Genetic toxicity: Bacterial reverse mutation test (OECD 471)

The basic assay is the Ames plate incorporation test. The test measures the rate of reverse mutations in several Salmonella typhimurium strains that carry different mutations in genes of the histidine operon. The bacteria are grown in the presence of the test material on a minimal agar containing just a trace of histidine, which is sufficient for a few cell divisions. Only those bacteria that have reverse mutation to histidine independence will grow and form colonies.

Multiple modes of mutation induction (e.g. base-substitution or frame-shift mutation) can be detected in the set of Salmonella strains. To detect mutations caused by oxidative agents, an Escherichia coli strain that carries the AT base pair mutation at a critical site of one of the genes in tryptophan biosynthesis pathway is included in the test. In this case, tryptophan independence is surveyed. Possible metabolic activation to a mutagenic compound is tested by including certain mammalian enzymes in the assay, e.g., in a form of liver S9-mix isolated from rats treated with Aroclor 1254 or phenobarbital/5,6-benzoflavone.

Biosafe Ltd follows the OECD Test Guideline 471 but does not currently provide the test according to the GLP requirements.

For screening purposes, or if only a limited amount of test material is available, Ames fluctuation test can be performed. The test material is mixed with the bacterial suspension (in histidine-limited growth medium with or without the S9-mix), which is then diluted in histidine-free medium (containing pH indicator) and distributed on a 96-well microplate. If reverse mutation occurs to enable histidine biosynthesis, the bacteria grow in those wells and the indicator changes colour because of pH change. The number of wells with bacterial growth is a measure of mutagenic activity of the test material.