Advances in genotoxicity testing : towards the development of high-throughput and animal-free alternatives in ecotoxicological assessments

  • Weiterentwicklung von Genotoxizitätstests: Entwicklung von Hochdurchsatzmethoden und tierversuchsfreien Alternativen bei ökotoxikologischen Bewertungen

Brendt, Julia; Hollert, Henner (Thesis advisor); Schäffer, Andreas (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


The rising usage and development of new chemicals, e.g. in the pharma industry, and as a consequence thereof an increasing need for further regulations for authorization lead to a rapidly increasing demand for high-throughput methods for assessment. An extensive risk- assessment is necessary to avoid adverse effects on the environment and simultaneously reduce the hazard for human health. The assessment of a chemical or sample should be determined using different levels of organisation, e.g. whole organisms or cellular level. Therefore, bioassays are conducted to investigate the risk for the environment as well as for human health. For lower level of organisation effect-based methods are used. Some of these effect-based methods require animal experiments. In consequence of the 3R (replace, reduce, refine) and subsequently due to ethical reasons animal experiments are being increasingly replaced with in vitro methods. However, as some of those in vitro methods still require animal compounds. Hence, there is a great need for development of animal free compounds that can be used as a replacement. One of those animal-derived compounds commonly used in in vitro bioassaying can be found in the external metabolization system S9, which is obtained from rats in an animal experiment. The so called S9-fraction is used to simulate the mammalian liver in vitro. Nevertheless, each S9-fraction varies indicated by the type of induction but also depending on the condition of the used animal. However, these animal components should also be replaced more and more by animal-free alternatives. One possible animal-free alternative can be found in the newly developed ewoS9R, that was developed recently by Dr. Andreas Schiwy and colleagues, RWTH Aachen University with support of the German Excellence Initiative (SeedFunds) and the BMWi Exist-project EWOMIS. The animal-free alternative should be able to fully replace the commonly used rat- derived S9 in order to avoid any differences to previous studies with commonly derived S9. A full replacement is needed as otherwise misleading comparisons could be drawn. Therefore, ewoS9R was compared with a rat-derived S9-fraction in two commonly used bioassays for genotoxicity, the Ames fluctuation assay and the micronucleus assay. The Ames fluctuation assay is the most popular test system to detect, the mutagenic potential of a substance or environmental sample using bacteria. A mutagenic potential could lead to changes in the DNA which can be passed to the next generation. The standard test strains Salmonella typhimurium TA 98 and TA 100 have been investigated in this thesis as this combination is able to detect most of the mutagenic samples. The micronucleus assay uses various primary cells and cell lines and aims to detect genotoxic effects on DNA, chromosomes and effects on cell division. In this study, V79 cells (derived from lung epithelia of the Chinese hamster (Cricetulus griseus)) have been used as it is a very common used cell line for the micronucleus assay. The comparison of the commonly used rat-derived S9-fraction with ewoS9R using 12 different known genotoxic or mutagenic substances showed promising results. Nevertheless, further research is necessary to fully replace the rat-derived product, as the animal-free ewoS9R was used in a lower protein-amount than the rat-derived product and therefore, a direct comparison of the results of both products is challenging. In addition, new variants of the Ames test as well as the micronucleus assay have been investigated to enable a more rapid high-throughput screening: the Ames Respiration Activity Monitoring System (RAMOS)-test and a high-throughput cost-efficient micronucleus assay. RAMOS uses the detection of oxygen consumption during the Ames test to monitor mutagenicity. The high-throughput low-budget micronucleus assay has been investigated on well-plates in order to increase the number of tested samples. To speed up the time needed for evaluation an automatic reader as well as automatic computer assisted counting were established. Both test systems are capable of more samples in less time compared to the commonly used test designs, while the RAMOS-technique additionally delivers a higher amount of information about bacterial growth than the Ames fluctuation assay. Nevertheless, further research is needed on both the test systems and the animal-free metabolization system in order to achieve a safer and less resource consummating future.