OECD 471: Bacterial Reverse Mutation Test (Ames Assay)

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Bacterial Reverse Mutation Assay


The purpose of this study is to evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation.

This protocol is based on:

OECD Guideline for Testing of Chemicals: No. 471 – Bacterial Reverse Mutation Test (July 1997) and U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 – Bacterial Reverse mutation Test (August 1998).

Assay Principle

The bacterial reverse mutation test evaluates the mutagenic potential of a test article by using Salmonella typhimurium and Escherichia coli strains to detect point mutations, which includes the substitution, addition or deletion of one or a few DNA base pairs, in the presence and absence of metabolic activation (S9).


  • Solubility will be tested in deionized water, dimethyl sulfoxide (DMSO), 95% ethanol or acetone.
  • A toxicity screen will be performed in S. typhimurium TA100 strain, using eight concentrations of the test article, one plate per concentration, both with and without exogenous metabolic activation (S9).
  • The highest dose will be the highest obtainable concentration in the vehicle, but will not exceed 5 mg/plate or 5 μl/plate.

Main Test:

  • The main assay is run using all five strains (E. coli WP2 and S. typhimurium TA97a, TA98, TA100 and TA1535) on triplicate plates. Positive and vehicle controls will be evaluated concurrently for all five strains, on six plates per strain. Five concentrations will be chosen based on solubility and screen toxicity results. All plating will be with and without exogenous metabolic activation (S9).
  • An independent repeat assay may need to be performed.


  • Bacterial colonies will be counted and the mean number of colonies of each dose will be divided by the mean for the negative (vehicle) control value to obtain a ratio to vehicle
  • Negative Results: Determined by the absence of a dose-related increase in all five tester strains, again taking into account toxicity of the test article and controls.
  • Positive Results: A ≥ 2-fold increase of bacterial colonies over the mean negative control value (vehicle), indicating that tested material induces point mutations in the genome of either Salmonella typhimurium and/or Escherichia coli.