[Code of Federal Regulations]

[Title 40, Volume 31]

[Revised as of July 1, 2006]

From the U.S. Government Printing Office via GPO Access

[CITE: 40CFR798.5265]



[Page 187-190]

 

                   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-[rarr] 

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



[[Page 188]]



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.

    (iii) Bacterial growth. Fresh cultures of bacteria should be grown 

up to the late exponential or early stationary phase of growth 

(approximately 10\8\-10\9\ 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.



[[Page 189]]



    (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 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.



[[Page 190]]



    (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.

    (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]