§ 798.5265 The salmonella typhimurium reverse mutation assay.

(a) Purpose. The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures his−→ his ⁼ reversion induced by chemicals which cause base changes or frameshift mutations in the genome of this organism.

(b) Definitions. (1) A reverse mutation assay in Salmonella typhimurium detects mutation in a gene of a histidine requiring strain to produce a histidine independent strain of this organism.

(2) Base pair mutagens are agents which cause a base change in the DNA. In a reversion assay, this change may occur at the site of the original mutation or at a second site in the chromosome.

(3) Frameshift mutagens are agents which cause the addition or deletion of single or multiple base pairs in the DNA molecule.

(c) Reference substances. These may include, but need not be limited to, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene, congo red, benzopurpurin 4B, trypan blue or direct blue 1.

(d) Test method—(1) Principle. Bacteria are exposed to test chemical with and without a metabolic activation system and plated onto minimal medium. After a suitable period of incubation, revertant colonies are counted and compared to the number of spontaneous revertants in an untreated and/or vehicle control culture.

(2) Description. Several methods for performing the test have been described. Among those used are:

(i) The direct plate incorporation method.

(ii) The preincubation method.

(iii) The azo-reduction method.

The procedures described here are for the direct plate incorporation method and the azo-reduction method.

(3) Strain selection—(i) Designation. At the present time four strains, TA 1535, TA 1537, TA 98 and TA 100 should be used. The use of other strains in addition to these four is left to the discretion of the investigator.

(ii) Preparation and storage. Recognized methods of stock culture preparation and storage should be used. The requirement of histidine for growth should be demonstrated for each strain. Other phenotypic characteristics should be checked using such methods as crystal violet sensitivity and resistance to ampicillin. Spontaneous reversion frequency should be in the range expected either as reported in the literature or as established in the laboratory by historical control values.