Objective of the study

The aim of genotoxicity testing is to:

• predict genotoxic potential
• identify genotoxic carcinogens at an early stage
• elucidate the mechanism of action of some carcinogens.

The bacterial reverse mutation test uses strains of Salmonella typhimurium and Escherichia coli that require amino-acids to grow. The principle of this test is that it detects mutations which revert mutations present in the test strains, and restore the functional capability of the bacteria to synthesize an essential amino acid.

Point mutations are the cause of many human genetic diseases and there is substantial evidence that point mutations in oncogenes and tumour suppressor genes of somatic cells are involved in tumour formation in humans and experimental animals.

The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation inducing activity. An extensive data base has demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests.

Although many compounds that are positive in this test are mammalian carcinogens, the correlation is not absolute. It is dependent on chemical class and there are carcinogens that are not detected by this test because they act through other, non-genotoxic mechanisms or mechanisms absent in bacterial cells. Mammalian mutation tests may thus be more appropriate for some pesticide groups.

Circumstances under which the study is recommended to be required

The bacterial reverse mutation assay is always required.

Test organism

At least five strains of bacteria should be used. The recommended combination of strains is:

1. S. typhimurium TA1535, and
2. S. typhimurium TA1537 or TA97 or TA97a, and
3. S. typhimurium TA98, and
4. S. typhimurium TA100, and
5. E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102

Test substance

• Technical grade active ingredient

Typical endpoints of the study

Data should be presented as the number of revertant colonies per plate.

There are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. Biological relevance of the results should be considered first; statistical methods may be used as an aid in evaluating the test results.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.

Test guidelines

The following test guidelines may be used for the bacterial reverse mutation study:
• OECD Guidelines for the Testing of Chemicals. Test No. 471: Bacterial reverse mutation test.
• US EPA Health Effects Test Guidelines. OPPTS 870.5100. Bacterial reverse mutation test
• EC Testing Method B.13/14. Mutagenicity - Reverse mutation test using bacteria. Council Regulation (EC) No 440/2008.