Table 6.

Assays used in genotoxicological studies.

Assay	Characteristics	Advantages	Disadvantages
Comet Assay	Sensitive and rapid method for DNA strand break detection in individual cells. Used in genotoxicological studies to determine oxidative DNA damage that has been implicated in several health conditions (in combination with certain bacterial enzymes), to show protective effects of different dietary factors in chemopreventive studies, to determine sequence or gene-specific damage and repair (in combination with fluorescence in situ hybridization) as well as of possible diagnostic use [160,161].	Identify DNA damage at the single-cell level Sensitivity for detecting low levels of DNA damage using a small number of cells per sample (<10,000) Eukaryote single cell population can be used both in vitro and in vivo Ease of application Low cost Short time needed to perform the assay [161,162]	Not a reliable biomarker for genotoxic effects in DNA damage The detected DNA damage does not correspond to fixed mutations [161,163,164,165]
Somatic Mutation and Recombination Test (SMART)	In vivo genotoxicity assays are performed on Drosophila melanogaster. It has been used extensively to analyze many chemicals with different action mechanisms, showing a vast ability to detect genotoxic effects. [160,166,167].	Easy to perform Inexpensive Shows high sensitivity, specificity, and accuracy Detects loss of heterozygosity by deletions, point mutations, mitotic recombination, and nondisjunction [166,168]	Time-consuming Delay in the development of the larvae [168,169]
Micronucleus Test	A small, chromatin-containing round-shaped body that is visible in the cytoplasm of cells. Can originate from acentric fragments, segregation of chromosomes, dicentric chromosome breakage, chromosome instability, or aggregation of double minutes. A rise in the frequency of micronucleated cells is a biomarker of genotoxic effect that can reflect exposure to agents with clastogenic (chromosome breaking; DNA as target) or aneugenic (aneuploidogenic; effect on chromosome number; mostly non-DNA target) modes of action. [160,170,171,172,173,174].	Reliable identification of cells that have completed only one nuclear division Easy to perform Less time required Inexpensive Allows the detection of chromosome and genome mutations and co-detection of apoptosis and necrosis, measuring the extent and progression of nuclear division in a dividing cell population Detects dicentric bridges, chromosome loss, nondisjunction, excision repair events, and Hypoxanthine guanine phosphoribosyl transferase variants Allows automatic scoring [175,176]	Low sensitivity Detects only acentric fragments Cell division is needed for effective use [176]
Chromosomal Aberration Test	It can be performed in primary peripheral blood lymphocytes or established cell lines, such as Chinese hamster ovary cells. Recognizes agents that cause structural chromosome aberrations (clastogenesis) produced by various mechanisms [177,178].	Allows identification of all chromosome mutation types and co-detection of mitotic indices [178]	Need for cell cultivation and highly skilled and experienced personnel Time-consuming Expensive Automatic scoring is not possible [176,179]
Bacterial Reverse Mutation Test	Uses amino acid-requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substituting, adding, or deleting one or a few DNA bases pairs. Detects chemicals that induce mutations that revert mutations present in the tester strains and rehabilitate the functional capability of the bacteria to synthesize an essential amino acid. [180,181,182,183].	Allows replicates to be made and results to be obtained relatively quickly Allows inexpensive study of a large number of test materials inexpensively Enables identification of the molecular mechanism effect of test material Very versatile test [183,184]	May not be appropriate for the evaluation of certain classes of pharmaceutical, for example, highly bactericidal compounds such as antibiotics, and those which are thought (or known) to interfere specifically with the mammalian cell replication system Can produce false positives Possible interference from biological samples, e.g., plant extracts that contain amino acids (histidine) with the test system [180,184]