ALTEX :: Alternatives to Animal Experiments

2001, VOLUME 2

Permanent Female and Male EG-Cell-Lines of Balb/CJ Mice--an Alternative Concept for Reproductive Toxicity Testing?

Martina Klemm, Elke Genschow, Manfred Liebsch and Horst Spielmann

ZEBET (BgVV), D-Berlin

SUMMARY

While several in vitro tests are available for replacing animal experiments in base-level screening of new substances, few have been defined adequately for quantitative studies in reproductive toxicology. In order to offer a sensitive and predictive in vitro method to assess the genotoxic potential of chemical agents on male and female reproduction, we established primordial germ (PG) cell-derived permanent female and male embryonic germ (EG) and embryonic stem (ES) cell lines of the mouse (strain Balb/cJ). All EG and ES cell lines were characterised (by PCR and karyotype) and periodically checked for quality criteria like alkaline phosphatase activity and mean generation time (MGT) to ensure clone stability. The differences in developmental sensitivity of EG cells, ES cells and differentiated fibroblast cells of the mouse cell line 3T3 regarding genotoxicants were comparatively tested under identical test conditions. Cytotoxicity assay was based upon determination of growth inhibition (MTT-test) and genotoxic effects were determined by sister chromatid exchanges (SCE) induced by standard reference mutagens like Ethylnitrosourea (ENU), Methylnitrosourea (MNU), Methylmethansulfonate (MMS), Hydroxyurea (HU) and Mitomycin C (MMC). After calibrating the in vitro EG cell assay by testing positive and negative control substances, we are now starting to clarify if our in vitro test system can reliably and reproducibly discriminate between known genotoxic and non toxic agents. Furthermore, a distinction between different degrees of genotoxic effects is mandatory for our assay. To classify the genotoxic potential of all tested chemicals we will develop a biostatistical prediction model on the basis of concentration-response curves and exclusion of a major impact of cytotoxicity. Finally, we would like to establish an additional in vitro test focusing on EG cell progression through meiotic prophase as predictive endpoint for sex-specific genotoxicity testing.