United States       Prevention, Pesticides      EPA712-C-98-247
          Environmental Protection    and Toxic Substances      August 1998
          Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.5100
           Bacterial Reverse
           Mutation Test

     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

     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

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.

    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-

    (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

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

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

    (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-

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

    (2) At least five different analysable concentrations of the test sub-
stance should be used with approximately half log (i.e.  VlO) intervals be-

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:
7 12-Dimethylbenzanthracene
Congo Red (for the reductive metabolic activation method) ...
Cyclophosphamide (monohydrate)

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:
(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




TA1537, TA97 and
WP2 ui/r/\andTA102
WP2, WP2 uvrA and
WP2 t/wrt(pKM101)
     (4) Other appropriate positive  control reference substances  may be
used. The  use of chemical class-related positive control  chemicals may
be considered, when available.

     (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.

     (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-

     (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.


     (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
     (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.

    (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.

    (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

    (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).


     (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
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