Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro.

AY 9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; (C) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 micrograms/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dose-dependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.     

The effect of the hypocholesteremic drug, AY 9944 on the synthesis of bile salts in rat liver

1. The compound trans-1,4 bis-(2-dichlorobenzylaminomethyl)cyclohexane dihydrochloride (AY9944) blocks cholesterol synthesis at a late stage. This leads to a decrease in cholesterol and accumulation of cholesta-5,7-diene-3-beta-ol (7-dehydrocholesterol) in tissues and plasma. 2. The effect of AY9944 on bile salt synthesis in rat liver was studied. The synthesis of conjugated cholic and chenodeoxycholic acids was measured in hepatocytes isolated from rats 2 h, 24 h and 48 h after administration of a single oral dose of AY9944. Production of the two bile salts was inhibited by 70-80% in hepatocytes from AY9944-treated as compared to untreated animals. 3. When AY9944 was added to the incubation medium in vitro of hepatocytes prepared from untreated rats the synthesis of conjugated cholic and chenodeoxycholic acids was not inhibited during the first hour of incubation, probably because of the presence of endogenous cholesterol. However when hepatocytes from untreated rats were incubated with AY9944 for periods of 2 h or longer, bile salt production was decreased markedly. 4. Bile salt synthesis is stimulated when rats are subjected to total biliary drainage for 24 h. The effect of AY9944 on this stimulation was studied. The content of conjugated cholic and chenodeoxycholic acid in the bile was measured as an indicator of bile salt synthesis. 5. In control animals the rate of secretion of biliary bile salts began to increase after about 24 h of total biliary drainage and reached a maximum after approximately 36 h. A single oral dose of AY9944 given 2 h after the start of total biliary drainage delayed and reduced this response. 6. The results show that inhibition of cholesterol synthesis by AY9944 resulting in the replacement of cholesterol by 7-dehydrocholesterol decreases but does not completely prevent bile salt synthesis.     

Combined embryotoxic action of toluene, a widely used industrial chemical, and acetylsalicylic acid (aspirin)

CFY rats were exposed to inhalation of fresh air at days 10-13 of gestation; at day 12 the dams were given 0, 125, 250, 500, or 1,000 mg/kg acetylsalicylic acid (ASA) by gavage. During the same period of gestation (days 10-13) further groups of rats were exposed to toluene at 1,000, 2,000, and 3,600 mg/m3 atmospheric concentration and were given 250 mg/kg ASA by gavage; two subgroups of animals treated with 250 mg/kg ASA in combination with 3,600 mg/m3 toluene inhalation were given 0, 2.5, or 5 gm/kg glycine 2 hours before the ASA dose. At day 21 the animals were killed and examined for teratogenic effects and histological changes. After 48 hours toluene exposure other groups of rats were treated with ASA or with ASA plus glycine (administered 2 hours earlier) on day 20 of gestation. These animals were killed 2 hours later and the salicylic acid concentration in maternal and embryonic plasma and in amniotic fluid was measured by gas chromatography. With the rising ASA doses both maternal toxicity (increased mortality, decreased food consumption, and weight gain) and embryonic toxicity (postimplantation loss, increased incidence of weight-retarded fetuses, increased minor anomalies and malformations, decreased average weight of fetuses) increased. Toluene was found to potentiate the toxic effect of ASA and to increase both maternal and embryonic toxicity. The type of ASA-induced minor anomalies and malformations was also found to be altered under the effect of toluene pretreatment. By raising the toluene concentration the salicylic acid level in the maternal and embryonic plasma and in the amniotic fluid was increased above the expected concentration. The mechanism of the potentiating interaction should be looked for in the depletion of the glycine pool by toluene (and its metabolites) and in the resultant increase of salicylic acid level. Increasing ASA embryotoxicity caused by toluene can be warded off by glycine administration.     

Teratogenic potentially of single dose of thalidomide in JW-NIBS rabbits

A single dose (500 mg/kg) of thalidomide was administered orally to pregnant JW-NIBS rabbits in various stages of organogenesis. Head anomalies in fetuses (anencephaly, holoprosencephaly and hydrocephaly) were induced at a high frequency by the maternal administration of thalidomide on day 7, and also in a few fetuses on day 8. These fetuses included those with an abnormal skull such as hypoplasia of cerebral and facial skull. Microphthalmia in fetuses was observed with a single administration from day 7 to 12 of gestation. Contracture of forearms and club foot in fetuses resulted from the maternal administration of thalidomide on day 8 or 9 of gestation, respectively. With a single administration on day 8 or 9 of gestation, kinky tail in fetuses resulted, and brachyury was observed with a high frequency from day 8 to 11 of gestation. Skeletal anomalies such as fusion or displacement of coccygeal vertebral bodies were observed at a high frequency with a single treatment from day 8 to 10 of gestation. Among the internal anomalies observed was abnormal lobation of the lung, resulting from a single treatment from day 6 to 15 of gestation (except for day 13), and abnormal lobation of the liver, induced from day 7 to 10. The cardiovascular anomalies were induced at a high frequency with a single treatment from day 7 to 9 of gestation. In the present experiment, the critical period for each anomaly produced by thalidomide in JW-NIBS rabbits was determined.     

Regulation of bile acid synthesis. V. Inhibition of conversion of 7-dehydrocholesterol to cholesterol is associated with down-regulation of cholesterol 7 alpha-hydroxylase activity and inhibition of bile acid synthesis.

In the chronic bile fistula rat, the administration of a bolus dose of mevinolinic acid, an inhibitor of HMG-CoA reductase, was followed by rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and a decrease in bile acid synthesis. These observations suggested that either newly synthesized cholesterol or some other metabolite of mevalonate may be involved in the regulation of bile acid synthesis. In order to distinguish between these two alternatives, we carried out experiments in which cholesterol synthesis was blocked by AY9944, a compound that inhibits the conversion of 7-dehydrocholesterol to cholesterol, a last step in the cholesterol biosynthesis pathway. Rats underwent biliary diversion for 72 h at which time they were given intravenously either a bolus dose of AY9944 (1 mg/kg) or control vehicle. At 0 (pre-treatment control), 0.5, 1.5, and 3 h post bolus, livers were harvested and specific activities of cholesterol 7 alpha-hydroxylase were determined. At 1.5, 3, and 6 h post bolus, AY9944 inhibited bile acid synthesis by 19 +/- 6%, 40 +/- 4%, and 41 +/- 6%, respectively, as compared to pretreatment baseline. Cholesterol 7 alpha-hydroxylase activity determined at 0.5, 1.5, and 3 h was decreased by 44 +/- 6%, 44 +/- 2%, and 36 +/- 2%, respectively, as compared to the control value. In in vitro experiments using microsomes from livers of control bile fistula rats, the addition of AY9944 (up to 100 microM) failed to inhibit cholesterol 7 alpha-hydroxylase activity. The results of this study demonstrate that, in the chronic bile fistula rat, acute inhibition of cholesterol synthesis at either early or late steps leads to a rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and decrease in bile acid synthesis.     

Cholesterol deficit but not accumulation of aberrant sterols is the major cause of the teratogenic activity in the Smith-Lemli-Opitz syndrome animal model

Low cholesterol and high 7-dehydrocholesterol (7DHC) levels are associated with a blockade of Delta7-reductase in the Smith-Lemli-Opitz syndrome (SLOS) and in the animals treated with the inhibitor AY9944. The impact of the cholesterol deficit and of the accumulation of 7DHC on the embryo were investigated in AY9944-treated pregnant rats receiving an enriched cholesterol or 7DHC diet. Sterol profiling was performed under the various nutritional conditions. AY9944 caused a severe decrease in the maternal and embryo cholesterol. The deficit in the embryo was sustained by the embryonic uptake of the inhibitor. A cholesterol-rich diet was efficient in restoring the maternal and embryonic cholesterol and phenotype but a 7DHC-rich diet did not modify the sterol status compared with dams treated with only AY9944. The offspring phenotype remained deleterious whether or not the dams received 7DHC-rich diet. Over 80% of the 7DHC was absorbed, as was cholesterol, which was not quantitatively influenced by AY9944. When cholesterol and 7DHC were simultaneously administered, a competition for intestinal absorption enhanced the lowering cholesterol effect of AY9944.Whether or not the dams received a 7DHC dietary supplement, the offspring's phenotype became normal when the diet was supplemented with cholesterol. Under conditions in which the ratio of cholesterol/7DHC is substantially varied, the normal development of embryos can be achieved as long as the cholesterol is sufficient. The phenotype is reversed in vivo by cholesterol which contrasts with the irreversible effects manifested in vitro by oxidized 7DHC by-products.     

Developmental toxicity evaluation of Bendectin in CD rats

Bendectin, composed of doxylamine succinate and pyridoxine HCl (1:1), is an antinauseant previously prescribed for nausea and vomiting during pregnancy. The present study examined the maternal and developmental effects of Bendectin (0, 200, 500, or 800 mg/kg/day, po) administered to timed-pregnant CD rats (36-41/group) during organogenesis (gestational days [gd] 6-15). At death (gd 20), all live fetuses were examined for external, visceral, and skeletal abnormalities. At 500 and 800 mg/kg/day, maternal toxicity included reduced food consumption during treatment and for the gestation period, increased water consumption in the posttreatment period, reduced weight gain during treatment, and sedation; water consumption was reduced during treatment and for the gestation period, and maternal mortality (17.1%) was observed only at the high dose. Developmental toxicity included reduced prenatal viability (800 mg/kg/day) and reduced fetal body weight/litter (500 and 800 mg/kg/day). In addition, reduced ossification of metacarpals (800 mg/kg/day), phalanges of the forelimbs (500 and 800 mg/kg/day), and of caudal vertebral centra (all doses) was observed. No increase in percent malformed live fetuses/litter was observed. The proportion of litters with one or more malformed fetuses was higher than vehicle controls only at 800 mg/kg/day, with short 13th rib (to which the test species is predisposed) as the predominant observation. By contrast, a positive control agent (nitrofen, 50 mg/kg/day, po, 14 dams) produced 85% malformed fetuses/litter with the predominant malformation being diaphragmatic hernia. In conclusion, the incidence of litters with one or more malformed fetuses was increased only at a dose of Bendectin which produced maternal mortality (17.1%) and other indices of maternal and developmental toxicity (see Discussion).     

Ameltolide. I: Developmental toxicology studies of a novel anticonvulsant

Ameltolide, a novel anticonvulsant agent, has been shown in animal models to be effective in controlling seizures. The developmental toxicity of ameltolide was evaluated in two species. Naturally mated rats and rabbits were dosed once daily by gavage on gestation days (GD) 6-17 and 6-18, respectively. Rats were given doses of 0, 10, 25, or 50 mg/kg; rabbits were given 0, 25, 50, or 100 mg/kg. Laparotomy was performed on rats on GD 20 and on rabbits on GD 28. In rats, maternal toxicity was indicated at the 25- and 50-mg/kg dose levels by depressed body weight gain. Fetal body weight was depressed at the 50-mg/kg dose level. Fetal viability and morphology were not affected. The no-observed effect levels (NOEL) for adult and developmental toxicity in the rat were 10 and 25 mg/kg, respectively. In rabbits, maternal toxicity was indicated by a net loss in body weight at the 50- and 100-mg/kg dose levels. Fetal viability and body weight were depressed at the 100 mg/kg dose level. Shortened digits occurred on the right forepaw of one fetus at the 50-mg/kg dose level (in conjunction with severe maternal toxicity) and on the hindpaws of two fetuses from separate litters at the 100-mg/kg dose level. Incomplete ossification of the phalanges occurred on the forepaws of nine fetuses from four litters at the 100-mg/kg dose level. Ameltolide was weakly teratogenic in the rabbit. The NOEL for adult and developmental toxicity in the rabbit was 25 mg/kg.     

Influence of maternal stress on uranium-induced developmental toxicity in rats

It has been demonstrated that uranium is an embryo/fetal toxicant when given orally or subcutaneously to pregnant mice. On the other hand, maternal stress has been shown to enhance the developmental toxicity of a number of metals. In this study, maternal toxicity and developmental effects of a concurrent exposure to uranyl acetate dihydrate (UAD) and restraint stress were evaluated in rats. Four groups of pregnant animals were given subcutaneous injections of UAD at 0.415 and 0.830 mg/kg/day on Days 6 to 15 of gestation. Animals in two of these groups were also subjected to restraint for 2 hr/day during the same gestational days. Control groups included restrained and unrestrained pregnant rats not exposed to UAD. Cesarean sections were performed on gestation Day 20, and the fetuses were weighed and examined for malformations and variations. Maternal toxicity and embryotoxicity were noted at 0.830 mg/kg/day of UAD, while fetotoxicity was evidenced at 0.415 and 0.830 mg/kg/day of UAD by significant reductions in fetal body weight and increases in the total number of skeletally affected fetuses. No teratogenic effects were noted in any group. Maternal restraint enhanced uranium-induced embryo/fetal toxicity only at 0.830 mg/kg/day, a dose that was also significantly toxic to the dams. As in previous studies with other metals, maternal stress enhances uranium-induced developmental toxicity at uranium doses that are highly toxic to the dams; however, at doses that are less acutely toxic the role of maternal stress would not be significant.     

Teratogenicity of the 13-cis and all-trans-isomers of the aromatic retinoid etretin: correlation to transplacental pharmacokinetics in mice during organogenesis after a single oral dose

NMRI mice were treated on day 11 (day 0 = plug day) of gestation with a single oral dose of 100 mg/kg of either all-trans-etretin (acitretin) or 13-cis-etretin. For teratology studies mice were sacrificed on day 18 of gestation, and the fetuses were examined for malformations. For pharmacokinetic studies, groups of 5 mice were sacrificed after 5, 10, and 30 min and 1, 2, 4, and 8 h. The concentrations of retinoids in maternal plasma and in embryos were determined by a newly developed HPLC gradient method. All-trans-etretin induced malformations in 94% of the fetuses, mainly in fore and hind limbs and cleft palate. 13-cis-etretin did not show any teratogenic or embryo-lethal effects at the dose level used. These findings could be explained by a transplacental pharmacokinetic study. The maximum peak level and also the AUC (area under the concentration-time curve) value of all-trans-etretin in the embryos was 7-8 times higher than corresponding values for 13-cis-etretin, probably due to extensive transport of the trans-isomer and limited transport of the cis-isomer from maternal plasma to the embryos. The concentration quotient between embryo and the maternal plasma was between 0.43 and 1.10 for all-trans-etretin, and only 0.16-0.31 for 13-cis-etretin over the time period studied. An in vivo isomerization of the compounds was also observed which was more extensive for 13-cis-etretin than for all-trans-etretin. Our results indicate that the low teratogenic potency of 13-cis-etretin is due to a limited placental transfer of this compound; on the other hand, the potent teratogen all-trans-etretin is rapidly and extensively transferred to the embryo.     

Embryotoxicity of oral administered chlorothalonil in mice

BACKGROUND: Chlorothalonil (2,4,5,6-tetrachloroisophthalonitril), the nephrotoxic fungicide, was examined for its potential to produce developmental toxicity in mice after oral administration. METHODS: Pregnant ICR (CD-1) mice were given sublethal doses of 0 (corn oil), 100, 400, and 600 mg/kg/day chlorothalonil by gavage on gestation days (GD) 6-15. RESULTS: Maternal effects in 400 and 600 mg/kg/day dose groups included signs of toxicity such as weakness and depression in the maternal activity, and reduction in body weight and weight gain. No maternal toxicity was apparent in the 100 mg/kg/day dose group. Maternal exposure to chlorothalonil during organogenesis significantly affected the number of live fetuses, early resorption, and mean fetal weight in the 400 and 600 mg/kg/day dose groups. No external, visceral, and skeletal abnormalities were observed among any of the treated groups compared to the control. CONCLUSIONS: On the basis of the present results chlorothalonil can produce clinical signs of toxicity and fetotoxicity without teratogenic effects at 400 and 600 mg/kg/day dose groups.     

Evaluation of the protective activity of deferiprone, an aluminum chelator, on aluminum-induced developmental toxicity in mice

BACKGROUND: Since deferiprone can be an effective chelating agent for the treatment of aluminum (Al) overload, in the present study we investigated whether this chelator could protect against Al-induced maternal and developmental toxicity in mice. METHODS: A single oral dose of Al nitrate nonahydrate (1,327 mg/kg) was given on gestation day 12, the most sensitive time for Al-induced maternal and developmental toxic effects in mice. At 2, 24, 48, and 72 hr thereafter, deferiprone was given by gavage at 0 and 24 mg/kg. Cesarean sections were performed on day 18 of gestation and fetuses were examined for malformations and variations. RESULTS: Aluminum-induced maternal toxicity was evidenced by significant reductions in body weight gain, corrected body weight change, and food consumption. Developmental toxicity was evidenced by a significant decrease in fetal weight per litter and an increase in the total number of fetuses and litters showing bone retardation. No beneficial effects of deferiprone on these adverse effects could be observed. By contrast, a more pronounced decrease in maternal weight gain and corrected body weight change, as well as a higher number of litters with fetuses showing skeletal variations was noted in the group exposed to Al nitrate and treated with deferiprone at 24 mg/kg. CONCLUSIONS: According to the current results, deferiprone would not be effective to prevent Al-induced maternal and embryo/fetal toxicity in mice.     

Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.     

Developmental toxicity of orally administered technical dimethoate in rats

BACKGROUND: Dimethoate (O,O-dimethyl-S-(N-methylcarbamoyl-methyl) phosphorodithioate), an organophosphate insecticide, was examined for its potential to produce developmental toxicity in rats after oral administration. METHODS: Pregnant Fischer 344 rats were given sublethal doses of 0 (corn oil), 7, 15, and 28 mg/kg/day dimethoate by gavage on gestation days (GD) 6-15. Maternal effects in 15 and 28 mg/kg/day dose groups included cholinergic signs such as tremors, diarrhea, weakness, and salivation, and depression in the maternal and fetal brain acetylcholinesterase (AChE) activities. Other maternal toxicity that included reduction in body weight and feed consumption was observed only in the treated group of 28 mg/kg/day. No maternal toxicity was apparent in the 7 mg/kg/day dose group. RESULTS: Maternal exposure to dimethoate during organogenesis significantly affected the number of live fetuses, early resorption, and mean fetal weight in the 28 mg/kg/day dose group. No external, visceral, and skeletal abnormalities were observed in any of the treated groups compared to the control. CONCLUSIONS: On the basis of the present results dimethoate can produce clinical signs of toxicity and significant inhibition of the maternal and fetal AChE activities in dose groups of 15 and 28 mg/kg/day and showed fetotoxicity without teratogenic effects at 28 mg/kg/day.     

Teratogenic effects of the plant hormone indole-3-acetic acid in mice and rats.

These studies evaluated the teratogenic potential of indole-3-acetic acid (IAA), a naturally occurring plant hormone, in CF-1 mice and Sprague-Dawley rats. Mice were given 5, 50, 200, or 500 mg IAA/kg/day by gavage on days 7 through 15 of gestation. Rats were given 50, 200, or 500 mg IAA/kg/day by gavage on days 7 through 15 of gestation. IAA was teratogenic in mice and rats at 500 mg/kg/day; cleft palate was induced in both species at this dose level. In mice, other malformations including exencephaly, ablepharia, dilated cerebral ventricles, and crooked tail were also observed. Mice given 500 mg/kg of IAA gained less than control mice during gestation; no evidence of maternal toxicity was observed in rats. IAA did not cause fetal resorptions in either species and was not teratogenic at dose levels below 500 mg/kg.     

Limb malformations of rat fetuses exposed to a distal inhibitor of cholesterol biosynthesis

Triparanol, an inhibitor of desmosterol Delta24 reductase, produces a high rate of limb malformations in rat fetuses exposed at gestational day 10 (gd 10) to a single oral dose (150-200 mg/kg) given to the pregnant dam. AY9944, another efficient distal inhibitor of cholesterol biosynthesis that blocks dehydrocholesterol Delta7 reductase, produces a similar degree of cholesterol depletion but fewer malformations. Gas liquid chromatography-mass spectrometry (GC-MS) profiling of the sterols in the serum of the dams and in extracted embryos shows that in addition to desmosterol Delta24 reductase inhibition the conversion of Delta8 to Delta7 unsaturated sterols is also blocked by Triparanol. Therefore, the inhibitor induces the accumulation of desmosterol (Delta8 cholesten-3beta-ol, 8-dehydrocholesterol) and zymosterol (Delta8, Delta24 cholestadien-3beta-ol) in embryo tissues. The high concentration of the teratogenic drug assayed in the embryos at three successive gestational days (10-30 micro g/g) is thought to cause the blockade in both Delta24 reductase and Delta8-Delta7 isomerase, which results in the particular profile of aberrant sterols. Comparison of the animal model with human syndromes, including limb osseous and skeleton perturbations, suggests a combination of desmosterol and Delta8 unsaturated sterols as being involved in the deleterious influence on limb bone formation.     

Effects of tert-butyl acetate on maternal toxicity and embryo-fetal development in Sprague-Dawley rats

This study investigated the potential adverse effects of tert-butyl acetate (TBAc) on maternal toxicity and embryo-fetal development after maternal exposure of pregnant rats from gestational days 6 through 19. TBAc was administered to pregnant rats by gavage at 0, 400, 800, and 1,600 mg/kg/day. All dams were subjected to a Caesarean section on day 20 of gestation, and their fetuses were examined for any morphological abnormalities. At 1,600 mg/kg, maternal toxicity manifested as increases in the incidence of clinical signs and death, lower body weight gain and food intake, increases in the weights of adrenal glands and liver, and a decrease in thymus weight. Developmental toxicity included a decrease in fetal weight, an increase in the incidence of skeletal variation, and a delay in fetal ossification. At 800 mg/kg, only a minimal developmental toxicity, including an increase in the incidence of skeletal variation and a delay in fetal ossification, were observed. In contrast, no adverse maternal or developmental effects were observed at 400 mg/kg. These results show that a 14-day repeated oral dose of TBAc is embryotoxic at a maternally toxic dose (i.e., 1,600 mg/kg/day) and is minimally embryotoxic at a nonmaternally toxic dose (i.e., 800 mg/kg/day) in rats. However, no evidence for the teratogenicity of TBAc was noted in rats. It is concluded that the developmental findings observed in the present study are secondary effects to maternal toxicity. Under these experimental conditions, the no-observed-adverse-effect level of TBAc is considered to be 800 mg/kg/day for dams and 400 mg/kg/day for embryo-fetal development.     

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.     

Developmental Toxicity of the HMG‐CoA Reductase Inhibitor (PPD10558) in Rats and Rabbits

PPD10558 is an orally active, lipid‐lowering 3–hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor (statin) being developed as a treatment for hypercholesterolemia in patients who have not been able to tolerate statins because of statin‐associated myalgia. We have studied the potential developmental toxicity effects of PPD10558 in pregnant rats and rabbits given daily oral doses during the period of organogenesis. Rats were dosed with 0, 20, 80, or 320 mg/kg/day from Gestation Day (GD) 6 to 17 and rabbits received dose levels of 0, 12.5, 25, or 50 mg/kg/day from GD 6 to 18. Additional groups in both studies served as toxicokinetic animals and received the PPD10558 in the same manner as the main study groups at the same dose levels. Blood samples were collected from toxicokinetic animals at designated time points on GD 6 and 17 in rats and GD 6 and 18 in rabbits. Fetal exposure in rats was assessed on GD 20. Maternal and developmental parameters were evaluated in rats and rabbits on GD 20 and GD 29, respectively. No maternal and developmental toxicity was observed at any of the dose levels used in the rat study. Evidence of fetal exposure was determined in fetal plasma with mean fetal concentrations of PPD10558 and the metabolite (PPD11901) found to be between 1 and 6% of the mean maternal concentrations. In rabbits, marked maternal toxicity including mortality (eight deaths; 1 dose at 25 and 7 at 50 mg/kg/day), abortions (2 at 25 mg/kg/day and 6 at 50 mg/kg/day) and reduction in gestation body weight, gestation body weight changes and decreased food consumption were observed. In addition, fetal body weights of the combined sexes were significantly reduced at 50 mg/kg/day in comparison with the controls. Mean peak exposure (Cmax) and total exposure (AUC(0–24)) of PPD11901 in both rats and rabbits were higher than that of PPD10558 on GD 6 and GD 17 at each of the three dose levels.. Based on the results of these studies, the no observed adverse effect level (NOAEL) for maternal and developmental toxicity in rats was considered to be ≥320 mg/kg/day, the highest dose level used in the study. The NOAEL for maternal and developmental toxicity in rabbits was 12.5 mg/kg/day and 25 mg/kg/day, respectively.