United States Prevention, Pesticides EPA712-C-96-219
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA 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 pub-
lished 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. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
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 "Environ-
mental 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 com-
ments. 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, Ar-
lington, 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 Bul-
letin 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 test-
ing 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 har-
monized OPPTS test guideline are OPPT 40 CFR 798.5265 The Sal-
monella 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 in-
duced 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 lim-
ited to, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthra-
cene, congo red, benzopurpurin 4B, trypan blue, or Direct Blue 1.
(e) Test method—(1) Principle. Bacteria are exposed to test chemi-
cal with and without a metabolic activation system and plated onto mini-
mal medium. After a suitable period of incubation, revertant colonies are
counted and compared to the number of spontaneous revertants in an un-
treated 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 incorpo-
ration 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 charac-
teristics 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 (approxi-
mately 108-109 cells/mL).
(4) Metabolic activation. Bacteria should be exposed to the test sub-
stance both in the presence and absence of an appropriate metabolic activa-
tion system. For the direct plate incorporation method, the most commonly
used system is a cofactor supplemented postmitochondrial fraction pre-
pared 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 con-
trols 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 sys-
tem. The positive control reference substance for tests including a meta-
bolic activation system should be selected on the basis of the type of acti-
vation 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 per-
formed 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 chem-
ical may be altered in the presence of metabolic activation systems. Tox-
icity 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 con-
sidered 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 chemical and 0.1 ml 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 ade-
quate 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 (iL 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 shak-
ing 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 trip-
licate.
(g) Data and report—(1) Treatment of results. Data should be pre-
sented 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 deter-
mining 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 sig-
nificant 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 re-
verse mutation assay indicate that, under the test conditions, the test sub-
stance induces point mutations by base changes or frameshifts in the ge-
nome 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 informa-
tion 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 ad-
ditional background material on this test guideline.
(1) Ames, B.N. et al. Methods for detecting carcinogens and mutagens
with the &z/mo«e//a/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).
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