Improved methodology for identifying the teratogenic potential in early drug development of hERG channel blocking drugs.

Drugs blocking the potassium current IKr of the heart (via hERG channel-inhibition) have the potential to cause hypoxia-related teratogenic effects. However, this activity may be missed in conventional teratology studies because repeat dosing may cause resorptions. The aim of the present study was to investigate an alternative protocol to reveal the teratogenic potential of IKr-blocking drugs. The IKr blocker astemizole, given as a single dose (80 mg/kg) on gestation day (GD) 13 to pregnant rats caused digital defects. In whole rat embryo culture (2h) on GD 13, astemizole caused a decrease in embryonic heart rate at 20 nM, and arrhythmias at 200-400 nM. Cetirizine, without IKr-blocking properties, did not affect the rat embryonic heart in vitro. The present study shows that single dose testing on sensitive days of development, together with whole embryo culture, can be a useful methodology to better characterize the teratogenic potential of IKr-blocking drugs.

Stage-specific skeletal and visceral defects of the I(Kr)-blocker almokalant: further evidence for teratogenicity via a hypoxia-related mechanism.

BACKGROUND: As a class effect, potent I(Kr)-blockers have been shown to induce stage-specific external malformations. The aim of this study was to investigate whether I(Kr)-blockers also induce stage-specific visceral and skeletal defects and to further elucidate a proposed arrhythmia-hypoxia hypothesis. METHODS: Single oral doses of the selective I(Kr)-blocker almokalant (ALM) 25-150 micromol/kg, 7-14 dams/group, were given to Sprague-Dawley rats on gestation days (GD) 10-14, and the fetuses were examined for malformations on GD 21. One group was pretreated with the spin-trapping agent, alpha-phenyl-N-t-butylnitrone (PBN), given intraperitoneally 1 hr before ALM on GD 11. RESULTS: Cardiac ventricular septum defects and vascular malformations were observed after dosing on GD 10-11 and, to a lesser degree, on GD 12-13. Urogenital defects, absence/malposition of the postcaval lung lobe, and attenuated diaphragm were observed mainly on GD 10-11. Skeletal examination showed a high incidence of vertebral abnormalities on thoracic level on GD 10, on lower thoracic to caudal level on GD 11, and sternebral defects were observed all days. On GD 13 brachy-, oligo-, and syndactyly of the forepaw were induced, and of the hindpaw on GD 14. PBN reduced the incidence of both visceral and skeletal defects. CONCLUSIONS: The stage specificity of observed visceral and skeletal defects correlates well with what has been reported in the literature after temporary interruption of oxygen supply during the same stages of development. The protective effect by PBN present further evidence that the teratogenicity of potent I(Kr)-blockers is related to induction of hypoxia- reoxygenation injury due to embryonic cardiac arrhythmia.

Class III antiarrhythmics and phenytoin: teratogenicity due to embryonic cardiac dysrhythmia and reoxygenation damage.

Class III antiarrhythmic drugs, like almokalant, dofetilide and ibutilide, cause a spectrum of malformations in experimental teratology studies. The pattern of developmental toxic effects is very similar to those reported for phenytoin, which is an established human and animal teratogen. The toxic effects are characterised by embryonic death, decreased fetal weights, and stage specific malformations, such as distal digital reductions, orofacial clefts and cardiovascular defects. Class III antiarrhythmics decrease the excitability of cardiac cells by selectively blocking the rapid component of the delayed rectified potassium channel (IKr), resulting in prolongation of the repolarisation phase of the action potential. Phenytoin, which decrease the excitability of neurones, has recently also been shown to block IKr, in addition to its known blockade of sodium channels. Animal studies indicate that IKr is expressed in the embryo and that the embryonic heart is extremely susceptible to IKr-blockers during a restricted period in early development. At concentrations not affecting the maternal heart, the embryonic heart reacts with bradycardia, arrhythmia and cardiac arrest when exposed to such drugs. Available studies strongly support the idea that birth defects after in utero exposure to both selective and non-selective IKr-blockers (like phenytoin) are initiated by concentration dependent embryonic bradycardia/arrhythmia resulting in 1) hypoxia; explaining embryonic death and growth retardation, 2) episodes of severe hypoxia, followed by generation of reactive oxygen species within the embryo during reoxygenation, causing orofacial clefts and distal digital reductions, and 3) alterations in embryonic blood flow and blood pressure, inducing cardiovascular defects.

Phenytoin-induced cleft palate: evidence for embryonic cardiac bradyarrhythmia due to inhibition of delayed rectifier K+ channels resulting in hypoxia-reoxygenation damage.

BACKGROUND: Phenytoin (PHT) teratogenicity has been related to embryonic arrhythmia due to the capacity of PHT to block I(K) channels pharmacologically, resulting in hypoxia-reoxygenation damage. The aim of this study was to further elucidate the proposed mechanism. METHODS: Pregnant CD-1 mice were given PHT (85 mg/kg) or saline intraperitoneally on gestational days 10-11. Embryonic heart rhythm and presence of hemorrhage in orofacial region was recorded on day 12, fetuses were examined for malformations on day 18. Embryonic heart rate was also recorded on individual days after dosing days 9-16. In addition, PHT was given at doses of 10, 25, or 85 mg/kg on day 12 for analysis of plasma concentrations. RESULTS: PTH-induced bradycardia and arrhythmia in approximately 20% of the embryos, 48% showed hemorrhage in the orofacial region; 39% of the fetuses had cleft palate. The region in which hemorrhages were visible in the embryo corresponded with the region where tissue deficiency (cleft palate) was visible in the fetus at term. None of the controls showed hemorrhages, dysrhythmia, or cleft palate. PHT affected embryonic heart rates on days 9-13, but not on days 14-16. Single dose administration on day 12, the most sensitive day, resulted in a dose-dependent decrease in embryonic heart rate (12-34%). Embryonic arrhythmia occurred at 25 and 85, but not at 10 mg/kg or in the controls. Mean maternal free plasma concentrations were 6 and 14 micromol/L in the 10- and 25-mg/kg groups, respectively. CONCLUSIONS: PHT-induced cleft palate was preceded by embryonic dysrhythmia and hemorrhage in the orofacial region. Embryonic heart rhythm was phase specifically affected, as described for selective I(Kr) channel blockers, at clinically relevant concentrations. The results support the idea that PHT teratogenicity is a consequence of pharmacologically induced dysrhythmia and hypoxia-related damage.

Developmental toxicity in the pregnant rabbit by the class III antiarrhythmic drug sotalol.

The purpose of this study was to investigate the potential of sotalol to cause developmental toxicity in the pregnant rabbit. Sotalol is a beta-adrenoceptor blocking drug which also has class III antiarrhythmic properties via Ikr channel blockade. EXPERIMENT 1: Nine pregnant New Zealand White rabbits were given doses of either 300, 225, or 150 mg/kg of sotalol during gestational days, called Days, 13-16 which resulted in total litter loss. EXPERIMENT 2: A single dose of sotalol, 100 or 150 mg/kg was administered during Days 8-17 to 15 rabbits. Dosing on Day 8, 9, or 10 resulted in a slightly higher incidence of embryonic death compared to historical controls. There was marked increased embryonic death of 55-90% (four does with total litter loss), decreased number of live foetuses per litter, and elevated mean foetal weight after dosing during Days 12-16. EXPERIMENT 3: 16 pregnant rabbits were administered single doses of sotalol of either 100, 85, 75, 60 or 50 mg/kg on Day 14. The main finding was increased embryonic death, which ranged from total litter loss to approximately 30% at 50 mg/kg. At 50 mg/kg, the maternal Cmax, AUC(1-24 hr), and t1/2 were approximately 45 microM, 340 micromol x hr/l, and 6 hr, respectively. In conclusion, sotalol treatment resulted in embryonic death in the rabbit in early pregnancy in the same way as has been seen for other drugs with Ikr blocking properties (class III antiarrhythmics) in rodents. The observed developmental toxicity in the rabbit is most likely secondary to embryonic arrhythmia as has been shown in rodent studies. The results may indicate that Ikr blocking agents are developmental toxicants across species including man.

Developmental toxicity of the class III antiarrhythmic agent almokalant in mice. Adverse effects mediated via induction of embryonic heart rhythm abnormalities.

Almokalant (ALM, CAS 123955-10-2), a class III antiarrhythmic drug, has been shown to be embryotoxic in rats. In the absence of human pregnancy outcome data, the human relevance of these findings in rats is unknown, and results from other species would indicate if these findings are of more universal interest. Therefore, this study was initiated to evaluate the potential effects in mice. ALM was given to three groups of pregnant mice (approximately 20 mice/group) during gestation days 6-15 at dose levels of 50, 125 and 300 mumol/kg. A fourth group served as a control. In addition, whole embryo culture was performed on gestation day 10 with doses of ALM ranging from 325-5200 nmol/l (approximately 17 embryos/group) in order to study if ALM had the potential to induce dysrhythmia in the embryonic mouse heart. ALM induced total embryonic death in the high dose group, and in the intermediate group the level of embryonic death was elevated and the mean foetal weights decreased. A slight increase in minor skeletal defects was observed, mainly consisting of reduced calcification of elements in the vertebral column and among the phalanges. ALM caused bradycardia in a concentration dependent manner (13-42% at 650-5200 nmol/l). Irregular heart rhythm and/or episodes of cardiac arrest were observed in one embryo at 2600 and in seven embryos at 5200 nmol/l. In conclusion, ALM caused embryotoxicity in the mouse, most likely secondary to adverse effects on the embryonic heart. The results may suggest that class III antiarrhythmics are embryotoxic also in humans.

Teratogenicity of the class III antiarrhythmic drug almokalant. Role of hypoxia and reactive oxygen species.

The class III antiarrhythmic drug almokalant (ALM) was given to pregnant rats on Gestation Day 11 (125 micromol/kg) or 13 (25 micromol/kg). Other groups were pretreated with alpha-phenyl-N-t-butylnitrone, (PBN; 850 micromol/kg intraperitoneally) 1 h before administration of ALM or given (-)-2-oxo-4-thiazolidine carboxylic acid (OTC; 250 micromol/kg subcutaneously) 4 h before administration of ALM. PBN is a spin-trapping agent that can capture reactive oxygen species (ROS), and OTC is an antioxidant. Controls received tap water only. All groups (eight in total) consisted of 7 to 10 pregnant rats. ALM induced cardiovascular defects, orofacial clefts, and tail defects after administration on Day 11, and reduced the size of digits on Day 13. Pretreatment with PBN prevented induction of all the above-mentioned malformations by ALM. The results also indicated that OTC may have some protective effect against ALM-induced teratogenicity but not to the same extent as PBN. The results support the hypothesis that almokalant induces malformations via induction of episodes of embryonic arrhythmia/cardiac arrest, which result in hypoxia followed by reoxygenation and generation of ROS.

Pharmacologically induced embryonic dysrhythmia and episodes of hypoxia followed by reoxygenation: a common teratogenic mechanism for antiepileptic drugs?

Antiepileptic drugs (AEDs), such as phenytoin (PHT), carbamazepine (CBZ), trimethadione (TMD), and phenobarbital (PB), have all been associated with a similar pattern of malformations, as well as growth retardation and developmental delay. Valproic acid (VPA) has been associated with a different pattern of malformations. Recent studies suggest that PHT's fetal adverse effect is related to its membrane stabilizing pharmacological properties (blockage of voltage-dependent ion channels). During a restricted sensitive period, this results in induction of concentration-dependent bradyarrhythmia in the embryo and episodes of hypoxia/reoxygenation. The aim of this study was to compare the potential of PHT, CBZ, PB, TMD, and dimethadione (DMD; the active metabolite of TMD) to cause bradyarrhythmias. All of these AEDs exert mainly their pharmacological effect via blockage of ion channels. VPA and vigabatrin (VGB), which are pharmacologically active mainly by other mechanisms, were also tested. C57 Bl/6J mouse embryos were cultured in vitro on gestation day 10 in vitro (in 20% rat serum). The drugs were suspended in either water or dimethylsulfoxide and administered into the culture medium in increasing concentrations up to 20 times the human therapeutic plasma concentration. A scoring system was employed in order to rank the drugs based on their potential to cause bradycardia, ventricular arrhythmia, and cardiac arrest in relation to human therapeutic concentrations. Based on this system, the drugs were ranked as follows: DMD = PHT >> PB = CBZ > TMD = VPA >> VGB (no potential). The results correlate well with the available clinical/experimental data of the tested AED's potential to induce hypoxia-related fetal adverse effects, such as oral clefts, distal limb defects, growth retardation, and developmental delay. The results support the idea that adverse fetal effects after in utero exposure to PHT, PB, CBZ, and TMD (via the active metabolite DMD) are initiated via a common pharmacological mechanism: blockage of ion channels in the developing heart in the early embryo resulting in bradyarrhythmias, hemodynamic alterations, and hypoxia/reoxygenation damage.

Initiation of phenytoin teratogenesis: pharmacologically induced embryonic bradycardia and arrhythmia resulting in hypoxia and possible free radical damage at reoxygenation.

The aim of this study was to investigate if phenytoin has the capacity to induce embryonic hypoxia mediated via adverse effects on the embryonic heart. Mouse embryos of different strains (CD-1, C57B1/6J and A/J) as well as Sprague Dawley (SD) rat embryos were cultured in vitro (in 75-80% rat serum) by the whole embryo technique. Effects on the heart were examined on gestational day 10 for mouse embryos and days 11 and 13 for rat embryos. Phenytoin was dissolved in water to give concentrations of 50-800 microM. In the mouse embryo studies, phenytoin caused a concentration-dependent decrease in embryonic heart rate in all three strains, with a slight decrease at 100 microM (2-7%) and a more pronounced effect at 200 microM (approximately 20%). Temporary or permanent cardiac arrest occurred in 86% of the CD-1 embryos at 500 microM, in 67% of the C57B1/6JM at 400 microM, and in all A/J embryos at 300 microM. Arrhythmias was observed in 8% in CD-1 embryos at 200 microM, in 18% at 150 microM in C57B1/6J embryos, and in 67% of the A/J embryos at 100 microM (lowest tested concentrations where arrhythmias occurred). In rat embryos, a concentration-dependent decrease in heart rate was observed on both days 11 and 13 at similar concentrations as in the mouse embryo studies. In a separate experiment, the effects on the heart rate of free phenytoin (not serum protein bound) were examined in rat embryos cultured in serum-free medium. Already at 12 microM a significant decrease in heart rate was observed. Altogether, the results support the hypothesis that phenytoin teratogenicity is initiated by pharmacologically induced embryonic hypoxia. A genetic susceptibility to the adverse effects of phenytoin on the embryonic heart may be of importance to explain strain and species differences in phenytoin teratogenicity.

[Epilepsy and pregnancy. Balancing between risks to the mother and child]. / Epilepsi och graviditet. En balansgång mellan risker för mor och barn.

Pregnancy may be especially problematic for the epileptic woman, obstetric complications tend to be more frequent, and seizure control and the pharmacokinetics of anticonvulsants may be affected. The risk of seizures is particularly high during labour and delivery-almost 10-fold higher than at other times during pregnancy. As uncontrolled generalised tonic-clonic seizures may be hazardous to both gravida and fetus, the use of anticonvulsants to prevent their occurrence is to be recommended during pregnancy even though all anticonvulsant drugs are potential teratogens. There is a 2- to 3-fold increase in the risk of birth defects in conjunction with fetal exposure to these drugs. Although the mechanisms mediating the teratogenic effects have not been identified, interference with folate metabolism, formation of toxic metabolites and drug-induced fetal hypoxia have been suggested. Despite the incompleteness of our knowledge, some recommendations can be made for the management of pregnant women with epilepsy. Pre-pregnancy counselling is important. Epileptic women contemplating pregnancy need to be informed of the pros and cons, and any major change in anticonvulsant therapy should be made before conception. Monotherapy is preferable, using the drug appropriate to seizure type and epilepsy syndrome at the lowest dosage and serum level that protects against tonic-clonic seizure. The clinical situation needs to be assessed and drug levels need to be monitored more frequently during pregnancy than otherwise. Patients on anticonvulsant treatment during pregnancy also need to be informed of the possibility of antenatal diagnosis. The use of new anticonvulsant drugs during pregnancy represents a particular challenge, since available clinical data may be insufficient to indicate their teratogenic potential Such a drug should be used in pregnancy only if essential to obtain seizure control. Moreover, the outcome of all such pregnancies needs to be carefully documented.

Toxicity of bupivacaine and ropivacaine in relation to free plasma concentrations in pregnant rats: a comparative study.

The relationship between free drug concentration and toxicity of bupivacaine and ropivacaine, a new local anaesthetic agent, was studied in a pregnant rat model. The compounds were given subcutaneously to rats in late pregnancy. Dose levels (bupivacaine 5.5 to 24 mg/kg and ropivacaine 5.3 to 26 mg/kg) were selected based upon the proposed human dosage and the known pharmacological activity of the compounds. Chewing, spasm, dyspnoea, drowsiness, salivation and convulsions were observed in a dose-dependent manner in the animals given 14 to 24 mg/kg of bupivacaine, while only a few animals receiving 26 mg/kg of ropivacaine showed less severe symptoms. Deaths from clonic convulsions were occasionally seen in animals receiving 14 mg/kg or more of bupivacaine. No animals receiving ropivacaine died. No effects on litter size offspring loss or weight of the offspring at birth were observed with one exception. After 24 mg/kg of bupivacaine an increased postnatal loss of the offsprings were noticed, most likely due to impaired maternal care. Protein binding, at expected Cmax, were significantly lower for ropivacaine (around 49%) compared with bupivacaine (around 69%) at dose levels. The results suggest an increased safety margin before onset of toxic side effects after treatment with ropivacaine, compared to bupivacaine, in pregnant rase.

Teratogenic potential of almokalant, dofetilide, and d-sotalol: drugs with potassium channel blocking activity.

Drugs with class III antiarrhythmic activity are potential human teratogens because of their ability to cause bradycardia in the embryo during the organogenic period. Three drugs with class III antiarrhythmic activity, almokalant, dofetilide and d-sotalol, were compared in vitro using rat embryo culture. Each of these drugs caused a concentration-dependent bradycardia in 11- or 13-day rat embryos. For each drug the effective concentration was considerably greater than the human therapeutic plasma concentration. The reproductive outcome was also compared in vivo in Sprague-Dawley rats by oral administration of almokalant or dofetilide on single days during the organogenic period. Both drugs caused increased resorptions and the same stage-dependent malformations. Dosing on gestational day (GD) 11 was associated with right-sided oblique cleft lip and short tail, while dosing on day 13 caused digital hypoplasia and/or amputation. Susceptibility to these drugs started on GD 9 when the embryonic heart starts beating and ended on GD 15. The malformations were preceded by hemorrhage; which is consistent with the proposed pathogenesis that the drug-induced bradycardia caused embryonic hypoxia/ischemia. This study indicates that the induction of malformations/embryonic death by class III antiarrhythmic drugs which inhibit Ikr is a class effect secondary to a common pharmacological action on the embryonic heart.

Prenatal coexposure to metallic mercury vapour and methylmercury produce interactive behavioural changes in adult rats.

Pregnant rats were 1) administered methyl mercury (MeHg) by gavage, 2 mg/kg/day during days 6-9 of gestation, 2) exposed by inhalation to metallic mercury (Hg degrees) vapour (1.8 mg/m3 air for 1.5 h per day) during gestation days 14-19, 3) exposed to both MeHg by gavage and Hg degrees vapour by inhalation (MeHg + Hg degrees), or 4) were given combined vehicle administration for each of the two treatments (control). The inhalation regimen corresponded to an approximate dose of 0.1 mg Hg degrees/kg/day. Clinical observations and developmental markers up to weaning showed no differences between any of the groups. Testing of behavioural function was performed between 4 and 5 months of age and included spontaneous motor activity, spatial learning in a circular bath, and instrumental maze learning for food reward. Offspring of dams exposed to Hg degrees showed hyperactivity in the motor activity test chambers over all three parameters: locomotion, rearing and total activity; this effect was potentiated in the animals of the MeHg + Hg degrees group. In the swim maze test, the MeHg + Hg degrees and Hg degrees groups evidenced longer latencies to reach a submerged platform, which they had learned to mount the day before, compared to either the control or MeHg groups. In the modified, enclosed radial arm maze, both the MeHg + Hg degrees and Hg degrees groups showed more ambulations and rearings in the activity test prior to the learning test. During the learning trial, the same groups (i.e., MeHg + Hg degrees and Hg degrees) showed longer latencies and made more errors in acquiring all eight pellets. Generally, the results indicate that prenatal exposure to Hg degrees causes alterations to both spontaneous and learned behaviours, suggesting some deficit in adaptive functions. Coexposure to MeHg, which by itself did not alter these functions at the dose given in this study, served to significantly aggravate the changes.

Drugs during pregnancy: an issue of risk classification and information to prescribers.

The Swedish system for the classification of fetal risk of drugs was the first of its kind and was implemented in 1978. Drugs for use in pregnant women are classified in 4 general categories--A to D. The US Food and Drug Administration (FDA) introduced a system in 1979 also using the letters A to D, together with an X category. However, the definitions differ considerably between the FDA system and the Swedish system, resulting in a very different allocation of drugs to the respective categories. In the Swedish system, category A includes drugs that have been extensively used and/or for which there are reliable clinical data indicating no evidence of disturbance of the reproductive process. Category B includes drugs for which data from pregnant women are insufficient for making any solid estimation of human teratogenic risk, and classification is therefore based on animal data, with allocation to 3 subgroups. For products in category C, the pharmacological action of the drug may have undesirable effects on the human fetus or newborn infant. Finally, category D contains drugs for which human data indicate an increased incidence of malformations. The categorisation statement is always followed by a short explanatory text. In contrast to the FDA system, the Swedish system has been well accepted, as judged by an interview study including 934 physicians and pharmacists. We believe that much of the American dissatisfaction may be a consequence of shortcomings in the category definitions of the FDA system. The FDA system requires an unrealistically high quality of data, e.g. the availability of controlled studies in pregnant women that fail to demonstrate a risk to the fetus are needed for a drug to be assigned to category A. Consequently, the majority of drugs on the US market are allocated to category C, interpreted as 'risk cannot be ruled out'. The distribution of drugs into the various categories is thus very different between the Swedish and FDA systems. We think that the issue of this debate reflects a fundamental problem related to public health information: how should a large, compounded, changing and difficult to evaluate databank be organised before it is made available to professionals and secondarily to lay people?

Phenytoin causes phalangeal hypoplasia in the rabbit fetus at clinically relevant free plasma concentrations.

New Zealand White rabbits were treated orally with 0 (controls), 50, 100, or 150 mg/kg phenytoin on days 14-16 of pregnancy. Total and free plasma concentrations of phenytoin were determined in maternal plasma 2, 6, and 24 hr after the final dose in all animals. In addition, after administration of 150 mg/kg maternal plasma concentrations were also determined after 12 and 48 hr, the concentrations in amniotic fluid after 6 hr, and those in fetal tissue 6 and 24 hr after the final treatment. A high degree of plasma protein binding was observed in maternal blood. Treatment with 50 mg/kg resulted in free plasma concentrations of up to 5.0 mumol/l during the 24 hr period following the final dose. Significantly higher free plasma concentrations were observed at the two higher dose levels; up to 9.7 mumol/l at 100 mg/kg and 12.7 mumol/l at 150 mg/kg. Digital hypoplasia was not seen in the control group or the animals treated with 50 mg/kg. However, treatment with 100 mg/kg resulted in hypoplasia in a single or a few digits in approximately 50% of the fetuses, and 150 mg/kg provoked hypoplasia in almost all digits in all fetuses. These results show that even though the doses which caused digital defects in rabbits are much higher than those used therapeutically, the resulting free concentrations of phenytoin are similar to those which are associated with the same type of defects in humans. These data indicate that the pharmacologically induced fetal hypoxia/ischemia and vascular disruption preceding malformations of this type, which were observed in a previous study in rabbits, may be of human relevance.

Behavioural effects of prenatal metallic mercury inhalation exposure in rats.

The effects of administration by inhalation of metallic mercury vapour (Hg0) to pregnant rats, approximately corresponding to doses of 0.2 mg Hg0/kg/day (high dose) or 0.07 mg Hg0/kg/day (low dose), on the developmental and behavioural repertoire of the offspring were studied. Exposure occurred during days 11-14 plus 17-20 of gestation. The dose levels were selected so as not to induce maternal toxicity. Maturation variables such as surface righting, negative geotaxis, pinna unfolding, and tooth eruption revealed no differences between Hg0-treated offspring and controls. Tests of spontaneous motor activity showed that the Hg0-treated offspring were hypoactive at 3 months of age but hyperactive at 14 months. In spatial learning tasks the prenatally exposed offspring showed retarded acquisition in the radial arm maze but no differences in circular swim maze. A simple test of learning, habituation to a novel environment (activity chambers), indicated a reduced ability to adapt. These data suggest that prenatal exposure to Hg0 vapour results in similar behaviour changes in the offspring as reported for methylmercury.

Reproductive toxicity studies of the antihypertensive agent felodipine in the rat.

Felodipine (4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridine dicarboxylic acid 5-ethyl 3-methyl ester, CAS 72509-76-3), a vascular selective dihydropyridine calcium antagonist, was tested in three different reproduction studies in the rat. In fertility, teratology and peri-postnatal studies Sprague-Dawley rats were dosed by gavage with 10, 25 and 70 mumol/kg (fertility and teratology studies) or 3, 10 and 30 mumol/kg (peri-postnatal study). There were no increased incidences of external, visceral or skeletal malformations in the teratology study in groups treated with felodipine during organogenesis, compared with the control group. In the fertility and peri-postnatal studies, prolonged parturition and increased incidences of stillborn fetuses and postnatal death were observed in groups given 10 mumol/kg or more, but not at 3 mumol/kg. Similar to the findings in this study, nifedipine, nitrendipine, nicardipine, diltiazem and isradipine have all been reported to induce increased incidences of perinatal death in offsprings in peri-postnatal studies. Thus, the increased incidences of perinatal death (most likely secondary to inhibition of the uterine activity) seems to be a class effect common to all calcium antagonists.

Altered behaviour in adult mice orally exposed to tri- and tetrachloroethylene as neonates.

Male NMRI mice were orally exposed to trichloroethylene (TRI, 50 and 290 mg/kg per day) and tetrachloroethylene (PER, 5 and 320 mg/kg per day) between days 10 and 16 postnatally. Spontaneous activity, observed as locomotion, rearing and total activity, was measured over three 20-min periods at an age of 17 and 60 days. Compared to controls, mice at 17 days of age were unaffected, while at 60 days of age mice exposed to PER showed changes in all three spontaneous motor activity variables, while TRI-exposed mice only were affected in rearing. This indicates a neonatal susceptibility of brain maturation to these chlorinated organic solvents in achieving long-lasting changes in adult behaviour.

Histopathological and hemodynamic studies supporting hypoxia and vascular disruption as explanation to phenytoin teratogenicity.

The limb plates and craniofacial regions in rabbit fetuses were examined shortly after the last dose of phenytoin on day 16 after daily administration by gavage with either 150 mg/kg on days 14-16 or 300 mg/kg on days 15-16. Both treatment regimens resulted in similar changes. Histologically, the digital areas of the limb plates showed extensive edema and dilated blood vessels within 2 h. After 8 h, vascular disruption occurred with hemorrhages. At 24-48 h after dosing, mesenchymal necrosis and, on some occasions, amputation of digits was observed. In the craniofacial region, well-defined superficial hemorrhage was seen in the frontal and nasal region at 8 h. Histologically, subectodermal hemorrhage caused by vascular disruption and microfocal mesenchymal necrosis was observed. At 48 h, some fetuses showed severe diffuse intracranial and superficial hemorrhage, resulting in massive tissue damage, also in the central nervous system (CNS). Maternal heart rate, blood pressure, PO2, and PCO2 were also measured in awake pregnant rabbits 6 h after the last dose on day 16 after daily administration with 150 mg/kg during gestational days 14-16. An attempt was also made to measure fetal heart rate in anesthetized rabbits. The maternal heart rate and blood pressure decreased with about 15% in phenytoin-treated animals, resulting in a decrease in PO2 (approximately 15%) and an increase in PCO2 (approximately 15%). A decrease in fetal heart rate was also registered. The results thus indicate that phenytoin exerts its teratogenic effects by inducing fetal hypoxia, leading to vascular disrupture and necrosis of existing and developing structures.