Hans Hummler

Periconceptional vitamin a use: How much is teratogenic?

The objective of the review is to determine whether preformed vitamin A (retinol and retinyl esters) is teratogenic at dosages commonly used by women living in industrialized countries. Published human and animal data and research developed by the authors are reviewed. It is well known that vitamin A is essential for normal reproduction and development. Although doses of 10,000 IU/d or less of preformed vitamin A (retinyl esters and retinol) are considered safe, doses > 10,000 IU/d as supplements have been reported to cause malformations in a single epidemiologic study. Nonhuman primate data show no teratogenicity at doses of 30,000 IU/d. Daily periconceptional exposures greater than 25,000 IU/d of preformed vitamin A have not been sufficiently studied to establish specific risk. Because no study reports adverse effects of 10,000 IU/d preformed vitamin A supplements and this dose is more than the Recommended Dietary Allowance for pregnant women (2670 IU or 800 RE/d), we recommend that women living in industrialized countries or who otherwise have nutritionally adequate diets may not need to ingest more than the Recommended Dietary Allowance of preformed vitamin A as supplements. If periconceptional vitamin A exposures to levels up to 30,000 IU/d (9,000 micrograms RE/d) do occur unintentionally, multiple animal studies do support only very low risk. Human epidemiologic studies do not establish at what level vitamin A becomes teratogenic; however, pharmacokinetic data presented in this paper indicate that blood levels of retinoids from women taking 30,000 IU/d of preformed vitamin A are not greater than retinoid blood levels in pregnant women during the first trimester who delivered healthy babies. Interestingly, neither teratogenicity nor vitamin A toxicity has been observed in multiple species exposed to high doses of beta-carotene.

Teratogenesis and reproductive safety evaluation of the retinoid etretin (Ro 10-1670).

Reproduction and teratology studies were performed with etretin, the free acid analog of the retinoid etretinate. The lowest teratogenic dose of etretin in mice, rats and rabbits was 3, 15 and 0.6 mg/kg, respectively. In all three species, the lowest dose which was embryolethal, fetolethal or reduced the survivability of the pups during lactation was 2–3 times higher than the above doses. In rats, the lowest effective dose of etretin was 3 mg/kg in both the study for fertility and general reproductive performance and the peri- and postnatal study. The main adverse effect in these two experiments was a reduced survival of the F1-generation.

The high sensitivity of the rabbit to the teratogenic effects of 13-cis-retinoic acid (isotretinoin) is a consequence of prolonged exposure of the embryo to 13-cis-retinoic acid and 13-cis-4-oxo-retinoic acid, and not of isomerization to all-trans-retinoic acid

Previous studies suggested that the rabbit is much more susceptible to the teratogenic action of 13-cis-retinoic acid (13-cis-RA) than the mouse or the rat, while the teratogenicity of all-trans-RA was comparable in these species. In the present study we investigated if pharmacokinetics can explain these species- and structure-related differences. The embryotoxic and teratogenic potential of all-trans-retinoid acid (all-trans-RA) and 13-cis-RA were evaluated in the Swiss hare rabbit after oral administration of daily doses of the two drugs throughout organogenesis, from gestation day (GD) 6 to 18 (plug day=GD 0). All-trans-RA was given at dose levels of 0.7, 2 or 6 mg/kg body weight per day and 13-cis-RA at 3, 7.5 or 10 mg/kg per day. The doses needed to elicit a minimum teratogenic response were found to be 6 mg/kg per day for all-trans-RA and 10 mg/kg per day for 13-cis-RA. Using these doses, transplacental pharmacokinetics of all-trans- and 13-cis-RA were performed. Pregnant rabbits were treated once daily from GD 7 to 12 and plasma and embryo samples were collected for HPLC analysis at various time intervals after the final dose. The main plasma metabolites of all-trans-and 13-cis-RA were all-trans-β-glucuronide (all-trans-RAG) and 13-cis-4-oxo-RA, respectively. The elimination of 13-cis-RA and its metabolites from maternal plasma were much slower than of all-trans-RA resulting in accumulation of the 13-cis-isomers in plasma. Marked differences in the placental transfer of the two drugs and their metabolites were observed. All-trans-RA and all-trans-4-oxo-RA were efficiently transferred to the rabbit embryo, reaching concentrations similar to the plasma levels. On the contrary, the 13-cis-isomers reached the embryo to a lesser extent. Despite its limited placental transfer, a considerable embryonic exposure to 13-cis-RA and 13-cis-4-oxo-RA was noticed after treatment with isotretinoin, as indicated by their area-under-the-concentration-time-curve (AUC) values in the embryo, which were in the same range as the corresponding AUC value of all-trans-RA after treatment with the all-trans-isomer. Our results suggest that the high sensitivity of the rabbit to the teratogenic effects of 13-cis-RA can be attributed mainly to the 13-cis-isomers and not to isomerization to all-trans-RA. The significant exposure of the rabbit embryo to 13-cis-RA and its 4-oxo metabolite is a result of their very slow excretion rates from the maternal organism. Furthermore, this study supports the view that embryonic AUC values should be considered as the most suitable pharmacokinetic correlate to retinoid induced teratogenesis.

Pathogenesis of retinoic acid-induced ear malformations in a primate model

13-cis retinoic acid (RA) is a causative agent for human/monkey retinoic acid embryopathy (RAE), in which the most common type of malformation is microtia or anotia. In the present study, malformed ears of monkey fetuses exposed to RA during early embryogenesis were analyzed and revealed a subtype of defects., i.e., apparent duplication of the external/middle ear. A part of the posterior auricle appeared to be ectopically formed in the anterior auricular region or in the region posterior to the auricle. Additionally, there was duplication of the zygomatic arch, malleus, and incus. In order to characterize possible pathogenetic events underlying these malformations, embryos at selected stages were collected after dosing dams with RA at 5 mg/kg/day during gestational days 12-27. Cellular retinoic acid binding protein I whole-mount immunostaining showed that RA induced specific alterations in the migration of cranial neural crest cells (NCC). NCC en route to the second pharyngeal arch were bifurcated, and some of these NCC migrated abnormally into the first and/or third arches, which may underlie external ear duplication. Scanning electron microscopy and neurofilament immunostaining provided evidence that there was partial duplication of trigeminal nerve/ganglion following RA insult. The duplication of NCC neuronal derivatives in the first pharyngeal arch is consistent with duplication of NCC mesenchymal components (zygomatic arch, malleus, and incus). Therefore, RA-induced alterations in cranial NCC migration patterns are likely to be a pathogenetic event underlying ear malformations (including duplication) of RAE in monkeys.

Dietary vitamin A and teratogenic risk: European Teratology Society discussion paper

This review discusses the predictive value of animal models in assessment of possible risk from excess vitamin A consumption during pregnancy and the human evidence concerning risk of congenital malformations from excess vitamin A in the diet, consumed either as a constituent of normal foods or in the form of dietary vitamin supplements. Other sources of exposure to vitamin A include medicines (dermal and oral preparations) and cosmetics, but these are not further considered here. Conservative estimates of the likely safe intake of vitamin A during pregnancy are available, based on results from scientific research to date. However, current uncertainties are such that further research is needed to more clearly define intakes which may be on the borderline between those which are beneficial and those which may pose a risk to the developing embryo and fetus.

Cardiovascular alterations in rat fetuses exposed to calcium channel blockers

Preclinical toxicologic investigation suggested that a new calcium channel blocker, Ro 40-5967, induced cardiovascular alterations in rat fetuses exposed to this agent during organogenesis. The present study was designed to investigate the hypothesis that calcium channel blockers in general induce cardiovascular malformations indicating a pharmacologic class effect. We studied three calcium channel blockers of different structure, nifedipine, diltiazem, and verapamil, along with the new agent. Pregnant rats were administered one of these calcium channel blockers during the period of cardiac morphogenesis and the offspring examined on day 20 of gestation for cardiovascular malformations. A low incidence of cardiovascular malformations was observed after exposure to each of the four calcium channel blockers, but this incidence was statistically significant only for verapamil and nifedipine. All four agents were associated with aortic arch branching variants, although significantly increased only for Ro 40-5967 and verapamil.

Vitamin A teratogenicity and risk assessment in the macaque retinoid model.

Studies were performed in the cynomolgus monkey (Macaca fascicularis) to provide risk assessment information on safe dose levels of Vitamin A during human pregnancy. Vitamin A palmitate was orally administered at 7500 IU/kg (2.25 mg/kg) to 80 000 IU/kg (24 mg/kg) body weight during early pregnancy (gestation day [GD] 16-27). The results indicated a dose-related increase in exposure (AUC) to retinyl esters and retinoic acids (RA) (all-trans-RA, all-trans-4-oxo-RA, 13-cis-RA, 13-cis-4-oxo-RA). There was also a dose-related increase in abortion and malformation that affected typical retinoid target tissues in the embryo, including the craniofacial region, heart, and thymus. The NOAEL and LOAEL for structural malformations were 7500 IU/kg and 20 000 IU/kg (6 mg/kg), respectively. A companion study involving oral administration of 13-cis-RA during the same gestational period established the NOAEL for malformations at 0.5 mg/kg/day, which is close to the human therapeutic dose range (0.5 to 1.5 mg/kg/day) associated with retinoid embryopathy. Based on the known similarities in teratogenic susceptibility to 13-cis-RA, the monkey NOAEL for Vitamin A (7500 IU/kg) was used to estimate safe levels of this nutrient in humans applying a safety factor of 10. This approach yielded safe levels of Vitamin A during human pregnancy in the range of approximately 25 000 to 37 000 IU/day.

Retinoid teratogenicity in the macaque: Verification of dosing regimen

Abstract: To further define teratogenicity associated with 13‐cis‐retinoic acid (13‐cis‐RA) in the cynomolgus monkey, the drug was orally administered on three different treatment regimens. Experiment (Exp.) 1 (2.5 mg/kg/day, gestational day [GD] 12–27, n = 11) investigated the teratogenicity of a single daily dose of 13‐cis‐RA administered shortly after embryo implantation. Pharmacokinetic sampling was done to determine retinoid profiles on the first (GD12) and last (GD27) days of treatment. Exposure to 13‐cis‐RA during early organogenesis in Exp. 2 (2.5 mg/kg/day, GD20–27, and 2 × 2.5 mg/kg/day, GD28–30, n = 5) investigated the potential adverse effects of 13‐cis‐RA on the developing limb. The use of multiple doses of 13‐cis‐RA in Exp. 3 (2 × 2.5 mg/kg/day, GD26–27, n = 5) investigated the necessity of double dosing on the induction of retinoid embryopathy in the macaque. Malformations of retinoid target organs as well as embryolethality were most prevalent when single daily doses of 13‐cis‐RA were administered during pre‐ and early organogenesis in Exp. 1. Moreover, multiple doses on GD26–27 failed to induce any manifestation of abnormal development in Exp. 3. These results confirm that the lowest observed adverse effect level (LOAEL) in macaques is 2.5 rather than 5.0 times greater than that observed in human pregnancies. Exposure during forelimb development (GD20–30) in Exp. 2 was unsuccessful in inducing defects of this skeletal region, although defects in several retinoid target organs (i.e., cerebellum and internal ear) were present, indicating that a teratogenic threshold was achieved. Pharmacokinetic analysis of 13‐cis‐RA and its metabolites on GD12 and 27 in Exp. 1 showed considerable exposure to the administered drug and its 4‐oxo‐metabolite. In contrast, the exposure to all‐trans‐RA was negligible. The results support the use of a specific treatment schedule in early gestation in the macaque as the most appropriate model for characterizing the teratogenic potential of retinoids in humans.

Retinoid metabolism and transplacental pharmacokinetics in the cynomolgus monkey following a nonteratogenic dosing regimen with all-trans-retinoic acid

Retinoids often exhibit a complex metabolic pattern and differential transplacental kinetics, which make it difficult to pinpoint the proximate compound responsible for the observed teratogenic effect. We have therefore studied the pharmacokinetics and metabolism of all-trans-retinoic acid (all-trans-RA) in cynomolgus monkeys following application of a nonteratogenic dosing regimen and compared the results with corresponding data from a previous study with a teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. All-trans-RA was administered to pregnant cynomolgus monkeys (Macaca fascicularis) by nasogastric intubation at a dose of 5 mg/kg body wt once daily from gestational day (GD) 16 to 26 and twice daily at 8-h intervals from GD 27 to 31. Examination of the fetuses of four dams on GD 100 +/- 2 showed no embryotoxic or teratogenic effects of the applied dosing regimen (Experiment 1). Maternal plasma retinoid pharmacokinetics on GD 16, 26, and 31 as well as embryonic retinoid profiles after the last drug administration on GD 31 were determined in thirteen further dams (Experiment 2). All-trans-RA reached much lower plasma concentrations after the last two treatments on GD 31 than after the first one on GD 16 and the eleventh one on GD 26 (0-24-h area-under-the-concentration-time-curve (AUC) values: 104 +/- 59 ng x h/ml (after the last treatment on GD 31), 189 +/- 110 (GD 16) and 393 +/- 305 ng x h/ml (GD 26). The predominant plasma metabolites of all-trans-RA were its beta-glucuronide and the beta-glucuronide of all-trans-4-oxo-RA. Both of these retinoids accumulated in the plasma during the period of treatment and displayed AUC values 5- to 30-fold higher than those of all-trans-RA. Embryonic concentrations of all-trans-RA were not increased over endogenous levels after the last administration on GD 31 when plasma concentrations were low. To evaluate the placental transport of all-trans-RA in the presence of high plasma concentrations, a further experiment was performed, in which a single dose of all-trans-RA (10 mg/kg body wt) was given to four pregnant monkeys on GD 31, and plasma pharmacokinetics as well as embryonic concentrations of retinoids at 4 h post-treatment were determined (Experiment 3). This dosing schedule yielded high plasma concentrations of all-trans-RA, while embryonic concentrations were about 40% of plasma levels. Based on the plasma AUC values on GDs 16 and 26 obtained in Experiment 2 and the degree of placental transfer, as determined on GD 31 in the presence of high plasma levels in Experiment 3, we estimated embryonic AUC values for the 24-h period following the nonteratogenic doses on GDs 16 and 26 in Experiment 2. These AUC values were similarly high to the embryonic AUC value of all-trans-RA obtained after application of the teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. In addition, plasma AUC values of all-trans-RA were 2- to 7-fold higher after all-trans-RA administration (present study) than after dosing with the teratogenic dose of 13-cis-RA. These results strengthen our recent suggestion that the teratogenic effects induced in cynomolgus monkeys by 13-cis-RA treatment cannot solely result from the action of all-trans-RA, but may involve 13-cis-RA and 13-cis-4-oxo-RA, which could act directly or function as transport vehicle.

A comparative study on the mutagenicity of ethylenethiourea in bacterial and mammalian test systems

Abstract The mutagenic effect of ethylenethiourea (ETU), a degradation product and metabolite of ethylenebisdithiocarbamates, which are widely used as fungicides, was studied in different test systems. ETU induced mutations of the base-pair substitution type in Salmonella typhimurium TA 1530 in vitro as well as in the host-mediated assay. In the host-mediated assay, a dose of 6000 mg/kg (LD50 = 5400 mg/kg) resulted in a slight but significant increase of the reversion frequency by a factor of 2.37. The results of the micronucleus test were negative after two-fold oral applications of 700, 1850 and 6000 mg/kg to Swiss albino mice. Thus it is concluded that ETU hardly induces any chromosomal anomality in the bone marrow. No dominant-lethal effect was observed after single oral doses of 500, 1000 and 3500 mg/kg given to male mice.