The bacterial reverse mutation test (also called Ames test) is used to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The test is performed according to OECD Guideline for Testing of Chemicals, Test No. 471 (adopted in 1997) and employs auxotrophic strains of Salmonella typhimurium and Escherichia coli. Point mutations were made in the histidine (S.typhimurium) or the tryptophan (E.coli) operon, thus blocking bacterial biosynthesis of the corresponding amino acids. A chemical’s mutagenic potential is evaluated by detecting the appearance of the reverse mutants of the auxotrophic strains, making them prototrophs, able to grow in corresponding deficient media. This test is commonly used as a quick screen to estimate the mutagenic potential of new chemicals and drug candidates.
Modified Ames test can be performed upon request. It includes addition of hepatic S9 fraction to simulate the effect of metabolism, since certain compounds, like benzopyrene, become mutagenic only after their metabolic conversion.
5 bacterial strains are used according to OECD Guideline for Testing of Chemicals: S.typhimurium TA98, TA100, TA1535, TA1537, and E.Coli wp2[pKM101] + wp2 uvrA mixed 1:2.
Chemicals are tested according traditional Ames test using standard incorporation protocol (Revised methods for the Salmonella mutagenicity test. Maron DM, Ames BN. //Mutat Res. 1983 May;113(3-4):173-215).
Three concentrations of a test agent, as well as a positive and negative control, are tested using 5 tester strains. The colonies are counted and the results are expressed as the number of revertant colonies per plate. Compounds with know mutagenic activity are used for positive control for each tester strain: TA98- 2-nitrofluorene (0.1 µg/plate), TA100- 4-nitroquinoline N-oxide (0.02 µg/plate), TA1535- NaN3 (0.15 µg/plate), TA1537- 9-aminoacridine (7.5 µg/plate), E.Coli strains- 4-nitroquinoline N-oxide (0.02 µg/plate). DMSO is used as a negative control.
Approximately 8 mg (or equivalent in solution) of dry compound is necessary for this test.