1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Cells
A variety of cell types are available for use in this test including subclones of L5178Y, CHO, CHO-AS52, V79 or TK6 cells. Cell types used in this test should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency. Cells should be checked for mycoplasma contamination and should not be used if contaminated.
The test should be designed to have a predetermined sensitivity and power. The number of cells, cultures and concentrations of test substance used should reflect these defined parameters (14). The minimal number of viable cells surviving treatment and used at each stage in the test should be based on the spontaneous mutation frequency. A general guide is to use a cell number, which is at least 10 times the inverse of the spontaneous mutation frequency. However, it is recommended to utilise at least 10
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cells. Adequate historical data on the cell system used should be available to indicate consistent performance of the test.
1.4.1.2. Media and culture conditions
Appropriate culture media, and incubation conditions (culture vessels, temperature, CO
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concentration, and humidity) should be used. Media should be chosen according to the selective systems and cell type used in the test. It is particularly important that culture conditions should be chosen that ensure optimal growth of cells during the expression period and colony forming ability of both mutant and non-mutant cells.
1.4.1.3. Preparation of cultures
Cell are propagated from stock cultures, seeded in culture medium and incubated at 37
o
C. Prior to use in this test, cultures may need to be cleansed of pre-existing mutant cells.
1.4.1.4. Metabolic activation
Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (15)(16)(17)(18) or a combination of phenobarbitone and ß–naphthoflavone (19)(20).
The post-mitochondrial fraction is usually used at concentrations in the range from 1-10 % v/v in the final test medium. The choice and condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases it may be appropriate to utilise more than one concentration of post-mitochondrial fraction.
A number of developments, including the construction of genetically engineered cell lines expressing specific activating enzymes, may provide the potential for endogenous activation. The choice of the cell lines used should be scientifically justified (e.g. by the relevance of the cytochrome P450 isoenzyme for the metabolism of the test substance).
1.4.1.5. Test substance/Preparation
Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the cells. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.
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