The Ames test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs.
1Mortelmans K and Zeiger E. (2000) Mutation Research 455; 29-60
|Method||Ames MPFTM 98/100|
|Strains||Salmonella typhimurium TA98 and TA100 (others available on request)|
|Test Article Concentrations||6 concentrations up to 2 mg/mL or the highest concentration at which the compound is soluble|
|Metabolizing System||Aroclor-1254 induced rat liver S9|
|Quality Controls||Vehicle control
|Number of Replicates||3 replicates per concentration|
|Test Article Requirements||50mg of solid compound|
|Data Delivery||Written report presenting protocol used and data|
What bacterial strains do you assess and why?
Cyprotex offer the Ames test as an early stage assessment of genotoxicity using a miniaturized screening version which requires less compound and evaluates two of the most common mutations, TA98 and TA100.
TA98 (frameshift mutation) and TA100 (base-pair substitution) are two common strains of Salmonella typhimurium strains assessed in Ames testing. Both strains have:
Why is it important to investigate mutagenicity of compounds?
Compounds that have been demonstrated to induce genetic mutations, chromosomal breaks, and/or rearrangements are considered genotoxic and have the potential to cause cancer in humans. In some cases it is not the compounds themselves that are genotoxic, but impurities generated in the manufacturing process; thus it is important to identify and test these impurities as well as the compounds.
Please provide an overview of the Cyprotex Ames test MPFTM mutagenicity assay.
We typically perform six concentrations with a top concentration of 2mg/mL depending on solubility constraints. The bacteria are exposed to the chemicals in the presence and absence of aroclor-induced rat S9. For the S9 incubations, an NADPH-regenerating system is also included. Exposure is over 90 min in medium containing sufficient histidine to support two cell divisions. The cultures are then diluted into pH indicator medium lacking histidine, and incubated for 48 hr. Cells that have undergone a reversion will grow in the well, resulting in a colour change during growth. The number of wells containing revertant colonies are counted and compared to the vehicle control.
What controls are included in the Ames assay?
Positive controls are known mutagens 2-nitrofluorene and 4-nitroquinoline-N-oxide for the assay without S9 and 2-aminoanthracene in the assay with S9. The negative control is the appropriate vehicle control.
What are the regulatory requirements for genotoxicity assessment?
Many different industries rely on genotoxicity testing for safety assessment of their products. These include the pharmaceutical and veterinary industries, the agrochemical and chemical industries, medical device industry and the cosmetics and personal care industries.
For the pharmaceutical industry, the general features of a standard test battery for regulatory genotoxicity studies include the assessment of mutagenicity in a bacterial reverse gene mutation test (e.g., Ames test) and the assessment of genotoxicity in mammalian cells in vitro and/or in vivo.
Two options are proposed in the 2011 ICH guidance S2(R1)2:
For compounds that give negative results, the completion of either option of the standard test battery, performed and evaluated in accordance with current recommendations, will usually provide sufficient assurance of the absence of genotoxic activity and no additional tests are warranted. Compounds that give positive results in the standard test battery might, depending on their therapeutic use, need to be tested more extensively.
In cases where compounds are highly toxic to bacteria (e.g., some antibiotics), the bacterial reverse mutation (Ames) test should still be carried out, just as cytotoxic compounds are tested in mammalian cells, because mutagenicity can occur at lower, less toxic concentrations. In such cases, any one of the in vitro mammalian cell assays should also be done, i.e., Option 1 should be followed.
For other industries (e.g., chemicals, pesticides and personal care products), the testing requirements and guidance tends to be country-specific. However, most industries have in vitro testing as the first stage in the testing battery for genotoxicity assessment, and typically this includes 3 tests covering 3 different endpoints. Further animal testing may be required for those test articles which are identified as genotoxic in the in vitro assays. However, in the cosmetics and personal care industry, animal testing is banned in many countries and so there is a total reliance on in vitro testing or other non-animal alternative methods.
1 Mortelmans K and Zeiger E. (2000) Mutation Research 455; 29-60
2 ICH guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1) November 2011
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