For the azo reduction method, a cofactor supplemented postmitochondrial fraction prepared from the livers of untreated hamsters is preferred. Positive controls should ensure both strain responsiveness and efficacy of the metabolic activation system. Strain specific positive controls should be included in the assay. Positive controls to ensure the efficacy of the activation system. The positive control reference substance for tests including a metabolic activation system should be selected on the basis of the type of activation system used in the test.
Congo red is an example of a positive control compound in the azo reduction method.
Other positive control reference substances may be used. The azo reduction method should include positive controls from the same class of compounds as the test agent wherever possible. Test chemicals and positive control reference substances should be dissolved or suspended in an appropriate vehicle and then further diluted in vehicle for use in the assay. The test should initially be performed over a broad range of concentrations. Among the criteria to be taken into consideration for determining the upper limits of test chemical concentration are cytotoxicity and solubility.