did not affect the number of implantations nor did it cause any resorptions. The body weights of fetuses were not significantly affected. MX did not cause external malformations or skeletal anomalies. Two fetuses at 60 mg/kg and one fetus at 30 mg/kg had major visceral malformations (persistent truncus arteriosus, diaphragmatic hernia, dilated aorta with a stenosis of pulmonary arteries) and two minor artery abnormalities were observed in those animals. The frequency of unilateral displaced testis was slightly higher (9.2%) in the 60-mg/kg dose group than in controls (1.6%). Since the abnormalities did not form a consistent pattern and occurred most at maternally toxic dose, we conclude that MX can be regarded as non-teratogenic.     

Developmental toxicity of pentostatin (2'-deoxycoformycin) in rats and rabbits

The developmental toxicity of the potent adenosine deaminase (ADA) inhibitor, pentostatin (2'-deoxycoformycin), was investigated in pregnant rats and rabbits administered daily iv doses during organogenesis. Rats received 0, 0.01, 0.10, or 0.75 mg/kg on gestation days 6-15 and rabbits received 0, 0.005, 0.01, or 0.02 mg/kg on gestation days 6-18 and maternal and fetal parameters were evaluated on gestation day 21 (rats) or 30 (rabbits). Live fetuses were examined for external, visceral, and skeletal malformations and variations. In rats, maternal body weight gain and food consumption were significantly suppressed at doses of 0.10 and 0.75 mg/kg during the treatment period but returned to control levels during posttreatment. Increased postimplantation loss and decreased numbers of live fetuses, litter size, and fetal body weight were observed at 0.75 mg/kg. A statistically significant increase in the incidence of vertebral malformations occurred at 0.75 mg/kg. The incidence of certain skeletal variations (extra presacral vertebrae, extra ribs, hypoplastic vertebrae) was also increased at 0.75 mg/kg. Ossification of cervical centra was reduced at 0.75 mg/kg compared with controls. In rabbits, marked maternal toxicity (death, body weight loss, and decreased food consumption) and reproductive toxicity (abortion and premature delivery) occurred in all pentostatin-treated groups. However, there were no significant effects on number of live fetuses, pre- or postimplantation loss, litter size, or fetal body weights in the animals with live litters. There was also no apparent increase in the incidence of malformations or variations in the live fetuses of pentostatin-treated rabbits. Thus, these studies demonstrate developmental toxicity of pentostatin in rats and rabbits, and teratogenicity in rats, at maternally toxic doses.     

Conduct and interpretation of a dermal developmental toxicity study with KBR 3023 (a prospective insect repellent) in the Sprague-Dawley rat and Himalayan rabbit

KBR 3023, 1-(1-methyl-propoxycarbonyl)-2-(2-hydroxyethyl)piperidine, a prospective insect repellent being developed by Bayer Corporation, was evaluated for developmental toxicity in the Sprague-Dawley rat and Himalayan rabbit. As the intended human usage of the test compound is topical, the test systems were exposed to the compound via the dermal route. Specifically, the animals were fitted with Elizabethan collars, to reduce the likelihood of oral ingestion, and dermally administered either 0, 50, 200, or 400 mg KBR 3023/kg (rat), and 0, 50, 100, or 200 mg KBR 3023/kg (rabbit) on gestation days 0-19 (rat) and 0-28 (rabbit). Maternal toxicity, as demonstrated by clinical signs and changes in body weight gain and food consumption during gestation, was characterized. Animals were sacrificed on gestation day 20 (rat) and 29 (rabbit), at which time fetuses were removed by cesarean section and a gross maternal necropsy was performed. All fetuses were evaluated for external anomalies. With rats, approximately half of each litter was examined for visceral effects; the other half underwent a skeletal examination. With rabbits, all fetuses underwent both visceral and skeletal examinations. No effects were observed on maternal body weight gain or food consumption in either the rat or rabbit. In the rat, dermal effects (scaling/sloughing), were observed at the dose site of all test substance-treated groups from approximately gestation day 7 until termination of the study. Also noted were an increase in both absolute and relative liver weights in rats in the 400-mg/kg dose group. In the rabbit, dermal effects (slight erythema, squamous and cracked skin) were noted at the dose site of virtually all does administered the test compound, from approximately gestation day 7 until termination. Also observed in the rabbits was a potentially compound-related increase in soft stool, particularly at the highest dose level. In both species, there were no statistically significant effects on any reproductive parameters, or any embryonic endpoints, including pre/post-implantation loss and resorptions. There were no statistically significant effects on litter size or fetal or placental weights. No test compound-related external, visceral, or skeletal findings were observed. No effect on the individual fetal or litter incidence of total malformations or variations was observed and there was no difference in the incidence of malformations between males and females. KBR 3023 Technical, administered as described in these studies, produced maternal effects in the rat (liver weight) at a dose of 400 mg/kg, and in the rabbit (soft stool) in the 200-mg/kg dose group. No developmental toxicity was observed at any dose level.     

Teratogenic effect of D-amphetamine sulphate: histodifferentiation and electrocardiogram pattern of mouse embryonic heart

Pregnant albino mice (ICR random-bred strain) received daily injections of 50 or 100 mg/kg body weight of D-amphetamine sulphate between days 9 and 11 of gestation. Parallel control animals were injected with saline solution. Treated mice were sacrificed on day 15 or 19 of gestation. The embryos were examined for gross malformations and direct embryonic ECG recordings were made; they were weighed, and their hearts were carefully dissected. Microscopic sections of the heart were stained with hematoxylin and eosin. It was found that high doses of D-amphetamine raised the incidence of mortality in the treated pregnant mice up to 40%. The resorption rate in the survivors was high (up to 58%) following the high dose of the drug. Up to 15% of the embryos from the treated groups showed gross malformations, including skeletal and eye malformations and exencephaly. The electrocardiogram (ECG) recordings in most 19-day-old embryos from both treated groups showed a pattern with prolonged Q-T interval (PQT), similar to that of control embryos in the intermediate developmental stages (days 14-16 of gestation). The ECG of control embryos (from day 17 on) resembled that of prenatal fetuses. Microscopically, the hearts of treated embryos showed a large number of undifferentiated cardiac myoblasts. It can be inferred that high doses of D-amphetamine affect embryonic development generally and delay the histodifferentiation of the myocardium, resulting in incomplete maturation of the cardiac muscles, thus leading to the immature ECG pattern, with PQT intervals.     

Changes in the placenta and in the rat embryo caused by the demethylating agent 5-azacytidine

DNA methylation is an important mechanism for regulation of gene expression during vertebrate development. 5-azacytidine is used as an experimental tool for demethylation. In this work, a single dose of 5-azacytidine (5 mg/kg body weight) was administered to rats at different stages of development. After 5-azacytidine administration on the first or third day of pregnancy, no changes were detected. After administration on the fourth day of pregnancy or later, a reduction in growth was observed. After treatment on day five and on any other day till day eleven of pregnancy, no living fetuses were found. Of those treated on day twelve, 24% of fetuses survived, but forelimb and hindlimb malformations were present. Administered on day thirteen, 5-azacytidine did not interfere with survival, but malformations were still present. From day fourteen on, 5-azacytidine caused no gross external malformations. Placentas were also influenced by 5-azacytidine. They were significantly smaller and histological evaluation showed the labyrinthine part to be severely reduced. In contrast, trophoblast giant cells were more abundant than in controls.     

Possible mechanism of congenital malformations induced by exposure of mouse preimplantation embryos to mitomycin C

ICR mice were treated intraperitoneally with mitomycin C at 5 mg/kg on day 3 of gestation. On day 18 of gestation, fetuses of treated dams were inspected for external, skeletal and visceral malformations. At 6 or 12 hr after mitomycin C treatment, the blastocysts were obtained from the uteri of treated dams and the degenerated cells within inner cell mass (ICM) and trophectoderm (TE) tissues were examined microscopically. On day 5, 8, 11, or 18 of gestation, the uteri of treated dams were obtained and those including embryos/fetuses and placentae were examined histologically. Finally, on each of gestational days 5-14, the blood of the treated dams was collected and the hematological parameters determined. Pre- and postimplantation losses in the dams treated with mitomycin C were significantly increased; increased frequency of abdominal wall defects and lumbar ribs in term fetuses, decreased fetal weight, and increased placental weight were noted as well. No significant increase in visceral malformations was found in term fetuses treated with mitomycin C. Frequency of degenerated cells within ICM and TE of blastocysts from dams treated with mitomycin C was significantly increased as compared with the controls. In dams treated with mitomycin C, decidua developed insufficiently and the trophoblast giant cell layer was not separated from the uterine lumen by maternal components; hemorrhage from the denuded trophoblast giant cell layer into the uterine lumen was noted. The number of erythrocytes, as well as hemoglobin concentration, hematocrit, and the percentage of reticulocytes in blood of dams treated with mitomycin C were significantly lower from days 6-12 of gestation, as compared with controls. The results of the present study showed that an increase in number of degenerated cells within blastocysts results in preimplantation loss and both maternal and embryonic hypoxia during major organogenesis results in postimplantation loss and congenital fetal malformations.     

Teratogenicity of zinc deficiency in the rat: study of the fetal skeleton

Zinc deficiency (ZD) is teratogenic in rats, and fetal skeletal defects are prominent. This study identifies fetal skeletal malformations that affect calcified and non-calcified bone tissue as a result of gestational zinc deficiency in rats, and it assesses the effect of maternal ZD in fetal bone calcification. Pregnant Sprague-Dawley rats (180-250 g) were fed 1) a control diet (76.4 micrograms Zn/g diet) ad libitum (group C), 2) a zinc-deficient diet (0 microgram/g) ad libitum (group ZD), or 3) the control diet pair-fed to the ZD rats (group PF). On day 21 of gestation, laparotomies were performed. Fetuses were weighed, examined for external malformations, and stained in toto with a double-staining technique for the study of skeletal malformations. Maternal and fetal tissues were used for Zn, Mg, Ca, and P determinations. Gross external malformations were present in 97% of the ZD fetuses. No external malformations were found in fetuses from groups C and PF. Ninety-one percent of cleared ZD fetuses had multiple skeletal malformations, whereas only 3% of the fetuses of group PF had skeletal defects; no skeletal malformations were found in fetuses from group C. Some of the skeletal malformations described in the ZD fetuses, mainly affecting non-calcified bone, were not mentioned in previous reports, thus stressing the importance of using double-staining techniques. Examination of stained fetuses and counting of ossification centers revealed important calcification defects in ZD fetuses. These effects were confirmed by lower Ca and P concentrations in fetal bone with alteration of the Ca:P ratio.     

The Potential of Cr3 [Triaqua‐μ3‐Oxo‐Hexa‐μ‐Propionatotrichromium(III) Chloride] to Reduce Birth Defects in the Offspring of Diabetic CD‐1 Mice

Diabetes mellitus is a growing concern worldwide and leads to multiple complications during pregnancy. Pharmacologic doses of chromium (Cr) have been linked with improving insulin sensitivity and other positive benefits in the treatment of diabetes in animal models. By using streptozotocin induced hyperglycemia in female CD‐1 mice, reproductive outcomes of diabetic and chromium‐dosed diabetic females were examined. After dosing 10 mg/kg Cr in the form of triaqua‐μ₃‐oxo‐hexa‐μ‐propionatotrichromium(III) chloride or Cr3 during gestation days 8–16 (GD8–GD16), all females were sacrificed on gestation day 17 (GD17) and examined for maternal weight gain. The fetuses were examined for gross malformations and for skeletal malformations. The offspring of Cr3‐dosed females tended to have a reduction in the incidence of supernumerary ribs. While hyperglycemia still had negative impacts on the health of dams and their offspring, administration of Cr led to an apparent trend in the reduction in the number of malformations and incidence of supernumerary ribs compared to those of untreated diabetic mothers     

Fetal pathology produced by ethylene oxide treatment of the murine zygote

Exposure of female mice to ethylene oxide by inhalation 1 or 6 h after mating produced not only multitemporal death of conceptuses but also high rates of abnormalities among surviving fetuses. In contrast, only marginal effects were observed when females were exposed 9 or 25 h after mating. The abnormalities found among 17 day gestation live fetuses were predominated by hydrops and eye defects, which, together, constitute 54% of all anomalies. Most of the remaining anomalies were distributed among 5 other types: small size, cleft palate, and cardiac, abdominal wall, or extremity and/or tail defects. In a follow-up study, the fetuses of females treated 6 h postmating were examined at 11-15 days gestation and the progression of fetal death and of malformations was studied. Results indicate that the expression of most fetal anomalies does not become apparent until late in gestation. Several of these induced anomalies are similar to common human sporadic birth defects. This new class of experimentally induced fetal anomalies provides a new avenue for investigating zygotic biology and a system for studying the progression of aberrant development.     

Teratogenic activity of trichloroacetic acid in the rat

Trichloroacetic acid (TCA) is a by-product of the chlorine disinfection of water containing natural organic material. It is detectable in finished drinking water at levels comparable to the trihalomethanes (30-160 micrograms/L). TCA is also formed in vivo after ingestion of hypochlorite and has been identified as a major metabolite of chlorinated hydrocarbons such as trichloroethylene. The developmental effects of TCA were evaluated in the pregnant Long-Evans rat. Animals were dosed by oral intubation on gestation days 6-15 (plug = 0) with 0, 330, 800, 1,200, or 1,800 mg/kg/day. The vehicle control was distilled water. Maternal observations included clinical signs, weight change, and gross evaluation of organ weights and uterine contents at necropsy (day 20). Live fetuses were examined for external, skeletal, and soft tissue malformation. There were no maternal deaths associated with toxicity prior to sacrifice. Weight gain during treatment was reduced at 800, 1,200, and 1,800 mg/kg. Spleen and kidney weights were increased in a dose-related manner. The mean percent of resorbed implants per litter was 34, 62, and 90 at 800, 1,200, and 1,800 mg/kg, respectively. Live fetuses showed dose-dependent reductions in weight and length. The mean frequency of soft tissue malformations ranged from 9% at the low dose to 97% at the high dose. These were principally in the cardiovascular system (interventricular septal defect, levocardia). Skeletal malformations were found only at 1,200 and 1,800 mg/kg and were mainly in the orbit. Based on these observations TCA was considered to be developmentally toxic in the pregnant rat at doses of 330 mg/kg and above.     

Reproductive toxicity of BioThrax® in rabbits

BACKGROUND: An increasing number of women are being vaccinated during child-bearing years, including vaccination with BioThrax^® (Anthrax Vaccine Adsorbed, or AVA). As only a limited number of studies exist in humans that have examined the effects of AVA on reproductive health, this study was conducted in order to evaluate the impact AVA vaccination may have on pregnant female rabbits and their offspring. METHODS: Two hundred female rabbits were vaccinated with saline, adjuvant, or AVA twice prior to mating and on one of two occasions during gestation, in order to have exposure to the antigen during organogenesis. Blood samples were collected from does and fetuses/kits to assess the development and in utero transfer of antibodies to Bacillus anthracisprotective antigen (anti-PA IgG). Half of the does underwent Caesarean-sectioning on gestation day 29 and a gross necropsy was performed on both the does and their fetuses. The other half were allowed to naturally deliver and gross necropsy of the does and their kits was performed on lactation day 29. RESULTS: ELISA results showed that anti-PA IgG was generated by the does and passed to the fetuses/kits at detectable levels. CONCLUSIONS: AVA directly, or indirectly through the production of anti-PA IgG, did not appear to have an adverse effect on the pregnant females or their offspring, as measured by mating and fertility indices, natural delivery observations, clinical signs, gross lesions, in utero growth and survival, morphological development, or kit viability. Birth Defects Res (Part B) 86:370–376, 2009. © 2009 Wiley-Liss, Inc.     

Potentiating effect of caffeine on the teratogenicity of acetazolamide in C57BL/6J mice

Pregnant C57BL/6J mice were treated with 0 or 50 mg of caffeine (CAFF) per kg, and 0, 200 mg/kg (L) or 1,000 mg/kg (H) of acetazolamide (ACZM) during day 9 of gestation (9DPC). Individual fetuses were examined for gross morphological abnormalities and skeletal variations. The increase in fetal malformations seen, especially right forelimb electrodactyly, was augmented at both dose levels of acetazolamide by concomitant exposure to caffeine. Both frequency and severity of ectrodactyly were potentiated by caffeine. Skeletal examination revealed a reduction of the number of ossified cervical and caudal vertebral centra among litters exposed to ACZM at either dose. In either case (ACZM-H, ACZM-L) that effect was augmented by co-administration of CAFF. The first cervical vertebra (C1) appeared to provide the most sensitive index of teratogenic exposure. This study provides evidence that a subteratogenic dose of caffeine can potentiate the teratogenic effect of acetazolamide in C57BL/6J mice when dams are treated on day 9 of gestation. In addition, skeletal examination provided evidence that simultaneous treatment with both agents delayed fetal development. Many litters exposed to ACZM or both agents displayed a reduction in skeletal ossification even in the absence of gross morphological abnormalities, suggesting that ossification can be used as an indicator of prenatal exposure to potentially harmful substances in the C57BL/6 mouse strain.