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The interaction of the DNA-alkylating model compounds, ethylmethanesulfonate (EMS) and methylnitrosourea (MNU), was studied in pregnant NMRI mice by measuring DNA adduction in vivo. Previously, large-scale dose-response studies on teratogenicity as well as on DNA modification were performed using these substances. In addition, the risk of low doses in mice was estimated by comparative use of several approaches including molecular dosimetry. The risk was further analysed by combination experiments on teratogenesis with EMS and MNU. This paper describes a further approach with regard to an interaction of these compounds: the formation of DNA adducts was determined using a combined treatment regimen of [(14)C]-labelled MNU and EMS. The mutual influence of EMS and MNU on the DNA alkylation rates was found to be moderate. The dramatic increase in the teratogenic outcome following combined treatment found in previous studies was obviously not due to a massive interaction regarding the initial DNA alkylation rates. It may be explained, however, by the concept of toxic equivalency. Teratogenesis Carcinog. Mutagen. 20:27-34, 2000., (Copyright 2000 Wiley-Liss, Inc.)

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.

Analogously to an earlier study using methylnitrosourea (MNU) the prenatal-toxic risk of low doses of ethylmethanesulfonate (EMS) was estimated using different procedures, comparatively. First, the risk of low doses was estimated using linear extrapolation to zero. When using the variable "all gross structural abnormalities" the lowest effective dose in the experiment was 150 mg/kg body wt (5.6% incidence), the additional risk over background was calculated to be 5.0%, and the hypothetical incidence 0.1% was associated with the dose 3 mg/kg EMS. When evaluating "gross structural limb abnormalities," which are not observed in controls, the dose associated with the hypothetical incidence 0.1% was 17.4 mg/kg EMS. Furthermore, derived from a dose-response study of teratogenicity extrapolation to the possible risk of low doses was performed using nonlinear mathematical models. In this case, the results obtained are dependent on the dose response variable as well as from the statistical approach which was chosen. As an example, the values obtained from one evaluation are given: all gross structural abnormalities, Weibull transformation, jackknife approach: ED0.1% = 72 mg/kg EMS. For comparison a "virtually safe dose" was calculated by use of the no-observed-adverse-effect-level (NOAEL) risk factor approach. The NOAEL under our experimental conditions was 100 mg per kg body wt. By using an arbitrarily chosen risk factor of 100 a "safe dose" of 1 mg EMS per kg body wt was obtained. In addition, molecular dosimetry of the DNA adduct rate of O6-ethylguanine in the 11-day-old embryos was used. Based on the assumption that a linear correlation exists between this specific adduct rate and the incidence of teratogenic effects, the hypothetical incidence of 0.1% was associated with a dose of 99 mg/kg EMS. This value is quite similar to that obtained by extrapolation using probit analysis which is in contrast to the results obtained with MNU.

In previous studies using methylating agents a correlation was found between the initial DNA adduct rate (O6-methylguanine) in the embryo and the teratogenic efficiency. This was shown by measuring DNA adduct rates in the teratogenic dose range which exhibited similar adduct rates at the equivalent teratogenic dose levels. A similar approach was performed using the ethylating agent ethylmethanesulfonate (EMS). In the teratogenic dose range (150-250 mg/kg bw) the adduct rates of O6-ethylguanine were similar compared to those of O6-methylguanine which were obtained with methylating agents. We conclude that a correlation between teratogenicity and adduct rate (O6-alkylguanine) exists for both methylating and ethylating agents. Furthermore, DNA adduct formation following doses at and below the no-observed-adverse-effect-level (NOAEL) of teratogenicity was determined. The lowest experimental dose was 45 mg/kg EMS. Substantial DNA adduct rates in the embryos were found. These data will be used for molecular dosimetry in a risk assessment of low doses.

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.

It has been reported that fractionated doses of 13-cis-retinoic acid are disproportionately more embryotoxic in pregnant mice than is the same dose given in a single bolus. Here, we examined limited pharmacokinetic profiles of a single (100 mg/kg dose given to NMRI mice on day 11 of gestation) versus multiple (3 x 100 mg/kg, 4 h apart) doses in an effort to assess the relative contribution to teratogenicity made by the drug and/or its metabolites. The major plasma metabolite of 13-cis-retinoic acid in the mouse was 13-cis-retinoyl-beta-glucuronide, followed by the 4-oxo metabolites and all-trans-retinoic acid. Transfer to the mouse embryo was very efficient for all-trans-retinoic acid, whereas, it was tenfold less efficient for 13-cis-retinoic acid and 100-fold less efficient for 13-cis-retinoyl-beta-glucuronide. The isomer all-trans-retinoic acid was found in the placenta at concentrations two- to three-fold higher than in the plasma, suggesting placental accumulation as well as placental cis/trans isomerization. Since 13-cis-retinoyl-beta-glucuronide and 13-cis- and all-trans-retinoic acid were detected in the embryo after this multiple dosing schedule, any of the three or their combinations may have been involved in the induction of malformations, but all-trans-retinoic acid, a well-known potent teratogen detected at concentrations of between 590 and 80 ng/g for 10 critical hours during gestation, could have been the major component.