Genotoxicity testing

Ames test

Many human genetic diseases are attributed to point mutations. There is also strong evidence that tumor formation in humans and experimental animals involve substitutions, additions or deletions of one or a few DNA base pairs in oncogenes and tumor suppressor genes of somatic cells. There are in vitro test systems to predict carcinogenicity. The Ames test is one of the most commonly used mutagenicity/genotoxicity methods. It is the bacterial reverse mutation assay using amino acid-requiring Salmonella typhimurium and Escherichia coli strains that carry mutations in genes involved in histidine/tryptophan biosynthesis. Ames test is one of the eight in vitro genotoxicity test methods adopted at the EU level with OECD guidelines.

Method: The reverse mutation Ames test is a standard bacterial assay which measures histidine/tryptophan reversion induced by chemicals causing base changes or frameshift mutations in the genome of bacteria tester strains.

ImageGenotoxicity evaluation by Ames test using Salmonella typhimurium histidine and Escherichia coli wp2 tryptophan reversion assay utilizes bacteria with point mutations in the histidine (S. typhimurium) or the tryptophan (E. coli) operon, rendering the bacteria incapable of producing the corresponding amino acid. These mutations result in his- or trp- organisms that cannot grow unless histidine or tryptophan is supplied. When a mutagenic event occurs, base substitutions or frameshifts within the gene may cause a reversion to amino acid prototrophy. These reverted bacteria then grow in histidine- or tryptophan-deficient media, respectively. A chemical’s mutagenic potential is assessed by exposing these amino acid-requiring organisms to varying concentrations of chemical and selecting for the reversion event. Media lacking the specific amino acid are used for this selection which allow only those cells that have undergone the reversion to histidine/tryptophan prototrophy to survive and grow. The Ames test includes using an extract of liver enzymes to simulate mammalian metabolic activity which may activate non-mutagenic chemicals to their mutagenic derivatives.

Strains used: The recommended by OECD 471 combination of strains in Ames test is:
  - S. typhimurium TA98
  - S. typhimurium TA100
  - S. typhimurium TA1535 
  - S. typhimurium TA1537 E. coli wp2 (uvrA and pKM101)

TA100, TA1535 and the E.coli strains are for the detection of base substitution mutations and TA98 and TA1537 are for the detection of frameshift mutations. The S. typhimurium strains have GC base pairs whereas the E.coli strains have an AT base pair at their primary reversion site and detect certain oxidizing mutagens, crosslinking agents and hydrazines.

Mouse Lymphoma Assay

The in vitro mammalian cell gene mutation test (OECD 476) is used to detect gene mutations induced by chemical substances in eukaryotic cells, thus constituting excellent supplement for Ames test.

The mouse lymphoma assay (MLA) is a short-term assay designed to detect forward gene mutations induced by mutagens at the heterozygous thymidine kinase (Tk) locus and is capable of quantifying genetic alterations. The system, recommended by ICH and others, employes L5178Y Tk+/- cells and the TK (thymidine kinase) locus. Cells deficient in thymidine kinase (TK) due to mutation TK+/- -> TK-/- , caused by the in vitro treatment with mutagenic and/or carcinogenic agents, are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain proper gene of thymidine kinase, are not.

Assay is performed in accordance with OECD 476 guideline and PN-EN ISO 10993-3:2008 norm with the use of mouse T cell lymphoma cell line – L5178Y TK+/-.

Micronucleus Assay

The in vitro Micronucleus Assay (MNvit, OECD 487) is a mutagenic test system for the detection of chemicals that induce the formation of small membrane-bound DNA fragments (micronuclei-MN) in the cytoplasm of interphase cells. The MNvit, used for regulatory purposes measures formation of chromosomal changes following DNA damage induced by the compounds under test, and is used to predict the genotoxic potential of pharmaceuticals, industrial chemicals, food additives and cosmetic ingredients. That is why, MNvit is one of the most frequently used profiling assays in the pharmaceutical industry.

Assay is performed with the use of CHO-K1 cell line and according to OECD 487 guideline and PN-EN ISO 10993-3:2008 norm.