§ 79.68 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 the microorganism Salmonella typhimurium.
(b) Definitions. For the purposes of this section, the following definitions apply:
Base pair mutagen means an agent which causes a base change in DNA. In a reversion assay, this change may occur at the site of the original mutation or at a second site in the chromosome.
Frameshift mutagen is an agent which causes the addition or deletion of single or multiple base pairs in the DNA molecule.
Salmonella typhimurium reverse mutation assay detects mutation in a gene of a histidine-requiring strain to produce a histidine independent strain of this organism.
(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. Motor vehicle combustion emissions from fuel or additive/base fuel mixtures are, first, filtered to trap particulate matter and, then, passed through a sorbent resin to trap semi-volatile gases. Bacteria are separately exposed to the extract from both the filtered particulates and the resin-trapped organics. Assays are conducted using both test mixtures with and without a metabolic activation system and exposed cells are plated onto minimal medium. After a suitable period of incubation, revertant colonies are counted in test cultures and compared to the number of spontaneous revertants in unexposed control cultures.
(2) Description. Several methods for performing the test have been described. The procedures described here are for the direct plate incorporation method and the azo-reduction method. Among those used are:
(i) Direct plate incorporation method;
(ii) Preincubation method;
(iii) Azo-reduction method;
(iv) Microsuspension method; and
(v) Spiral assay.
(3) Strain selection—(i) Designation. Five tester strains shall be used in the assay. At the present time, TA1535, TA1537, TA98, and TA100 are designated as tester strains. The fifth strain will be chosen from the pool of Salmonella strains commonly used to determine the degree to which nitrated organic compounds, i.e., nitroarenes, contribute to the overall mutagenic activity of a test substance. TA98/1,8–DNP6 or other suitable Rosenkranz nitro-reductase resistant strains will be considered acceptable. The choice of the particular strain is left to the discretion of the researcher. However, the researcher shall justify the use of the selected bacterial tester strains.
(ii) Preparation and storage of bacterial tester strains. Recognized methods of stock culture preparation and storage shall be used. The requirement of histidine for growth shall be demonstrated for each strain. Other phenotypic characteristics shall be checked using such methods as crystal violet sensitivity and resistance to ampicillin. Spontaneous reversion frequency shall be in the range expected as reported in the literature and as established in the laboratory by historical control values.