Standard Ames Test
The Ames test is a widely utilized assay for detecting mutagenic potential in chemical compounds, including N-Nitrosamines. Despite its significance in toxicological screening, the standard Ames test has several limitations, particularly when evaluating compounds requiring complex metabolic activation.
Limitations of the Standard Ames Test
While the Ames test remains a cornerstone in mutagenicity testing, it has certain limitations:
- Limited Metabolic Activation:
The standard Ames Test typically uses rat liver S9, which lacks certain human-relevant cytochrome P450 (CYP) enzymes. This can lead to underactivation of procarcinogens (e.g., some N-nitrosamines) or overactivation of non-human hazards. Example: Rat S9 poorly metabolizes N-nitrosodiethylamine (NDEA) compared to human enzymes, risking false negatives.
- Limited Mutation Spectrum Detection:
Standard strains (e.g., TA98, TA100) detect only specific mutation types (frameshifts, base-pair substitutions), missing clastogens or epigenetic carcinogens.
- Over-Reliance on Bacterial Systems:
Since the test uses Salmonella typhimurium, it does not account for DNA repair mechanisms present in mammalian cells, potentially missing some mutagenic effects.
- False Positives and Negatives:
Some non-mutagenic compounds may yield positive results due to bacterial stress responses, while some mutagens requiring complex metabolic activation may go undetected.
The Enhanced Ames Test
EMA's latest release Q&As Guidance states that due to the low sensitivity of some N-nitrosamines (e.g., NDMA) under the conditions of the standard Ames test, the conditions of the Enhanced Ames Test provided by FDA's National Center for Toxicological Research (NCTR) are recommended. The FDA has concluded that the standard methods used for the Ames Test may not be sufficient to characterize the mutagenic potential of N-nitrosamines, and in some cases may produce negative results for known mutagenic nitrosamines. In response, FDA's National Center for Toxicological Research has been testing different conditions to develop the Enhanced Ames Test, which is intended to provide a more reliable assessment of the mutagenic potential of N-nitrosamine impurities.
The following Enhanced Ames Test (EAT) conditions are provided by the FDA
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Test strains |
Includes Salmonella typhimurium TA98, TA100. TA1535,TA1537 and Escherichia coli WP2 uvrA (pKM101) test strain |
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Test method and pre-insulation time |
Pre-insulation and non-flatbedding methods should be used, with a recommended pre-insulation time of 30 minutes. |
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S9 Type and Concentration |
The Enhanced Ames test should be performed in containing 30% rat liver S9 and 30% Hamster Liver S9. Rat and hamster desmosomal supernatants (S9s) should be prepared from rodent livers treated with cytochrome P450 enzyme-inducing substances (e.g., a combination of phenobarbital and β-naphthoflavone). |
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Negative (solvent/excipient) control |
The solvents used should be compatible with the Ames test according to OECD 471 guidelines. Available solvents include, but are not limited to: (1) Water; (2) Organic solvents such as acetonitrile, methanol, and dimethyl sulfoxide (DMSO). When organic solvents are used, the lowest possible volume in the pre-holding mixture should be used and it should be demonstrated that the amount of organic solvent used does not interfere with the metabolic activation of N-nitrosamines. |
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Positive Control |
According to OECD 471 guidelines, strain-specific positive controls should be performed at the same time. In the presence of S9, the two N-nitrosamines known to be mutagenic should also be used as positive controls. Available N-nitrosamine positive controls include: NDMA, 1-cyclopentyl-4-nitrosopiperazine NDSRIs。 |
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All other recommendations on Ames determination should follow OECD 471 guidelines |
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Unique Advantage of Hamster Liver S9 Fraction
Hamster liver S9 fraction is particularly valuable in Ames testing due to its superior ability to activate certain N-Nitrosamines compared to rat liver S9. It shares a closer metabolic profile to human liver enzymes, making it more relevant for human risk assessment. Additionally, hamster liver S9 contains higher levels of specific cytochrome P450 enzymes, which are crucial for the bioactivation of mutagens. This results in improved sensitivity and reduced false-negative rates, allowing better detection of mutagens that may otherwise go undetected with rat liver S9 alone.
Conclusion
While the standard Ames test remains a fundamental tool for assessing mutagenicity, its limitations necessitate improvements for better accuracy. The enhanced Ames test, particularly with the inclusion of hamster liver S9 fraction, significantly enhances the detection of complex mutagens like N-Nitrosamines, bridging the gap between bacterial and mammalian metabolism. This advancement provides a more reliable method for evaluating potential human carcinogens and supports better regulatory decision-making.
Keywords: N-Nitrosamines, NDSRIs, OECD 471, Enhanced Ames test, Hamster liver S9, Cytochrome P450 enzymes, Mutation Test
Post time: 2025-03-12 09:22:09