United States Prevention, Pesticides EPA712-C-98-247
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5100
Bacterial Reverse
Mutation Test
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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.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 870.5100 Bacterial reverse mutation test.
(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 798.5100 Escherichia
coli WP2 and WP2 uvrA reverse mutation assays, OPPTS 40 CFR
798.5265 The salmonella typhimurium reverse mutation assay and OECD
471 and 472, Bacterial Reverse Mutation Test.
(b) Purpose. (1) The bacterial reverse mutation test uses amino-acid
requiring strains of Salmonella typhimurium (S. typhimurium) and
Escherichia coli (E. coli) to detect point mutations, which involve substi-
tution, addition or deletion of one or a few DNA base pairs (see references
in paragraphs (g)(l), (g)(2), and (g)(3) of this guideline). The principle
of this bacterial reverse mutation test is that it detects mutations which
revert mutations present in the test strains and restore the functional capa-
bility of the bacteria to synthesize an essential amino acid. The revertant
bacteria are detected by their ability to grow in the absence of the amino
acid required by the parent test strain.
(2) Point mutations are the cause of many human genetic diseases
and there is substantial evidence that point mutations in oncogenes and
tumour suppressor genes of somatic cells are involved in tumour formation
in humans and experimental animals. The bacterial reverse mutation test
is rapid, inexpensive and relatively easy to perform. Many of the test
strains have several features that make them more sensitive for the detec-
tion of mutations, including responsive DNA sequences at the reversion
sites, increased cell permeability to large molecules and elimination of
DNA repair systems or enhancement of error-prone DNA repair processes.
The specificity of the test strains can provide some useful information on
the types of mutations that are induced by genotoxic agents. A very large
data base of results for a wide variety of structures is available for bacterial
reverse mutation tests and well-established methodologies have been de-
veloped for testing chemicals with different physico-chemical properties,
including volatile compounds.
(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.
Reverse mutation test in either Salmonella typhimurium or
Escherichia coli detects mutation in an amino-acid requiring strain (histi-
dine or tryptophan, respectively) to produce a strain independent of an
outside supply of amino-acid.
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Base pair substitution mutagens are agents that cause a base change
in DNA. In a reversion test this change may occur at the site of the original
mutation, or at a second site in the bacterial genome.
Frameshift mutagens are agents that cause the addition or deletion
of one or more base pairs in the DNA, thus changing the reading frame
in the RNA.
(d) Initial considerations. (1) The bacterial reverse mutation test uti-
lizes prokaryotic cells, which differ from mammalian cells in such factors
as uptake, metabolism, chromosome structure and DNA repair processes.
Tests conducted in vitro generally require the use of an exogenous source
of metabolic activation. In vitro metabolic activation systems cannot mimic
entirely the mammalian in vivo conditions. The test therefore does not pro-
vide direct information on the mutagenic and carcinogenic potency of a
substance in mammals.
(2) The bacterial reverse mutation test is commonly employed as an
initial screen for genotoxic activity and, in particular, for point mutation-
inducing activity. An extensive data base has demonstrated that many
chemicals that are positive in this test also exhibit mutagenic activity in
other tests. There are examples of mutagenic agents which are not detected
by this test; reasons for these shortcomings can be ascribed to the specific
nature of the endpoint detected, differences in metabolic activation, or dif-
ferences in bioavailability. On the other hand, factors which enhance the
sensitivity of the bacterial reverse mutation test can lead to an overestima-
tion of mutagenic activity.
(3) The bacterial reverse mutation test may not be appropriate for
the evaluation of certain classes of chemicals, for example highly bacteri-
cidal compounds (e.g., certain antibiotics) and those which are thought
(or known) to interfere specifically with the mammalian cell replication
system (e.g., some topoisomerase inhibitors and some nucleoside ana-
logues). In such cases, mammalian mutation tests may be more appro-
priate.
(4) Although many compounds that are positive in this test are mam-
malian carcinogens, the correlation is not absolute. It is dependent on
chemical class and there are carcinogens that are not detected by this test
because they act through other, non-genotoxic mechanisms or mechanisms
absent in bacterial cells.
(e) Test method—(1) Principle, (i) Suspensions of bacterial cells are
exposed to the test substance in the presence and in the absence of an
exogenous metabolic activation system. In the plate incorporation method,
these suspensions are mixed with an overlay agar and plated immediately
onto minimal medium. In the preincubation method, the treatment mixture
is incubated and then mixed with an overlay agar before plating onto mini-
mal medium. For both techniques, after 2 or 3 days of incubation, revertant
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colonies are counted and compared to the number of spontaneous revertant
colonies on solvent control plates.
(ii) Several procedures for performing the bacterial reverse mutation
test have been described. Among those commonly used are the plate incor-
poration method (see references in paragraphs (g)(l), (g)(2), (g)(3), and
(g)(4) of this guideline), the preincubation method (see references in para-
graphs (g)(2), (g)(3), (g)(5), (g)(6), (g)(7), and (g)(8) of this guideline),
the fluctuation method (see references in paragraphs (g)(9) and (g)(10)
of this guideline), and the suspension method (see reference in paragraph
(g)(ll) of this guideline). Suggestions for modifications for the testing of
gases or vapours have been described (see reference in paragraph (g)(12)
of this guideline).
(iii) The procedures described in this guideline pertain primarily to
the plate incorporation and preincubation methods. Either of them is ac-
ceptable for conducting experiments both with and without metabolic acti-
vation. Some compounds may be detected more efficiently using the
preincubation method. These compounds belong to chemical classes that
include short chain aliphatic nitrosamines, divalent metals, aldehydes, azo-
dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds and
nitro compounds (see reference in paragraph (g)(3) of this guideline). It
is also recognized that certain classes of mutagens are not always detected
using standard procedures such as the plate incorporation method or
preincubation method. These should be regarded as "special cases" and
it is strongly recommended that alternative procedures should be used for
their detection. The following "special cases" could be identified (to-
gether with examples of procedures that could be used for their detection):
azo-dyes and diazo compounds (see references in paragraphs (g)(3), (g)(5),
(g)(6), and (g)(13) of this guideline), gases and volatile chemicals (see
references in paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this guide-
line), and glycosides (see references in paragraphs (g)(17) and (g)(18) of
this guideline). A deviation from the standard procedure needs to be sci-
entifically justified.
(2) Description—(i) Preparations—(A) Bacteria. (7) Fresh cultures
of bacteria should be grown up to the late exponential or early stationary
phase of growth (approximately 109 cells per ml). Cultures in late station-
ary phase should not be used. The cultures used in the experiment should
contain a high titre of viable bacteria. The titre may be demonstrated either
from historical control data on growth curves, or in each assay through
the determination of viable cell numbers by a plating experiment.
(2) The culture temperature should be 37 °C.
(3) At least five strains of bacteria should be used. These should in-
clude four strains of S. typhimurium (TA1535; TA1537 or TA97a or
TA97; TA98; and TA100) that have been shown to be reliable and
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reproducibly responsive between laboratories. These four S. typhimurium
strains have GC base pairs at the primary reversion site and it is known
that they may not detect certain oxidising mutagens, cross-linking agents
and hydrazines. Such substances may be detected by E.coli WP2 strains
or S. typhimurium TA102 (see reference in paragraph (g)(19) of this guide-
line) which have an AT base pair at the primary reversion site. Therefore
the recommended combination of strains is:
(/) S. typhimurium TA1535.
(//) S. typhimurium TA1537 or TA97 or TA97a.
(///) S. typhimurium TA98.
(iv) S. typhimurium TA100.
(v) E. coli WP2 uvrA, or E. coli WP2 uvrA (pKMlOl), or S.
typhimurium TA102.
In order to detect cross-linking mutagens it may be preferable to include
TA102 or to add a DNA repair-proficient strain of E.coli [e.g., E.coli WP2
or E.coli WP2 (pKMlOl).]
(4) Established procedures for stock culture preparation, marker ver-
ification and storage should be used. The amino-acid requirement for
growth should be demonstrated for each frozen stock culture preparation
(histidine for S. typhimurium strains, and tryptophan for E. coli strains).
Other phenotypic characteristics should be similarly checked, namely: the
presence or absence of R-factor plasmids where appropriate [i.e. ampicillin
resistance in strains TA98, TA100 and TA97a or TA97, WP2 uvrA and
WP2 uvrA (pKMlOl), and ampicillin + tetracycline resistance in strain
TA102]; the presence of characteristic mutations (i.e. rfa mutation in S.
typhimurium through sensitivity to crystal violet, and uvrA mutation in
E. coli or uvrB mutation in S. typhimurium, through sensitivity to ultra-
violet light) (see references in paragraphs (g)(2) and (g)(3) of this guide-
line). The strains should also yield spontaneous revertant colony plate
counts within the frequency ranges expected from the laboratory's histori-
cal control data and preferably within the range reported in the literature.
(B) Medium. An appropriate minimal agar (e.g., containing Vogel-
Bonner minimal medium E and glucose) and an overlay agar containing
histidine and biotin or tryptophan, to allow for a few cell divisions, should
be used (see references in paragraphs (g)(l), (g)(2), and (g)(9) of this
guideline).
(C) Metabolic activation. Bacteria should be exposed to the test sub-
stance both in the presence and absence of an appropriate metabolic activa-
tion system. The most commonly used system is a cofactor-supplemented
post-mitochondrial fraction (S9) prepared from the livers of rodents treated
with enzyme-inducing agents such as Aroclor 1254 (see references in para-
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graphs (g)(l) and (g)(2) of this guideline) or a combination of
phenobarbitone and p-naphthoflavone (see references in paragraphs
(g)(18), (g)(20), and (g)(21) of this guideline). The post-mitochondrial
fraction is usually used at concentrations in the range from 5 to 30 percent
v/v in the S9-mix. The choice and condition of a metabolic activation sys-
tem may depend upon the class of chemical being tested. In some cases
it may be appropriate to utilize more than one concentration of post-
mitochondrial fraction. For azo-dyes and diazo-compounds, using a reduc-
tive metabolic activation system may be more appropriate (see references
in paragraphs (g)(6) and (g)(13) of this guideline).
(D) Test substance/preparation. Solid test substances should be dis-
solved or suspended in appropriate solvents or vehicles and diluted if ap-
propriate prior to treatment of the bacteria. Liquid test substances may
be added directly to the test systems and/or diluted prior to treatment.
Fresh preparations should be employed unless stability data demonstrate
the acceptability of storage.
(ii) Test conditions—(A) Solvent/vehicle. The solvent/vehicle should
not be suspected of chemical reaction with the test substance and should
be compatible with the survival of the bacteria and the S9 activity (see
reference in paragraph (g)(22) of this guideline). If other than well-known
solvent/vehicles are used, their inclusion should be supported by data indi-
cating their compatibility. It is recommended that wherever possible, the
use of an aqueous solvent/vehicle be considered first. When testing water-
unstable substances, the organic solvents used should be free of water.
(B) Exposure concentrations. (7) Amongst the criteria to be taken
into consideration when determining the highest amount of test substance
to be used are cytotoxicity and solubility in the final treatment mixture.
It may be useful to determine toxicity and insolubility in a preliminary
experiment. Cytotoxicity may be detected by a reduction in the number
of revertant colonies, a clearing or diminution of the background lawn,
or the degree of survival of treated cultures. The cytotoxicity of a sub-
stance may be altered in the presence of metabolic activation systems. In-
solubility should be assessed as precipitation in the final mixture under
the actual test conditions and evident to the unaided eye. The rec-
ommended maximum test concentration for soluble non-cytotoxic sub-
stances is 5 mg/plate or 5(il/plate. For non-cytotoxic substances that are
not soluble at Smg/plate or 5(il/plate, one or more concentrations tested
should be insoluble in the final treatment mixture. Test substances that
are cytotoxic already below Smg/plate or 5(il/plate should be tested up
to a cytotoxic concentration. The precipitate should not interfere with the
scoring.
(2) At least five different analysable concentrations of the test sub-
stance should be used with approximately half log (i.e. VlO) intervals be-
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tween test points for an initial experiment. Smaller intervals may be appro-
priate when a concentration-response is being investigated.
Testing above the concentration of 5 mg/plate or 5(il/plate may
be considered when evaluating substances containing substantial amounts
of potentially mutagenic impurities.
(C) Controls. (7) Concurrent strain-specific positive and negative
(solvent or vehicle) controls, both with and without metabolic activation,
should be included in each assay. Positive control concentrations that dem-
onstrate the effective performance of each assay should be selected.
(2) For assays employing a metabolic activation system, the positive
control reference substance(s) should be selected on the basis of the type
of bacteria strains used. The following chemicals are examples of suitable
positive controls for assays with metabolic activation:
Chemical
9,10-Dimethylanthracene
7 12-Dimethylbenzanthracene
Congo Red (for the reductive metabolic activation method) ...
Benzo(a)pyrene
Cyclophosphamide (monohydrate)
2-Aminoanthracene
781-43-1]
57_97_6]
573-58-0]
50-32-8]
50-18-0]
6055-19-2]
613-13-81
CAS number
2-Aminoanthracene should not be used as the sole indicator of the efficacy
of the S9-mix. If 2-aminoanthracene is used, each batch of S9 should
also be characterised with a mutagen that requires metabolic activation
by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene.
(3) For assays performed without metabolic activation system, exam-
ples of strain-specific positive controls are:
Chemical
(a) Sodium azide
(b) 2-Nitrofluorene
(c) 9-Aminoacridine or ICR 191
(d) Cumene hydroperoxide
(e) Mitomycin C
(f) N-Ethyl-N-nitro-N-nitrosoguanidine or
4-nitroquinoline 1-oxide
(g) Furylfuramide (AF-2)
CAS number
26628-2,
607-57-
90-45-9]
17070-4
80-15-9]
50-07-7
70-25-7
[56-57-5]
3688-53-
?-8]
3]
5-0]
-7]
Strain
TA1535andTA100
TA98
TA1537, TA97 and
TA97a
TA102
WP2 ui/r/\andTA102
WP2, WP2 uvrA and
WP2 t/wrt(pKM101)
Plasmid-containing
strains
(4) Other appropriate positive control reference substances may be
used. The use of chemical class-related positive control chemicals may
be considered, when available.
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(5) Negative controls, consisting of solvent or vehicle alone, without
test substance, and otherwise treated in the same way as the treatment
groups, should be included. In addition, untreated controls should also be
used unless there are historical control data demonstrating that no delete-
rious or mutagenic effects are induced by the chosen solvent.
(3) Procedure—(i) Treatment with test substance. (A) For the plate
incorporation method (see references in paragraphs (g)(l), (g)(2), (g)(3),
and (g)(4) of this guideline), without metabolic activation, usually 0.05
ml or 0.1 ml of the test solutions, 0.1 ml of fresh bacterial culture (contain-
ing approximately 108 viable cells) and 0.5 ml of sterile buffer are mixed
with 2.0 ml of overlay agar. For the assay with metabolic activation, usu-
ally 0.5 ml of metabolic activation mixture containing an adequate amount
of post-mitochondrial fraction (in the range from 5 to 30 percent v/v in
the metabolic activation mixture) are mixed with the overlay agar (2.0
ml), together with the bacteria and test substance/test solution. The con-
tents of each tube are mixed and poured over the surface of a minimal
agar plate. The overlay agar is allowed to solidify before incubation.
(B) For the preincubation method (see references in paragraphs (g)(2),
(g)(3), (g)(5), and (g)(6) of this guideline) the test substance/test solution
is preincubated with the test strain (containing approximately 108 viable
cells) and sterile buffer or the metabolic activation system (0.5 ml) usually
for 20 min. or more at 30-37 °C prior to mixing with the overlay agar
and pouring onto the surface of a minimal agar plate. Usually, 0.05 or
0.1 ml of test substance/test solution, 0.1 ml of bacteria, and 0.5 ml of
S9-mix or sterile buffer, are mixed with 2.0 ml of overlay agar. Tubes
should be aerated during pre-incubation by using a shaker.
(C) For an adequate estimate of variation, triplicate plating should
be used at each dose level. The use of duplicate plating is acceptable when
scientifically justified. The occasional loss of a plate does not necessarily
invalidate the assay.
(D) Gaseous or volatile substances should be tested by appropriate
methods, such as in sealed vessels (see references in paragraphs (g)(12),
(g)(14), (g)(15), and (g)(16) of this guideline).
(ii) Incubation. All plates in a given assay should be incubated at
37 °C for 48-72 hours. After the incubation period, the number of
revertant colonies per plate is counted.
(f) Data and reporting—(1) Treatment of results, (i) Data should
be presented as the number of revertant colonies per plate. The number
of revertant colonies on both negative (solvent control, and untreated con-
trol if used) and positive control plates should also be given.
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(ii) Individual plate counts, the mean number of revertant colonies
per plate and the standard deviation should be presented for the test sub-
stance and positive and negative (untreated and/or solvent) controls.
(iii) There is no requirement for verification of a clear positive re-
sponse. Equivocal results should be clarified by further testing preferably
using a modification of experimental conditions. Negative results need to
be confirmed on a case-by-case basis. In those cases where confirmation
of negative results is not considered necessary, justification should be pro-
vided. Modification of study parameters to extend the range of conditions
assessed should be considered in follow-up experiments. Study parameters
that might be modified include the concentration spacing, the method of
treatment (plate incorporation or liquid preincubation), and metabolic acti-
vation conditions.
(2) Evaluation and interpretation of results, (i) There are several
criteria for determining a positive result, such as a concentration-related
increase over the range tested and/or a reproducible increase at one or
more concentrations in the number of revertant colonies per plate in at
least one strain with or without metabolic activation system (see reference
in paragraph (g)(23) of this guideline). Biological relevance of the results
should be considered first. Statistical methods may be used as an aid in
evaluating the test results (see reference in paragraph (g)(24) of this guide-
line). However, statistical significance should not be the only determining
factor for a positive response.
(ii) A test substance for which the results do not meet the above cri-
teria is considered non-mutagenic in this test.
(iii) Although most experiments will give clearly positive or negative
results, in rare cases the data set will preclude making a definite judgement
about the activity of the test substance. Results may remain equivocal or
questionable regardless of the number of times the experiment is repeated.
(iv) Positive results from the bacterial reverse mutation test indicate
that a substance induces point mutations by base substitutions or
frameshifts in the genome of either Salmonella typhimurium and/or
Escherichia coll. Negative results indicate that under the test conditions,
the test substance is not mutagenic in the tested species.
(3) Test report. The test report should include the following informa-
tion:
(i) Test substance:
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
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(D) Stability of the test substance, if known.
(ii) Solvent/vehicle:
(A) Justification for choice of solvent/vehicle.
(B) Solubility and stability of the test substance in solvent/vehicle,
if known.
(iii) Strains:
(A) Strains used.
(B) Number of cells per culture.
(C) Strain characteristics.
(iv) Test conditions:
(A) Amount of test substance per plate (mg/plate or ml/plate) with
rationale for selection of dose and number of plates per concentration.
(B) Media used.
(C) Type and composition of metabolic activation system, including
acceptability criteria.
(D) Treatment procedures.
(v) Results:
(A) Signs of toxicity.
(B) Signs of precipitation.
(C) Individual plate counts.
(D) The mean number of revertant colonies per plate and standard
deviation.
(E) Dose-response relationship, where possible.
(F) Statistical analyses, if any.
(G) Concurrent negative (solvent/vehicle) and positive control data,
with ranges, means and standard deviations.
(H) Historical negative (solvent/vehicle) and positive control data,
with e.g., ranges, means and standard deviations.
(vi) Discussion of the results.
(vii) Conclusion.
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(g) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting
Carcinogens and Mutagens With the Salmonella/Mammalian-Microsome
Mutagenicity Test. Mutation Research 31, 347-364 (1975).
(2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella
Mutagenicity Test. Mutation Research 113, 173-215 (1983).
(3) Gatehouse, D. et al. Recommendations for the Performance of
Bacterial Mutation Assays. Mutation Research 312, 217-233 (1994).
(4) Kier, L.D. et al. The Salmonella Typhimurium/Mammalian
Microsomal Assay: A Report of the U.S. Environmental Protection Agency
Gene-Tox Program. Mutation Research 168, 69-240 (1986).
(5) Yahagi, T. et al. Mutagenicity of Carcinogen Azo Dyes and Their
Derivatives. Cancer Letters 1, 91-96 (1975).
(6) Matsushima, M. et al. Factors Modulating Mutagenicity Microbial
Tests. (Ed). Norpoth, K.H. and Garner, R.C. Short-Term Test Systems for
Detecting Carcinogens. (Springer, Berlin-Heidelberg-New York, 1980) pp.
273-285.
(7) Gatehouse, D.G. et al. Bacterial Mutation Assays. (Ed). Kirkland,
D.J. Basic Mutagenicity Tests. UKEMS Part 1 Revised. (Cambridge Uni-
versity Press, 1990) pp. 13-61.
(8) Aeschbacher, H.U., Wolleb, U., and Porchet, L.J. Liquid
Preincubation Mutagenicity Test for Foods. Food Safety 8, 167-177
(1987).
(9) Green, M.H.L., Muriel, W.J., and Bridges, B.A. Use of a Sim-
plified Fluctuation Test to Detect Low Levels of Mutagens. Mutation Re-
search 38, 33-42 (1976).
(10) Hubbard, S.A. et al. The Fluctuation Test in Bacteria. (Ed).
Kilbey, B.J., Legator, M., Nichols, W., and Ramel C. Handbook of Muta-
genicity Test Procedures. 2nd Edition. (Elsevier, Amsterdam-New York-
Oxford, 1984) pp. 141-161.
(11) Thompson, E.D. and Melampy, P.J . An Examination of the
Quantitative Suspension Assay for Mutagenesis With Strains of Salmonella
Typhimurium. Environmental Mutagenesis 3, 453-465 (1981).
(12) Araki, A. et al. Improved Method for Mutagenicity Testing of
Gaseous Compounds by Using a Gas Sampling Bag. Mutation Research
307, 335-344 (1994).
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(13) Prival, M.J. et al. Mutagenicity of Benzidine and Benzidine-Con-
gener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay.
Mutation Research 136, 33-47 (1984).
(14) Zeiger, E. et al. Salmonella Mutagenicity Tests. V. Results from
the Testing of 311 Chemicals. Environmental and Molecular Mutagenesis
19, 2-141 (1992).
(15) Simmon, V., Kauhanen, K., and Tardiff, R.G. Mutagenic Activ-
ity of Chemicals Identified in Drinking Water. (Ed). Scott, D., Bridges,
B., and Sobels, F. Progress in Genetic Toxicology. (Elsevier, Amsterdam,
1977) pp. 249-258.
(16) Hughes, T.J. et al. Vaporization Technique to Measure Muta-
genic Activity of Volatile Organic Chemicals in the Ames/Salmonella
Assay. Environmental Mutagenesis 9, 421-441 (1987).
(17) Matsushima, T. et al. Mutagenicity of the Naturally Occurring
Carcinogen Cycasin and Synthetic Methylazoxy Methane Conjugates in
Salmonella Typhimurium. Cancer Research 39, 3780-3782 (1979).
(18) Tamura, G. et al. Fecalase: A Model for Activation of Dietary
Glycosides to Mutagens by Intestinal Flora. Proceedings of the National
Academy of Sciences USA 77, 4961-4965 (1980).
(19) Wilcox, P. et al. Comparison of Salmonella Typhimurium TA
102 With Escherichia Coli WP2 Tester Strains. Mutagenesis 5, 285-291
(1990).
(20) Matsushima, T. et al. A Safe Substitute for Fob/chlorinated
Biphenyls as an Inducer of Metabolic Activation Systems. (Ed). F.J. de
Serres et al. In Vitro Metabolic Activation in Mutagenesis Testing.
(Elsevier, North Holland, 1976) pp. 85-88.
(21) Elliott, B.M. et al. Alternatives to Aroclor 1254-Induced S9 in
In Vitro Genotoxicity Assays. Mutagenesis 7, 175-177 (1992).
(22) Maron, D., Katzenellenbogen, J., and Ames, B.N. Compatibility
of Organic Solvents With the Salmonella/Microsome Test. Mutation Re-
search 88, 343-350 (1981).
(23) Claxton, L.D. et al. Guide for the Salmonella Typhimurium/
Mammalian Microsome Tests for Bacterial Mutagenicity. Mutation Re-
search 189, 83-91 (1987).
(24) Mahon, G.A.T. et al. Analysis of Data from Microbial Colony
Assays. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing
Part II. (Ed). Kirkland, D.J. Statistical Evaluation of Mutagenicity Test
Data. (Cambridge University Press, 1989) pp. 28-65.
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