Developments and sustainability in genetic toxicology: in vitro methodologies for the evaluation of the genetic damage

The impact that mutations may have on human health can be vast and profound and includes cancer, cardiovascular and neurodegenerative disorders. For this reason, it is crucial to have genetic toxicology tests that allow a rapid and accurate identification of the mutagenic potential of a xenobiotic. In particular, a promising method is represented by a published flow cytometric (FCM) version of the “In vitro mammalian cell micronucleus test” (MNvit), an assay that allows to highlight both structural and numerical chromosomal aberrations. Therefore, the research conducted in this thesis had as its objective the implementation of this method performed on TK6 cells and the evaluation of its versatility and reliability. For this purpose, the protocol was tested on a variety of samples, including more complex and potentially debris-rich ones (e.g., botanicals). Besides, the velocity of analysis typical of FCM was exploited in genotoxicological screenings of large classes of molecules (e.g., Novel Psychoactive Substances). Moreover, to test chemicals that are suspected to be mutagenic not only as such but also after metabolic activation the protocol was tested in the presence of an extrinsic metabolizing system (S9 mix) as well. Thereafter, to deepen additional possible metabolic activation systems, inherently metabolically competent hepatic spheroids that express high levels of human metabolizing enzymes were exploited in the genotoxicity assessment of naturally occurring molecules by means of the microscopy version of the MNvit. Lastly, a screening of different known mutagens was carried out by means of the FCM MNvit protocol. The results obtained represented a necessary step to create a broader reference dataset for positive and negative controls and to support and track the proficiency of the laboratory in performing this particular protocol.