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6-Mercaptopurine riboside (6-MPr) is used as a cytostatic chemotherapeutic. The teratogenic potential in rodents has been well known for several decades. In this study, the teratogenic risk of low doses of 6-MPr in NMRI mice was estimated based on a dose-response study. The effective doses corresponding to the incidences of 5%, 1%, and 0.1% were calculated using the probit and Weibull model. The evaluation was performed on the basis of both the fetus and the litter by evaluating the variable all gross structural abnormalities. From these experiments, benchmark doses were obtained which were used in low-dose risk assessment to define a reference dose. Depending on the biometrical model and the statistical unit used, values between 1.9 and 5.2 mg/kg (benchmark ED1) and 3.8 and 6.7 mg/kg (benchmark ED5) were obtained. These values were compared to the no observed adverse effect level (NOAEL) which was determined experimentally. The NOAEL was found to be 5 mg/kg, which is quite similar to the ED5 benchmark doses.

In previous studies the direct-acting alkylating model compounds methylnitrosourea (MNU) and ethylmethanesulfonate (EMS) were investigated with regard to dose-response of teratogenicity as well as DNA adduct formation in mice. In this study the teratogenic effects induced by combined treatment with these substances were analyzed using doses which, following single treatment with either substance, were around the threshold level, i.e., no adverse effect level (NOAEL) and lowest observed adverse effect level (LAOEL). Combined treatment of LAOELs resulted in a threshold-like response, while the combination of the NOAEL of one substance with the LAOEL of the other increased the response rate dramatically to nearly 100%. This phenomenon was further evaluated using biometrical methods. The dose-response surface as well as isobolograms were calculated in order to describe the combination effect. In addition, a dose-response model was fitted to the data. In conclusion, the initially surprising high combination effect revealed to be not so extraordinary when considering the steepness of the dose-response relationships of the single substances.

Prenatal-toxic risk estimation for the alkylating model compound methylnitrosourea (MNU) was performed using different procedures. Risk of low doses was estimated using linear extrapolation to zero (estimated ED0.1%: 0.1 mg/kg body wt MNU) as well as extrapolation by probit analysis based on a dose-response study (estimated ED0.1%: 1.6 mg/kg body wt). Furthermore, a "virtually safe dose" was established by means of the NOAEL risk factor approach (e.g., factor 30:0.03 mg MNU per kg body wt). In previous studies in murine embryos using MNU, we combined dose-response data and DNA adduct rate measurements and deduced that O6-methylguanine is a suitable variable for molecular dosimetry. In a tentative approach, we estimated the teratogenic risk of low doses based on the adduct rates of O6-methylguanine in the DNA of the embryos. It is concluded that in the case of steep dose-response relationships, which are typical for the majority of teratogenic effects, the NOAEL risk factor approach is more conservative than extrapolation based on probit analysis. Risk estimation using dosimetry with this model compound yields estimated incidences similar to linear extrapolation.

Possible effects on the next generation after long-term exposure (subcutaneous administration) of male rats to very high doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. Two dose regimes were applied: TCDD-25 (initial dose: 25 micrograms/kg body wt; maintenance dose: 5 micrograms/kg body wt, once weekly) and TCDD-75 (initial dose: 75 micrograms/kg body wt; maintenance dose: 15 micrograms/kg body wt). Male rats were treated for 10 weeks before mating and then throughout the entire 12 week mating period. They were mated to unexposed virgin females. One group of pregnant females was used for teratological evaluations, and another group was allowed to deliver. No significant differences were observed in the number of implantations or fetuses per litter, and resorption rate, and fetal weight between the controls and TCDD-treated groups. No gross-structural anomalies occurred in any of the fetuses sired by TCDD-treated males. In the TCDD-25 group an increased frequency of two types of variations was observed which also occur in controls: incompletely ossified fingers (TCDD-25 = 5.1%, controls = 2.6%), and incompletely ossified ossa zygomatica (TCDD-25 = 1.8%, controls = 0.5%). In the TCDD-25 group a slight but statistically significant increase was observed in the rate of stillbirths (TCDD-25 = 1.3%, controls = 0.1%), apparently due to an unusually low frequency occurring in the controls (overall historical controls = 0.6%). There was no difference in postnatal mortality (TCDD-25 = 1.3%, controls = 1.3%). Taken together, despite the very high doses of TCDD used, the data do not provide evidence for biologically significant paternally-mediated developmental toxicity in the fetuses and newborn.

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.

The alkylating agent acetoxymethyl-methylnitrosamine (DMN-OAc) triggers preferential left-sided paw defects in mice following IP administration on either day 11 or 12 of pregnancy. Predominantly, ectrodactyly and hypoplasia of the left paws were found. In an organ culture system, using limb buds of 11-day-old mouse embryos, differentiation is severely impaired following addition of 2 microM DMN-OAc to the culture medium. Left and right limbs are equally affected. In contrast, when DMN-OAc is administered in vivo to the dams with subsequent culturing of the limb buds, growth and differentiation of the left limb buds is more affected when compared to the right. Furthermore, DNA alkylation experiments were performed: in vitro, following addition of (14C)-DMN-OAc (2.3 microM) to the culture medium, the DNA alkylation rate of the limb bud DNA is determined. In vivo, following IP administration of 10 mg/kg DMN-OAc to the dams on day 11 of pregnancy, the extent of DNA alkylation of whole-embryo DNA is similar. However, the DNA alkylation rate of separately pooled left and right limb buds exhibits a two-fold difference according to the different teratogenic susceptibility. The results obtained with both in vivo and in vitro systems are consistent with the thesis that a certain amount of DNA alkylation in the tissue of the embryos is the initial step of alkylating agent-induced teratogenicity.

Pregnant mice received 10 or 100 mg retinol/kg body wt. by gavage on day 11 of gestation (plug day = day 0). One group of animals was used for a pharmacokinetic study. At various times after dosing, plasma and tissue samples were collected and analyzed by HPLC for retinyl esters, retinol, 13-cis- and all-trans-retinoic acid and 13-cis-4-oxo and all-trans-4-oxoretinoic acid. In the other group the fetuses were removed on day 18 and examined for malformations. After 10 mg/kg retinol, no teratogenic effect was observed. The pharmacokinetic investigation revealed a moderate increase of retinyl esters, retinol and all-trans-retinoic acid in plasma, embryonic tissue, placenta, yolk sac membranes and extraembryonic fluid. A high incidence of severe fetal malformations occurred after 100 mg/kg retinol. These malformations included limb defects (81% of fetuses) and cleft palate (55% of fetuses) which are characteristically found after administration of a single teratogenic dose of an active retinoid on day 11 of gestation. The concentration-time profile of retinoids after 100 mg/kg on day 11 showed a pronounced increase of retinyl esters and retinol in all compartments including the embryo and a massive generation of the polar metabolites all-trans-retinoic acid and all-trans-4-oxoretinoic acid. These polar metabolites were found in the embryo with peak concentrations of 327 +/- 115 and 143 +/- 20.7 ng/g (mean +/- SE) wet tissue, respectively. It is likely that all-trans-retinoic acid and all-trans-4-oxoretinoic acid, both well-known teratogens, largely contributed to the teratogenic outcome. The in-vivo oxidation of retinol may be an important factor in the teratogenic activity of high doses of vitamin A.