Deciphering the Impact of a Positive Ames Test on Your Program
The bacterial reverse mutation, commonly known as the Ames test, detects mutations, which are the cause of many human genetic diseases and play an important role in tumor initiation and development. The bacterial strains have various mutations that inactivate a gene involved in the synthesis of an essential amino acid, either histidine (Salmonella) or tryptophan (E. coli), so they can only grow in the culture medium that is supplemented with that amino acid.
When the bacteria are exposed to a mutagen, mutation(s) occur that may restore/reverse the ability of the bacteria to synthesize the amino acid and to continue growth once the limited amount of amino acid in the agar is depleted. Relevant mutations involve substitution of individual base pairs or frameshift mutations caused by addition or deletion of a stretch of DNA.
Ames Assay Screening
Given the importance placed on the outcome of the bacterial mutation test by regulatory authorities, many companies screen their compounds for microbial genotoxicity at a very early stage of the development. At this point, test item availability and resources are often limited, so it is not necessary or feasible to run a full standard Ames test.
For these cases we can use screening versions of the Ames test, such as testing only in the presence of metabolic activation, using a reduced top dose level, fewer replicate plates (singe plates per dose), reducing the number of bacterial strains examined (often using only TA98 and TA100), or a combination of these. The advantage of this approach is that results can be directly extrapolated to what might be expected in the subsequent GLP test.
The disadvantage is that there will be some difficulty in detection of weak mutagens or, in the case of reducing the number of strains, that some mutagens will be missed. However, a positive result in the Ames assay is sufficient to categorize the chemical as a mutagen.
When to Perform the Ames Test
- IND-enabling studies
- As part of the ICH S2(R1) standard battery (Option 1 or 2)
- REACH requirements
- As part of Annex VII testing
- Impurity assessment
- Required when an impurity, degradant or process intermediate has been identified as a possible mutagen during in silico analysis as per ICH M7 guideline
Ames Test Procedure
- Typically, four tester strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and one strain of Escherichia coli (WP2uvrA) are used in each assay
- Others are available, including TA97a, TA102, and WP2uvrA (pKM101)
- Each tester strain is incubated for 2-3 days with several dose levels of the test compound in the presence and absence of metabolic activation (S9)
- Positive outcome is characterized by a dose-dependent increase in revertant frequency that exceeds strain-specific limits
What is an Ames test?
The Ames test, (i.e., Salmonella typhimurium and/or Escherichia coli reverse mutation assay) offered at Charles River is a bacterial short-term test for the identification of carcinogens that measures mutations in the DNA in bacteria.
What is the difference between the plate incorporation and pre-incubation method in an Ames assay?
In the plate incorporation method the test item is mixed with the top agar and bacteria and is immediately plated. In the pre-incubation method the bacterial strains are mixed with the test item in the presence or absence of S9-mix for 20-30 minutes at 37 °C (with shaking). Thereafter, this mixture is added to top agar and plated. The pre-incubation method is generally considered the most sensitive method for detecting mutagens but is also more susceptible to cytotoxicity.
How are toxic effects of the Ames test item evaluated?
Cytotoxicity of the test item is normally indicated by the partial or complete absence of a bacterial background lawn or a substantial dose-related reduction in revertant colony counts, compared with the lower dose levels and concurrent vehicle control accounting for the laboratory historical control range.
Why are 5 tester strains required for mutagenicity testing in the Ames assay?
Every bacteria strain detects a certain type of damage. To cover a wide range of mutational events, five tester strains are included in the Ames assay.
Strain Detects TA98 frameshift TA100
WP2 uvrA, or
WP2 uvrA (pKM101)
base substitution, x-linker