In vitro assessment of genotoxicity as an early warning tool for carcinogenicity mainly relies on recording cytogenetic damages (micronuclei, nucleoplasmic bridges) in tumour-derived mammalian cell lines like V79 or CHO. The forecasting power of the corresponding standardised test is based on epidemiological evidence between micronuclei frequencies and cancer incidence. As an alternative to destructive staining of nuclear structures a fish stem cell line transgenic for a fusion protein of histone 2B (H2B) and enhanced green fluorescent protein (eGFP) was established. The cells are derived from koi carp brain (KCB) and distinguish from mammalian culturable cells by non-tumour-driven self-renewal. This technology enables the analysis of genotoxic-and malign downstream effects in situ in a combined approach. In proof-of concept-experiments, we used known carcinogens (4-Nitroquinoline 1-oxide, colchicine, diethylstilbestrol, ethyl methanesulfonate) and observed a significant increase in micronuclei (MNi) frequencies in a dose-dependent manner. The concentration ranges for MNi induction were comparable to human/mammalian cells (i.e. VH-16, CHL and HepG2). Cannabidiol caused the same specific cytogenetic damage pattern as observed in human cells, in particular nucleoplasmic bridges. Metabolic activation of aflatoxin B1 and cyclophosphamide could be demonstrated by pre-incubation of the test compounds using either conventional rat derived S9 mix as well as an in vitro generated biotechnological alternative product ewoS9R. The presented high throughput live H2B-eGFP imaging technology using non-transformed stem cells opens new perspectives in the field of in vitro toxicology. The technology offers experimental access to investigate the effects of carcinogens on cell cycle control, gene expression pattern and motility in the course of malign transformation. The new technology enables the definition of Adverse Outcome Pathways leading to malign cell transformation and contributes to the replacement of animal testing. Summary: Complementation of genotoxicity testing by addressing initiating events leading to malign transformation is suggested. A vertebrate cell model showing "healthy" stemness is recommended, in contrast to malign transformed cells used in toxicology/oncocology.

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Only few information on the potential toxic effectiveness of biofuels are available. Due to increasing worldwide demand for energy and fuels during the past decades, biofuels are considered as a promising alternative for fossil fuels in the transport sector. Hence, more information on their hazard potentials are required to understand the toxicological impact of biofuels on the environment. In the German Cluster of Excellence “Tailor-made Fuels from Biomass” design processes for economical, sustainable and environmentally friendly biofuels are investigated. In an unique and interdisciplinary approach, ecotoxicological methods are applied to gain information on potential adverse environmental effects of biofuels at an early phase of their development. In the present study, three potential biofuels, ethyl levulinate, 2-methyltetrahydrofuran and 2-methylfuran were tested. Furthermore, we investigated a fossil gasoline fuel, a fossil diesel fuel and an established biodiesel. Two in vitro bioassays, one for assessing cytotoxicity and one for aryl hydrocarbon receptor agonism, so called dioxin-like activity, as measured by Ethoxyresorufin-O-Deethylase, were applied using the permanent fish liver cell line RTL-W1 (Oncorhynchus mykiss). The special properties of these fuel samples required modifications of the test design. Points that had to be addressed were high substance volatility, material compatibility and low solubility. For testing of gasoline, diesel and biodiesel, water accommodated fractions and a passive dosing approach were tested to address the high hydrophobicity and low solubility of these complex mixtures. Further work has to focus on an improvement of the chemical analyses of the fuel samples to allow a better comparison of any effects of fossil fuels and biofuels.

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