United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–219

June 1996

Health Effects Test

Guidelines

OPPTS 870.5265

The Salmonella

typhimurium Reverse

Mutation Assay

‘‘Public Draft’’
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INTRODUCTION

This guideline is one of a series of test guidelines that have been

developed by the Office of Prevention, Pesticides and Toxic Substances,

United States Environmental Protection Agency for use in the testing of

pesticides and toxic substances, and the development of test data that must

be submitted to the Agency for review under Federal regulations.

The Office of Prevention, Pesticides and Toxic Substances (OPPTS)

has developed this guideline through a process of harmonization that

blended the testing guidance and requirements that existed in the Office

of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,

Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the

Office of Pesticide Programs (OPP) which appeared in publications of the

National Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development

(OECD).

The purpose of harmonizing these guidelines into a single set of

OPPTS guidelines is to minimize variations among the testing procedures

that must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

Public Draft Access Information: This draft guideline is part of a

series of related harmonized guidelines that need to be considered as a

unit. For copies: These guidelines are available electronically from the

EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP

Public Docket at (703) 305–5805 or by e-mail:

guidelines@epamail.epa.gov.

To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office

of Pesticide Programs, Field Operations Division (7506C), Environmental

Protection Agency, 401 M St. SW., Washington, DC 20460. In person:

bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending

electronic mail (e-mail) to: guidelines@epamail.epa.gov.

Final Guideline Release: This guideline is available from the U.S.

Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp:

fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0132 for disks

or paper copies. This guideline is also available electronically in ASCII

and PDF (portable document format) from the EPA Public Access Gopher

(gopher.epa.gov) under the heading ‘‘Environmental Test Methods and

Guidelines.’’
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OPPTS 870.5265 The Salmonella typhimurium reverse mutation

assay.

(a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and

Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances

Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source materials used in developing this harmonized OPPTS test guideline are OPPT 40 CFR 798.5265 The Salmonella typhimurium Reverse Mutation Assay and OECD 471 Genetic

Toxicology: Salmonella typhimurium Reverse Mutation Assay.

(b) 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.

(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR

Part 792—Good Laboratory Practice Standards (GLP) apply to this test

guideline. The following definitions also apply to this test guideline.

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.

Frameshift mutagens are agents which cause the addition or deletion

of single or multiple base pairs in the DNA molecule.

A reverse mutation assay in S. typhimurium detects mutation in a

gene of a histidine requiring strain to produce a histidine independent

strain of this organism.

(d) 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.

(e) 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.
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The procedures described in this guideline 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/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 should be included in each

experiment. Positive controls should ensure both strain responsiveness and

efficacy of the metabolic activation system.

(ii) Strain specific positive controls. Strain specific positive controls

should 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 posi-
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tive 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 five different amounts of test substance should

be tested with adequate intervals between test points.

(C) When appropriate, a single positive response should be confirmed

by testing over a narrow range of concentrations.

(f) 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 should be mixed and poured over the surface of a selective agar plate.

Overlay agar should be allowed to solidify before incubation. At the end

of the incubation period, revertant colonies per plate should be counted.

(2) Azo reduction method. (i) For this test with metabolic activation,

0.5 mL of S–9 mix containing 150 μL 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 °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 should be allowed to
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solidify before incubation. At the end of the incubation period, revertant

colonies per plate should 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 should

be the same as those used for the test with metabolic activation.

(3) Media. An appropriate selective medium with an adequate overlay

agar should be used.

(4) Incubation conditions. All plates within a given experiment

should be incubated for the same time period. This incubation period

should be for 48–72 h at 37 °C.

(5) Number of cultures. All plating should be done at least in triplicate.

(g) Data and report—(1) Treatment of results. Data should 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 should also be presented. Individual

plate counts, the mean number of revertant colonies per plate and standard

deviation should 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 doserelated 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.
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(5) Test report. In addition to the reporting recommendations as

specified under 40 CFR part 792, subpart J, the following specific information should 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.

(h) References.The following references should be consulted for additional background material on this test guideline.

(1) Ames, B.N. et al. Methods for detecting carcinogens and mutagens

with the Salmonella/mammalian-microsome mutagenicity test. Mutation

Research 31:347–364 (1975).

(2) de Serres, F.J. and Shelby, M.D. The Salmonella mutagenicity

assay: recommendations. Science 203:563–565 (1979).

(3) Prival, M.J. and 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. and Bonner, D.M. Acetylornithinase of E. coli: partial

purification and some properties. Journal of Biological Chemistry. 218:97–

106 (1956).