Q：How to select solvent/excipient for Ames test?

A：The premise of Ames test is to first have samples of different doses and concentrations. There are also some industry requirements for preparing samples that meet the test requirements. First, the selection principle of solvent/excipient is that it does not react with the test substance, has no toxicity to bacterial strain and S9, and has no mutagenicity; second, distilled water is preferred. Other solvents/excipients can be selected for the test substance that is insoluble in water. If the test substance is unstable to water, water free organic solvents or excipients should be selected; third, if organic solvent is selected, DMSO is preferred, and other solvents can also be selected. If uncommon solvent or excipient is selected, should provide some reference materials. If organic solvent is selected, the maximum amount added per plate should not exceed 0.1mL.

Q：How to define the highest dose group of Ames test?

A：The determination of the highest dose group of Ames test depends on the toxicity of the test substance to the test strain and the solubility of the test substance. It is recommended to conduct a preliminary experiment to determine the toxicity and solubility of the test sample. The highest dose group of the test sample shall be determined according to the toxicity of the test sample to bacteria and the solubility in the final mixture. The indications of toxicity: reduction of the number of revertant colonies, reduction of the size of the background lichen or reduction of the bacterial survival rate after treatment.

Q: How to set Ames test group?

A：Ames test usually includes four groups: untreated control group (spontaneous control group), positive control group (standard mutagen control group), solvent control group and sample dose group. Please note that if the solvent used for dissolving samples is different from that used for standard mutagens, DMSO control should also be added, and there will be some differences in the requirements of different areas in the number of sample dose groups. It is necessary to refer to specific reference standards or guidelines.

Q：Why should we test with or without metabolic activation system?

A：The metabolic activation system is the mixing solution of S9 and cofactors. Induction S9 itself is a component of animal liver after drug induction, and the purpose of induction is to amplify the enzyme activity. The group with metabolic activation system evaluated the genotoxicity of metabolites, while the group without metabolic activation system evaluated the genotoxicity of API. For unknown compounds, it is uncertain whether they are their own or whether their metabolites will have genotoxicity, so it is necessary to conduct tests in both cases.

Q: How many samples can Ames Kit detect?

A：Ames Kit can complete the detection of 1 sample. The specification of Ames Kit (5 bacterial versions/formal test) is 250 dishes/box, The genotoxicity test can be carried out for the control group (untreated control group, vehicle control group, positive control group) and the five dose groups of samples with and without metabolic activation system, five strains and three parallel conditions; The specification of Ames Kit (2 bacterial versions/pre test) is 150 dishes/box, The genotoxicity test can be carried out in the control group (untreated control group, vehicle control group, positive control group) and the 8 dose groups of samples with and without metabolic activation system, 2 strains, and 3 parallel conditions; The specification of the micro fluctuation Ames Kit is 96 holes * 16 plates, which can be used for genotoxicity testing of the control group (untreated control group, solvent control group, positive control group) and five dose groups of samples with or without metabolic activation system, two strains, 48 holes/treatment group.

Q: Can the strains in the kit be subcultured?

A：It is not recommended. Reasons: First, the strains in Ames Kit is frozen bacterial solution, which can meet the Ames test requirements. No other treatment is required after receiving the goods, and can be use directly; Second, the strains in Ames Kit are all nutrient deficient strains, frequent treatment may lose characteristics; Third, Ames test requires not only the concentration of strains, but also the number of spontaneous and positive mutant colonies. Although the national standard or guiding principles are relatively simple, it is difficult to meet the requirements.

Q:Can Ames Kit be used multiple times?

A: Not recommended. If multiple tests are required, it is suggested that different strains should be tested in different times. The strains in Ames Kit are nutrient deficient strains, It is also provided in the form of frozen bacterial solution. Repeated freezing and thawing may lead to the loss of some of its characteristics, On the other hand, it will also directly lead to the spontaneous and positive mutagen revertants failing to meet the requirements of standards or guidelines. S9 complex is a metabolic activation system in Ames test, and the metabolism is mainly caused by the participation of enzymes. Repeated freezing and thawing will reduce the enzyme activity, which may lead to false negative test results.

Q：Does Ames test still need to be conducted when the test substance (such as some antibiotics) is highly toxic to bacteria? Are there any other verification methods?

A：Ames test should still be performed when the test substance is highly toxic to bacteria, because mutagenicity may occur at a lower concentration with less toxicity. In order to reduce the risk of false negative results of compounds with potential genotoxicity, genotoxicity research usually requires a combination of multiple tests. The positive result of any genotoxicity test does not indicate that the compound is truly genotoxic or carcinogenic to humans. Therefore, gene mutation test and chromosome aberration test can be added.

Q: How to deal with the indistinguishable colony or precipitation caused by the poor solubility of the compound?

A：The conventional requirement is that there can be undissolved compounds in the plate, but the presence of precipitation cannot affect the colony count. If it is unavoidable, the colony can be observed and counted after 48h of culture, and continue to culture until 72h. If it grows, it can be judged as a colony, or it can be picked up and inoculated to the culture medium to observe whether it grows.

Q：There are more colonies in the group with S9 than in the group without S9. Is it normal?

A：It is normal, but even if it is too high, it will be within the requirements of national standards or guidelines. It may be that the liver S9 itself contains trace amino acid components, which can provide nutrition for bacteria.

Q: Is there no genotoxicity detected or no genotoxicity itself when the concentration of the test substance is low? How to judge?

A: On the one hand, the Ames test requires that if the test result is negative, verification should be carried out, that is, the test conditions should be changed to repeat the test. On the other hand, in order to reduce the risk of false negative results, genotoxicity research usually requires a combination of multiple tests, which can complement other tests such as gene mutation, chromosome aberration and micronucleus.

Q：Why choose water as the extraction solvent? Normal saline or DMSO should be selected according to the standard.