Richard Vogel

Animal testing and alternative approaches for the human health risk assessment under the proposed new European chemicals regulation

During the past 20xa0years the EU legislation for the notification of chemicals has focussed on new chemicals and at the same time failed to cover the evaluation of existing chemicals in Europe. Therefore, in a new EU chemicals policy (REACH, Registration, Evaluation and Authorisation of Chemicals) the European Commission proposes to evaluate 30,000 chemicals within a period of 15xa0years. We are providing estimates of the testing requirements based on our personal experiences during the past 20xa0years. A realistic scenario based on an in-depth discussion of potential toxicological developments and an optimised “tailor-made” testing strategy shows that to meet the goals of the REACH policy, animal numbers may be significantly reduced below 10xa0million if industry would use in-house data from toxicity testing, which are confidential, if non-animal tests would be used, and if information from quantitative structure activity relationships (QSARs) would be applied in substance-tailored testing schemes. The procedures for evaluating the reproductive toxicity of chemicals have the strongest impact on the total number of animals bred for testing under REACH. We are assuming both an active collaboration with our colleagues in industry and substantial funding of the development and validation of advanced non-animal methods by the EU Commission, specifically in reproductive and developmental toxicity.

Cytotoxicity test using blastocyst-derived euploid embryonal stem cells: a new approach to in vitro teratogenesis screening.

To develop a mammalian in vitro system for teratogenicity testing, cytotoxicity of xenobiotics was evaluated in pluripotent euploid embryonal stem cells (ESC) derived from mouse blastocysts. The dimethyl-thiazol-diphenyl tetrazolium bromide (MTT) assay was the most appropriate test system for cytotoxicity determinations with ESC. Only compounds that do not require metabolic activation were selected for testing from the database for validation of in vitro teratogenesis assays by Smith et al. Results obtained with ESC were compared to corresponding data from fibroblasts from day-14 mouse embryos to detect differences in sensitivity between undifferentiated and differentiated cells. ESC showed a higher sensitivity to known teratogens than fibroblast cultures, which allows calculation of a sensitivity ratio of adult cells (differentiated fibroblasts) to embryonal cells (undifferentiated ESC) in a mammalian system similar to the hydra assay. Although some xenobiotics had to be classified as false negatives in our system, the ESC cytotoxicity assay holds promise as a new in vitro screening assay in teratology.

Unique role of studies on preimplantation embryos to understand mechanisms of embryotoxicity in early pregnancy.

Resume de la connaissance de base sur laction des produits chimiques lors de la gestation precoce. Criteres toxicologiques devaluation des effets embryotoxiques apres exposition des embryons de preimplantation in vivo et in vitro. Utilisation des cultures dembryon pour letude du metabolisme des medicaments et de la pharmacocinetique

Genotoxic and embryotoxic effects of gonadotropin-hyperstimulated ovulation of murine oocytes, preimplantation embryos, and term fetuses.

Compared to spontaneous ovulation, gonadotropin hyperstimulated ovulation (superovulation) in mice resulted in a fourfold increase in the number of preimplantation embryos three days post coitum 50% of which will die before term. Both in vitro development of embryos during the preimplantation period and transfer of morulae from superovulated females to pseudopregnant untreated foster mothers indicate that the prenatal loss occurring shortly before implantation up to term is due to maternal factors rather than to direct hormonal effects on oocytes or early embryos. Indeed, no genotoxic events could be observed in 4-cell to blastocyst stage embryos from superovulated female mice as revealed by the chromosomal aberration test and the sister chromatid exchange assay. Chromosome analysis of the pronuclei from mouse zygotes showed an increased rate of aberrations in oocyte derived nuclei after superovulation in comparison to spontaneous ovulation. The present data suggest that aberrant murine oocytes may be fertilized, but they do not survive the first cleavage stages. The result is discussed with respect to the high incidence of chromosomal abnormalities found in human oocytes after gonadotropin-hyperstimulated ovulation.

Abnormal chromosome behavior in human oocytes which remained unfertilized during human in vitro fertilization.

Chromosomal abnormalities and abnormal embryonic development have previously been observed after human in vitro fertilization (IVF). Chromosomal abnormalities may arise not only after fertilization but even earlier during meiotic maturation of human oocytes in culture. Since chromosomal analysis is simple in oocytes during meiotic maturation, the chromosomal status was analyzed in oocytes which remained unfertilized in a human in vitro fertilization program. In 50 fertilization attempts the chromosomes of 62 unfertilized oocytes could be analyzed; 45 of them were in the process of meiotic maturation. In three oocytes two small polar bodies were observed 16–18 hr after insemination in the absence of fertilization. In one oocyte abnormal chromosome behavior was found during the first meiotic division, and in four oocytes during metaphase of the second meiotic division. These data suggest that chromosomal analysis of unfertilized oocytes in human IVF may improve the understanding of human oocyte maturation and fertilization.

Embryologic and cytogenetic effects of ethanol on preimplantation mouse embryos in vitro

Ethanol and its primary metabolite acetaldehyde were studied in cultured preimplantation mouse embryos with respect to embryotoxicity, embryolethality, chromosome breaking activities, and ability to induce sister chromatid exchange (SCE). Analysis of differentiation and cell number of mouse morulae and blastocysts show that acetaldehyde is three orders of magnitude more toxic than ethanol, indicating that the metabolite is responsible for the embryotoxicity of ethanol in preimplantation embryos. Concentrations of ethanol that do not inhibit growth induce SCEs and chromosome aberrations. The SCE-inducing effect of ethanol disappears in the presence of 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase (ADH). These data suggest that preimplantation embryos are able to convert ethanol to acetaldehyde and that ADH is the enzyme involved. It is, furthermore, shown histochemically that mouse oocytes as well as morulae and blastocysts are able to oxidize ethanol in the presence of NAD+.

Maternal factors influencing development of embryos from mice superovulated with gonadotropins

In NMRI mice superovulation with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) increased mating rate, number of implantation sites, rate of advanced and delayed resorptions, as well as retarded sternebral ossification and cleft palate. On day 3 of gestation in preimplantation embryos, cell number and mitotic index were lower after superovulation than after spontaneous ovulation. However, when preimplantation embryos from superovulated and control females were transferred on day 3 of pregnancy to pseudopregnant recipients (10 embryos per female) no differences could be detected between the two groups of fetuses at term. The results of the embryo transfer experiments indicate that abnormal embryonic development after superovulation with gonadotropins is predominantly induced by effects of the hormone treatment on the maternal uterine environment.

In vitro approach to fertility research: Genotoxicity tests on primordial germ cells and embryonic stem cells

In vitro screening tests for reproductive toxicology are required by the 7th amendment of the directive 67/548 EEC and the OECD-programme on existing chemicals. Unfortunately, appropriate methods for testing developmental toxicity or impairment of fertility are not at hand. Therefore, we have tried to design test method based on mutagenic effects in germ cells that may be used as a test of fertility impairment as well. Embryonic stem cells (ESC) derived from mouse blastocysts can be kept in culture routinely. Establishment of ESC and improvement of their culture conditions are described and special properties of ESC are discussed in relation to germ cells. Because some properties of germ cells and pluripotent embryonic stem cells (ESC) are found to be comparable, permanent lines of ESC hold promise to be used as an in vitro test of impairment of fertility.

Cytotoxic and genotoxic effects of bromodeoxyuridine during in vitro labelling for sister-chromatid differentiation in preimplantation mouse embryos

Exposure of preimplantation mouse embryos in culture to bromodeoxyuridine (BrdU) in the concentration range of 10(-9) to 2 x 10(-6) M allows sister-chromatid differentiation at the morula and blastocyst stage. The same BrdU concentrations induced no chromosomal aberrations, but a prolongation of the cell cycle and an increase of the SCE frequency. Even at the lowest BrdU concentration for sister-chromatid differentiation (10(-9) M the background level for SCE was found to be significantly higher in early embryos than in fetal or adult tissues of the mouse. Therefore, the high SCE frequency seems to be characteristic of undifferentiated embryonic cells. Methodological recommendations are also given for SCE assay in preimplantation mouse embryos.

Abnormal development of mouse embryos exposed to methylnitrosourea before implantation

On day 2 of gestation mice were exposed to single i.p. injections of 5, 10, 20, and 40 mg/kg methylnitrosourea (MNU). Evaluation at term revealed 100% embryolethality in the 40 mg/kg group but no signs of maternal toxicity (LD50 = 400 mg/kg). In mice treated with 5 and 10 mg/kg, no malformations could be detected at term. In contrast, 40% of the live fetuses exposed to 20 mg/kg MNU showed developmental abnormalities of vertebrae, ribs, long bones, and kidneys. Analysis of postnatal development 3 weeks after birth indicated a significant increase in mortality in the offspring of all animals exposed to MNU on day 2 of pregnancy. Further developmental or morphologic anomalies could not be detected in the offspring up to the age of 6 months, when autopsy was performed. The data show that exposure to MNU before implantation has embryolethal and teratogenic effects in a dose range one order of magnitude lower than the toxic dose range for adult animals.