[Code of Federal Regulations]
[Title 40, Volume 28]
[Revised as of July 1, 2002]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.5265]

[Page 186-189]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 798--HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
 
                       Subpart F--Genetic Toxicity
 
Sec. 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:

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    (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.
    (iii) Bacterial growth. Fresh cultures of bacteria should be grown 
up to the late exponential or early stationary phase of growth 
(approximately 108-109 cells per ml).
    (4) Metabolic activation. Bacteria should be exposed to the test 
substance both in the presence and absence of an appropriate metabolic 
activation system. For the direct plate incorporation method, the most 
commonly used system is a cofactor supplemented postmitochondrial 
fraction prepared from the livers of rodents treated with enzyme 
inducing agents such as Aroclor 1254. For the azo-reduction method, a 
cofactor supplemented postmitochondrial fraction prepared from the 
livers of untreated hamsters is preferred. For this method, the cofactor 
supplement should contain flavin mononucleotide, exogenous glucose 6-
phosphate dehydrogenase, NADH and excess of glucose-6-phosphate.
    (5) Control groups--(i) Concurrent controls. Concurrent positive and 
negative (untreated and/or vehicle) controls shall be included in each 
experiment. Positive controls shall ensure both strain responsiveness 
and efficacy of the metabolic activation system.
    (ii) Strain specific positive controls. Strain specific positive 
controls shall be included in the assay. Examples of strain specific 
positive controls are as follows:
    (A) Strain TA 1535, TA 100, sodium azide.
    (B) TA 98, 2-nitrofluorene.
    (C) TA 1537, 9-aminoacridine.
    (iii) 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. 2-Aminoanthracene is an example 
of a positive control compound in plate-incorporation tests using 
postmitochondrial fractions from the livers of rodents treated with 
enzyme inducing agents such as Aroclor-1254. Congo red is an example of 
a positive control compound in the azo-reduction method. Other positive 
control reference substances may be used.
    (iv) Class-specific positive controls. The azo-reduction method 
should include positive controls from the same class of compounds as the 
test agent wherever possible.
    (6) Test chemicals--(i) Vehicle. 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.
    (ii) Exposure concentrations. (A) 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. Cytotoxicity of 
the test chemical may be altered in the presence of metabolic activation 
systems. Toxicity may be evidenced by a reduction in the number of 
spontaneous revertants, a clearing of the background lawn or by the 
degree of survival of treated cultures. Relatively insoluble compounds 
should be tested up to the limits of solubility. For freely soluble 
nontoxic chemicals, the upper test chemical concentration should be 
determined on a case by case basis.
    (B) Generally, a maximum of 5 mg/plate for pure substances is 
considered acceptable. At least 5 different amounts of test substance 
shall be

[[Page 188]]

tested with adequate intervals between test points.
    (C) When appropriate, a single positive response shall be confirmed 
by testing over a narrow range of concentrations.
    (e) Test performance--(1) Direct plate incorporation method. For 
this test without metabolic activation, test chemica1 and 0.1 m1 of a 
fresh bacterial culture should be added to 2.0 ml of overlay agar. For 
tests with metabolic activation, 0.5 ml of activation mixture containing 
an adequate amount of postmitochondrial fraction should be added to the 
agar overlay after the addition of test chemical and bacteria. Contents 
of each tube shall be mixed and poured over the surface of a selective 
agar plate. Overlay agar shall be allowed to solidify before incubation. 
At the end of the incubation period, revertant colonies per plate shall 
be counted.
    (2) Azo-reduction method. (i) For this test with metabolic 
activation, 0.5 ml of S-9 mix containing 150 ul of S-9 and 0.1 ml of 
bacterial culture should be added to a test tube kept on ice. One-tenth 
milliliter of chemical should be added, and the tubes should be 
incubated with shaking at 30 [deg]C for 30 min. At the end of the 
incubation period, 2.0 ml of agar should be added to each tube, the 
contents mixed and poured over the surface of a selective agar plate. 
Overlay agar shall be allowed to solidify before incubation. At the end 
of the incubation period, revertant colonies per plate shall be counted.
    (ii) For tests without metabolic activation, 0.5 ml of buffer should 
be used in place of the 0.5 ml of S-9 mix. All other procedures shall be 
the same as those used for the test with metabolic activation.
    (3) Other methods. Other methods may also be appropriate.
    (4) Media. An appropriate selective medium with an adequate overlay 
agar shall be used.
    (5) Incubation conditions. All plates within a given experiment 
shall be incubated for the same time period. This incubation period 
shall be for 48-72 hours at 37 [deg]C.
    (6) Number of cultures. All plating should be done at least in 
triplicate.
    (f) Data and report--(1) Treatment of results. Data shall be 
presented as number of revertant colonies per plate for each replicate 
and dose. The numbers of revertant colonies on both negative (untreated 
and/or vehicle) and positive control plates shall also be presented. 
Individual plate counts, the mean number of revertant colonies per plate 
and standard deviation shall be presented for test chemical and positive 
and negative (untreated and/or vehicle) controls.
    (2) Statistical evaluation. Data should be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of revertants. Another 
criterion may be based upon detection of a reproducible and 
statistically significant positive response for at least one of the test 
substance concentrations.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of revertants or a 
statistically significant and reproducible positive response at any one 
of the test points is considered nonmutagenic in this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) Positive results from the S. typhimurium 
reverse mutation assay indicate that, under the test conditions, the 
test substance induces point mutations by base changes or frameshifts in 
the genome of this organism.
    (ii) Negative results indicate that under the test conditions the 
test substance is not mutagenic in S. typhimurium.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported:
    (i) Bacterial strain used.
    (ii) Metabolic activation system used (source, amount and cofactor); 
details of preparations of S-9 mix.
    (iii) Dose levels and rationale for selection of dose.

[[Page 189]]

    (iv) Positive and negative controls.
    (v) Individual plate counts, mean number of revertant colonies per 
plate, standard deviation.
    (vi) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Ames, B.N., McCann, J., Yamasaki, E. ``Methods for detecting 
carcinogens and mutagens with the Salmonella/ mammalian-microsome 
mutagenicity test,'' Mutation Research 31:347-364 (1975).
    (2) de Serres, F.J., Shelby, M.D. ``The Salmonella mutagenicity 
assay: recommendations,'' Science 203:563-565 (1979).
    (3) Prival, M.J., Mitchell, V.D. ``Analysis of a method for testing 
azo dyes for mutagenic activity in Salmonella typhimurium in the 
presence of flavin mononucleotide and hamster liver S-9,'' Mutation 
Research 97:103-116 (1982).
    (4) Vogel, H.J., Bonner, D.M. ``Acetylornithinase of E. coli: 
partial purification and some properties,'' Journal of Biological 
Chemistry. 218:97-106 (1956).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19078, May 20, 1987]