The term genotoxicity refers to the ability of agents to alter the genetic information within a cell. This DNA alteration can be in the form of single- and double- strand (ssDNA and dsDNA) breaks, point mutations, deletions, chromosomal aberrations, micronuclei formation, DNA repair and cell-cycle interactions. Genotoxicity testing for hazard identification and risk assessment is crucial for evaluating the ability of a chemical agent to induce genetic alterations. Currently, there is no single validated test able to provide information on the three genotoxicity critical end-points, which are mutation induction (permanent, transmissible changes in the DNA), clastogenicity (structural chromosomal damage), and aneugenicity (numeric chromosomal abnormalities).

Genotoxicity assessment can be determined through computational in silico methods that predict biological activities of a molecule from its physicochemical properties. These methods are based on computational tools, mathematical calculation, and analysis of predicted or experimental data through computer-based models. Thus, the in silico approach is widely used to assess toxicological effects relevant to human health as well as to prioritize chemicals for in vitro or in vivo testing.

A battery of in vitro genotoxicity assays provides simple, robust and time- and cost-effective testing of toxicity and the subsequent underlying mechanisms. To assess the risk of cancer, the most commonly utilized methods to determine genetic damage include the bacterial Ames test, DNA strand break measurements in cells (e.g. comet assay, alkaline unwinding and hydroxyapatite chromatography, alkaline elution), and cytogenetic assays (micronucleus and chromosomal aberration assays). Three in vitro genotoxic tests, the Ames test, the Comet assay and the Chromosomal aberration test, are explained in Table 2.3.2 and more 2.5.5 where all tests are summarized as well.

Consequently, in silico analysis as well as in vitro genotoxicity testing, provide valuable preliminary information, evaluating the initial safety of a substance; however, in vivo testing is also required. In vivo testing is conducted using animal tissues to verify the potential safety or the mutagenic effect of a substance in the animal's whole physiological system. In 1970, the two-year chronic exposure bioassay in laboratory rodents became the gold standard method for the identification of chemicals or physical agents with carcinogenic activity. To date, it is still the primary method requested by regulatory organizations across the world. In these long-term bioassays, rodents, usually rat and mouse, are exposed to multiple doses of the agent over their life span and are observed for the development of neoplastic lesions.

Nevertheless, the carcinogenicity testing conducted in rodents is not necessarily translated into humans. For instance, these studies usually use higher doses of substances in comparison with those to which humans would normally be exposed. Additionally, the standard carcinogenicity studies utilize a large number of animals, approximately 400-500 of each species, with increasing concern surrounding the principles of 3Rs (Reduction, Replacement and Refinement) of animals in research. Therefore, alternative animal models are under investigation for evaluating their utility in safety assessment of nutritional supplements and/or hazard identification. For example, distinct genetically modified mouse strains have been generated that develop tumors in a rapid way compared to wild type ones through genetic modifications in genes critical to the carcinogenic process. Commonly used strains that are under evaluation include the p53+/- hemizygous knockout mouse, the rasH2 model, the Tg:AC skin model as well as the Xpa-/- and Xpa-/- p53 +/- transgenic mouse models. However, yet none of them has been defined as a genuine alternative to 2-year rodent bioassay.