OECD 471

Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system.

In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium. In the preincubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after two or three days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.

Several procedures for performing the bacterial reverse mutation test have been described. Among those commonly used are the plate incorporation method, the preincubation method the fluctuation method and the suspension method. Modifications for the testing of gases or vapours have been described. The procedures described in this guideline pertain primarily to the plate incorporation and preincubation methods. Either of them is acceptable for conducting experiments both with and without metabolic activation. Some compounds may be detected more efficiently using the preincubation method. These compounds belong to chemical classes that include short chain aliphatic nitrosamines, divalent metals, aldehydes, azo-dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds and nitro compounds It is also recognised that certain classes of mutagens are not always detected using standard procedures such as the plate incorporation method or preincubation method. These should be regarded as “special cases” and it is strongly recommended that alternative procedures should be used for their detection. The following “special  cases” could be identified (together with examples of procedures that could be used for their detection): azo-dyes and diazo compounds gases and volatile chemicals), and glycosides .

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