Genotoxicity protocols

The aim of this section is not to provide detailed protocols of the range of prokaryotic and eukaryotic assays used to detect the presence or absence of a genotoxic response. Such descriptions can be found in the published literature—for example, in the series of reports of the US EPA s Gene-Tox Committees. Rather, the principles that need to be addressed by the assays and the current approaches for obtaining underlying mechanistic data are presented. 

Although a great deal of effort was put into battery harmonization, there is no universal agreement on the best combination of tests for all purposes. There are other mutagenicity tests in use that may also be useful (Table 1). The choice of additional test(s) or protocol modification(s) depends on various factors. Compounds that contain structural alerts for genotoxicity are usually detectable in the standard test battery. However, compounds bearing... 

Koppen, G. (1996) Protocol of the alkaline comet test on plant cells. Comet Newsl., 4, 2-4. Koppen, G. and Verschaeve, L. (1997) The alkaline comet test on plant cells. A new genotoxicity test for DNA strand breaks in Viciafaba. Mutat. Res., 360, 193-200. 

The list of alternative tests for reproductive toxicity, at variable stage of development, is fairly long given the complexity of the reproductive cycle and the multiple cell types and functions involved. An official source of information on alternative test development is the website of the European Centre for the Validation of Alternative Methods (ECVAM) (http //ecvam-dbalm.jrc.ec. europa.eu/ updated to 15 June 2013). Forty methods are listed for the area of reproductive toxicity. They are split into four categories effects on female fertility (n = 8), effects on male fertility n= 10), developmental toxicity (n = 21), and genotoxicity-mutagenicity (n= 1). Only eight of these methods have been developed up to fully defined protocols that can be downloaded from the same website ... 

A detailed discussion of the test procedures employed is contained within Galloway et al. (1994). Detectable levels of chromosome aberrations are often found only at doses that induce some evidence of cytotoxicity, and consequently most current recommended protocols require that the maximum test concentration should induce >50% reduction in cell number or culture confluency for cell lines, or an inhibition of mitotic index by >50% for lymphocytes. However, there are growing concerns as to the relevance of genotoxic effects that are found only at highly cytotoxic concentrations (Galloway 2000), and this may be reflected by the poor specificity of mammalian cells tests (Kirkland et al. 2005). 

As discussed above, luminescent bacteria have been used successfully to measure acute toxicity of chemicals and oivironmental samples. In addition to acute toxicity testing, chemicals and environmental samples are often tested for potential genotoxicity. There are several validated methods in routine use for assessing genotoxicity of chemicals but they have been practical limitations. These limitations include high cost per test sample and complex protocols which necessitate highly trained personnel to obtain reproducible test data, and to provide reliable interpretation. 

A second genotoxicity test, the comet assay, detects a broad spectrum of DNA damage. A comet assay protocol developed and evaluated using the EpiDerm model demonstrated good interlaboratory reproducibility and concordance with in vivo data (Reus et al., 2013). Additional in vitro RhE comet assay development and evaluation of intra- and interlaboratory reproducibihty with EpiDerm, Realskin, and the Phenion model are currently ongoing. 

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