How is genotoxicity defined?
In general the term genotoxicity refers to the ability to induce structural changes in genes via interaction with DNA and/or non-DNA targets (1). The hazard classification "is primarily concerned with chemicals that may cause mutations in the germ cells of humans that can be transmitted to the progeny" (2). The classification system "is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germ cells" and "is not meant for the quantitative risk assessment of substances" (2). This classification system defines two hazard categories and two subcategories for germ cell mutagens:
|1||Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans.|
|1A||Substances known to induce heritable mutations in germ cells of humans.|
|1B||Substances which should be regarded as if they induce heritable mutations in the germ cells of humans.|
|2||Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans (2).|
Which non-animal alternative methods are validated and accepted?
So far, eight in vitro methods for genotoxicity testing have been adopted at the
EU level. The classical
in vitro genotoxicity tests, i.e. the
gene mutation test in mammalian cells (OECD 476), the chromosome aberration
test (OECD 473) and the
micronucleus test (OECD 487) are commonly used to predict the intrinsic
potential of substances to induce mutations. Mutations and chromosomal aberrations are
strongly associated with carcinogenic processes. Only
in vitro genotoxicity tests which measure a mutation endpoint are
qualified for identifying potential carcinogens. So far, none of the
in vitro genotoxicity tests are formally
validated with the exception of the
micronucleus test, although OECD guidelines
exist for all of these tests. Nevertheless,
genotoxicity tests are scientifically accepted and widely used (3).
For the improvement of the predictive value of a genotoxicity assessment the features of reconstructed skin models offer a more physiologically relevance regarding metabolic properties for dermal exposure. So far, reconstructed skin models can be used for two genotoxicity tests: The comet assay and the micronucleus assay. Both assays are under prevalidation to prove whether they have sufficient sensitivity and specificity (3). Both tests are not designed as standalone methods but should be used in addition to the standard battery of in vitro genotoxicity tests.
Table 1: Overview of existing OECD Test Guidelines for genotoxicity and mutagenicity testing
|TG 471||Bacterial Reverse Mutation Test|
|TG 472||Genetic Toxicology: Escherichia coli, Reverse Assay|
|TG 473||In Vitro Mammalian Chromosome Aberration Test|
|TG 474||Mammalian Erythrocyte Micronucleus Test|
|TG 475||Mammalian Bone Marrow Chromosome Aberration Test|
|TG 476||In Vitro Mammalian Cell Gene Mutation Test|
|TG 477||Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster|
|TG 478||Genetic Toxicology: Rodent Dominant Lethal Test|
|TG 479||Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells|
|TG 480||Genetic Toxicology: Saccharomyces cerevisiae , Gene Mutation Assay|
|TG 481||Genetic Toxicology: Saccharomyces cerevisiae , Mitotic Recombination Assay|
|TG 482||Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro|
|TG 483||Mammalian Spermatogonial Chromosome Aberration Test|
|TG 484||Genetic Toxicology: Mouse Spot Test|
|TG 485||Genetic Toxicology: Mouse Heritable Translocation Assay|
|TG 486||Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vitro|
|TG 487||In Vitro Mammalian Cell Micronucleus Test|
How does the micronucleus assay (for reconstructed skin models) work?
The micronucleus (MN) is formed during metaphase/anaphase transition of mitosis and represents broken fragments of daughter chromosomes outside of the nucleus. The micronucleus formation can be used as a diagnostic tool for chromosomal DNA damage and is monitored via staining of DNA, cell membrane and nuclear membranes followed by microscopic analyses (4).
After 24 hrs of culture (equilibration), the epiCS tissues are exposed to the test substances for two days with and without metabolic activation. To block cytokinesis during the exposure, cytochalasin B is added to the culture medium which enables the formation of micronuclei in binucleated interphase cells. After exposure to the substances the epidermal cells are isolated out of the tissues, followed by staining, fixation and microscopical analyses including scoring of micronucleated cells.
At the day of receipt epiCS are conditioned with fresh epiCS Culture Medium by pre-incubation for 24 hrs or at least overnight.
epiCS are exposed to Culture Medium containing Cytochalasin B (3 µg/ml). 10 µl of the test substance is applied topically onto the epiCS followed by 24 hrs incubation (37±1°C, 5±1% CO2, 95% RH).
Exchange the Culture Medium (containing Cytochalasin B) of the tissues and apply 10 µl of the test substance topically onto the epiCS. After the application tissues are incubated (37±1°C, 5±1% CO2, 95% RH) for another 24 hrs.
The epidermis is detached from the insert membrane after Trypsin-EDTA treatment. Single cells are harvested by a follow up treatment with Trypsin-EDTA. After cell separation the cells are counted and viability is determined.
Cells are fixed via KCl and MeOH/acetic treatment. After fixation cells are attached onto microscopy slides and stained with acridine orange solution.
counting and interpretation of results
For evaluation and analysis of the test results, at least 1000 binucleated cells per duplicate are required. Cells are scored and classified depending on the number of nuclei (mono- and binucleates) and binucleated cells containing micronuclei. Trinucleated or multinucleated cells are not scored. Binucleated cells are only scored if the nuclei are separated and of approximately equal size, even if they overlap, as long as nuclear boundaries can be distinguished. (see Fig.2)
If a test substance reproducibly increases the number of cells containing micronuclei compared to negative controls it is classified as a genotoxic substance.
For further Information, check the epiCS Manual.
Fig. 2 Binucleated cells containing a micronucleus (indicated by white arrows). Single cells were isolated from epiCS after treatment with Mitomycin C.
- Maurici D., Aardema M., Corvi R., et al. (2005). Genotoxicity and mutagenicity. ATLA, 33, Suppl 1, 117-130.
- United Nations Economic Commission for Europe (UNECE). (2011). Globally Harmonized System of classification and labeling of chemicals (GHS). Fourth revised edition. Part 3. Health hazards. Chapter 3.5. Germ cell mutagenicity.
- Adler S., Basketter D., Creton S., et al. (2011). Alternative (non-animals) methods for cosmetics testing: current status and future prospects-2010. Arch Toxicol 85, 372-379.
- Cherie Musgrove and Manel Camps (2012). Models for Detection of Genotoxicity in vivo: Present and Future, Mutagenesis, Dr. Rajnikant Mishra (Ed.), ISBN: 978-953-51-0707-1, InTech, DOI: 10.5772/50554.