United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-84-003 Mar. 1984
&EPA Project Summary
In Vitro and In Vivo Mutagenicity
Studies of Environmental
Chemicals
David C.L. Jones, Vincent F. Simmon, Kristien E. Mortelmans,
Ann D. Mitchell, Elizabeth L. Evans, Mary M. Jotz
Edward S. Riccio, Douglas E. Robinson, and Barbara A. Kirkhart
The objectives of this project were to
evaluate the mutagenicity of various
compounds, mostly pesticides, using
microbial and mammalian cell in vitro
techniques, as well as in vivo techniques
in Drosophila and mice, and to further
develop and refine these procedures for
application as test batteries.
Seventy-nine compounds were evalu-
ated for mutagenicity in one or more of
11 test systems: S. typhimurium plate in-
corporation assay; E. coll WP-2 reverse
mutation assay; S. cerevisiae D3 mitotic
recombination assay; S. cerevisiae D7
assays; E. coli, B. subtilis, and S.
typhimurium relative toxicity assays;
sister-chromatid exchange in Chinese
hamster ovary cells assay; L5178Y
mouse lymphoma cell forward mutation
assay; unscheduled DNA synthesis
assay; mouse micronucleus assay;
Drosophila sex-linked recessive lethal
assay; and mouse dominant lethal
assay.
The data from the evaluation of 41
pesticides and 10 industrial chemicals
are presented, and qualitative inter-
pretations of these data and of data ob-
tained under a previous contract, in-
cluding those for an additional 28
pesticides, are summarized.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory, Research Triangle Park, NC,
to announce key findings of the re-
search project that is fully documented
in a separate report of the same title (see
Project Report ordering information at
back).
Introduction
Under contract to the U.S. Environmental
Protection Agency (EPA), SRI International
evaluated the mutagenicity of various com-
pounds using microbial and mammalian cell
in vitro techniques, as well as in vivo techni-
ques in Drosophila and mice. Simulta-
neously, these test procedures were further
developed and refined.
This report includes a compilation of all the
quantitative data and a description of the test
results for each of the 51 chemicals (41
pesticides and 10 industrial chemicals)
evaluated under the contract, together with
a summary table listing the qualitative inter-
pretation of the results. In addition, the sum-
mary table includes the qualitative interpreta-
tions of the results of a previous contract in
which 27 of these 51 chemicals and 28 other
chemicals were evaluated.
The qualitative interpretations for all 79
chemicals are based on a review of all of the
data generated under both contracts, using
the criteria described in the methods section
of this report. In some cases, this process
resulted in qualitative interpretations that dif-
fered from those in previous reports. For ex-
ample, in previous reports, results for the
relative toxicity studies in £ co//and B. sub-
tilis were scored as positive when there was
a clear compound effect or as negative when
there was not. In the present evaluation,
positive or negative interpretation was limited
to clear effect or no-effect results, which any
other finding interpreted as inconclusive.
Those cases in which retesting and/or
reevaluation was done are specified in the
test and in the summary table.
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Materials and Methods
Battelle Memorial Laboratories (Colum-
bus, Ohio) obtained the pesticides from the
manufacturers and subsequently provided
samples to SRI International (Menlo Park,
California) or to WARF Institute, Inc.
(Madison, Wisconsin) for the tests reported
here. A few of the chemicals were obtained
from the manufacturers by the EPA Office
of Pesticide Programs, Washington, D.C.
Each pesticide was a "technical grade" pro-
duct or its equivalent. Monocrotophos was
tested as a formulated product, Azodrin 5
(Shell). Mancozeb was tested as two dif-
ferent products: Dithane M-45 (Rohm and
Haas) and Manzate 200 (Du Pont). Maneb
was also tested as two different products:
Dithane M-22 (Rohm and Haas) and Man-
zate D (Du Pont).
Seventy-nine compounds (69 pesticides
and 10 industrial chemicals) were examined
for mutagenicity in one or more of 11 test
systems. The pesticides are listed
alphabetically in Table 1 by their common
name, action, and chemical class. Nearly a
third of the compounds are organo-
phosphate insecticides; other classes include
carbamates, chlorinated hydrocarbons,
halogenated aromatics, arsenic compounds,
urea derivatives and natural plant products,
and synthetic derivatives. The mode of ac-
tion of these compounds ranges from
general contact and systemic poisons to
specific preemergence and postemergence
herbicides.
The 11 assay systems employed are listed
in Table 2, along with references to complete
descriptions of the assay procedures. The
following bioassays were carried out both in
the presence and absence of an Aroclor-1254
induced rat liver metabolic activation system:
S. typhimurium plate incorporation, £. coli
WP-2 uvrA reverse mutation, S. cerevisiae
D7 reverse mutation, mouse lymphoma
L5178Y cell forward mutation, S. cerevisiae
D3 enhanced mitotic recombination, S.
cerevisiae D7 gene conversion and mitotic
crossing-over, human lung fibroblast (WI-38)
unscheduled DNA synthesis, and Chinese
hamster ovary cell sister-chromatid ex-
change. All experiments were performed
with concurrent positive (known mutagen)
and negative (solvent) control chemicals.
Results and Discussion
Table 3 summarizes the test results for
each of the 79 chemicals evaluated. Cases
involving a retest are identified by footnotes,
as are cases in which the reexamination of
previous data resulted in a different inter-
pretation. The S. typhimurium plate incor-
poration results were scored as positive
when the results of any of the individual
Table 1. Pesticides Assayed for Genotoxic Effects"
Name (action) Chemical class
Acephate (II
Allethrin (I)
Aspon (I)
Azinphos-methyl (I)
Benomyl (Ft
Biphenyl (Fl
Botran (Ff
Bromacil !H)
sec-Butylamine AB (F)
sec-Butylamine AB»H3/W (F)
Cacodylic acid (HI
Captan IF)
Carbofuran (I)
Chlordimeform III
Chlorpyrifos (II
Creosote Pf
Creosote Pf
Crotoxyphos (I)
2,4-D acid (H)
2,4-DB acid (HI
Demeton (II
Diallate (H)
Diazinon (I)
Dicamba (H)
m-Dichlorobenzene (II
o-Dichlorobenzene (I)
p-Dichlorobenzene (I)
Dinoseb (HI
Disulfoton (II
O£-cis/trans chrysanthemic
acid (I)
DSMA (HI
Endrin (I)
Ethion (PI
Ethyl chrysanthemate II)
Fensulfothion (I)
Fenthion (II
Folpet (F)
Fonofos (I)
Formetanate hydrochloride (I)
Ma lath ion (I)
Mancozeb (Ff
Maneb IFF
Methomyl (II
Methoxychlor (I)
Monocrotophos (if
Monuron (H)
MSMA (H)
Parathion (I)
Parathion-methyl (I)
PCNB (F)
Pentachlorophenol (H)
Permethrin (I)
Phorate (I)
Polyram (F)
Propanil (H)
Ftesmethrin (I)
Rotenone (I)
Siduron (HI
Simazine (HI
Sumithrin (I)
2,4,5-T(H)
Triallate (H)
Trichlorfon (II
Jrifluralin (HI
Vegadex (Hf
Thio/dithiophosphoramidate
Pyrethroid
Organo thio I dithiophospha te
Organothio/dithiophosphate
Carbamate
Aromatic
Chlorinated nitroaniline
Diazine
Aliphatic amine
Aliphatic amine
Organoarsenical
Phthalimide
Carbamate
Haloaromatic amidate
Organothio/dithiophosphate
Organothio/dithiophosphate
Halophenoxy
Halophenoxy
Organothio/dithiophosphate
Jhiocarbamate
Organo thio /dithiophospha te
Halophenoxy
Haloaromatic
Haloaromatic
Haloaromatic
Dinitrophenol
Organothio/dithiophosphate
Pyrethroid
Organoarsenical
Chlorinated hydrocarbon
Organothio/dithiophosphate
Pyrethroid
Organothio/dithiophosphate
Organo thio /dithiophospha te
Phthalimide
Phosphonate/thiophosphonate
Carbamate
Organothio/dithiophosphate
Ethylenebisdithiocarbamate
Ethylenebisdithiocarbamate
Carbamate
Aromatic
Organophosphate
Urea
Organic arsenical
Organothio/dithiophosphate
Organothio/dithiophosphate
Haloaromatic
Haloaromatic
Pyrethroid
Organothio/dithiophosphate
Ethylenebisdithiocarbamate
Haloaromatic
Pyrethroid
Hydrocarbon
Urea
Triazine
Pyrethroid
Halophenoxy
Jhio/dithiocarbamate
Phosphonate/thiophosphonate
Nitroaromatic
Thio/dithiocarbamate
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Table 1.
(Continued)
Name {action)
Chemical class
Zineb IF)
Ethylenebisdithiocarbamate
' I = insecticide, F= fungicide, H = herbicide.
6 Botran is the chemical name used in report; dichloran is the common name.
c Compound is actually a complex coal tar mixture containing phenol, creosote, and other compounds.
d Tested as two products, Dithane M-45 and Manzate 200.
e Tested as two products, Dithane M-22 and Manzate D.
' Azodrin 5 is the chemical name used in this report; su/fa/late is the common name.
9 Vegadex is the chemical name used in this report; sulfallate is the common name.
Table 2. Assay Systems Used for Genotoxic Studies
Assay
Reference to
method employed
S typhimurium plate incorporation
E. coli WP-2 uvrA reverse mutation
S. cerevisiae D3 mitotic recombination
S. cerevisiae D7
Reverse mutation
Gene conversion
Mitotic crossing-over
Relative toxicity
E. coli, strains W3110 and p3478
B. subtilis, strains HJ7 and M45
S. typhimurium, strains SL4525 free I,
SL4700 free ~ iTA 1978 (uvrB +), and
TA1538 fuvrB")
Sister-chromatid exchange in CHO cells
L5178Y TK / ~~ Mouse lymphoma cell
forward mutation
Unscheduled DNA synthesis in W/-38 cells
Mouse micronuc/eus
Drosophila sex-linked recessive
Mouse dominant lethal
Ames et al. (1)
Bridges 12)
Brusick and Mayer 13)
Zimmerman 14)
Zimmerman (5)
Zimmerman (5)
Slater et al. (6)
Kada (7)
Ames et al. (11
Perry and Evans (8)
Stetka and Wolff (91
Cfive et al. (10)
Simmon (11, 12)
Schmid (13)
Wurgler et al. (14)
Simmon (15)
strains were positive. The totals indicate the
numbers of tests conducted, excluding in-
conclusive tests, and the numbers of tests
with positive results. For computing the
totals, tests with and without activation were
scored as positive when either result was
positive. For purposes of this analysis, each
of the three parameters in the S. cerevisiae
D7 system and each of the four strain pairs
in the relative toxicity assays was counted
as a separate test.
The general problem addressed by this
research was the classification of pesticides
according to their genotoxic effects.
Genotoxicity was assessed by prokaryotic
and eukaryotic test systems that measured
gene mutation, DNA damage, or chromo-
somal effects. The chemicals studied can be
divided into two groups: those that displayed
no genotoxic response and require little fur-
ther testing, and those that displayed some
positive response and require further evalua-
tion. The chemicals that elicited positive
responses in several kinds of genetic
bioassays are of greatest concern, particu-
larly as regards their potential effect on
humans.
Attempts to relate the results of in vitro
and in vivo bioassays to potential human
health hazards lead naturally to a more
specific classification or ranking of individual
chemicals. The present assessment falls
short of a definitive ranking of the chemicals
studied for the following reasons:
Table 3. Summary Data for 79 Chemicals
Relative Toxicity
S.lv/,/, F r-.nl! K ™» S- <=ere O7 f rnl, R Sllh, S typh
PESTICIDES PI. Inc WP-2 D3 MCO MGC RM PolA Rec uvrB Rec
Acephate + +" ++++++-+ ?b 7 -
Al/ethnn - + ? -
Aspon — 7 7
Azinphos-Methyl - c - c + c - rf 7 7 -
Benomyl
Bipheny/ 7 7
Botrarf
sec Butylamme 2AB + -
sec Butylamme 2AB H3P04
Cacodylic Acid +c + ':-t-+-t-+++ ? 7 - -
Captan + c + c + + +c + c + + + +
Carbofuran 7 7
Ch/ordtmeform 7 -
Chlorpyrrfos -c_c + + + +
Creosote PI - + - -
Creosote P2 - + - _
Crotoxyphos — ++ ? 7 - _
2,4-D Acid ^_____ _rf +c
2,4-DB Acid ____ + _c
Demeton + + + + ++++++++ -d + - -
Dial/ate -c + c — -+
Diazmon 7 7
Dicamba + + - _
m-Dichlorobenzene ++ + —
• in most cases tecnnicai graae cnemicais
were used. While this level of purity is
SC£ L5178Y UDS Mouse Dros Mouse No. ± No.
CHO Lymph WI-38 Micro SLRL DL Tests Pos.
+ ++++- - 12 8
51
40
7- -+ -c-d - 13 2
++ ++* + 33
40
5 0
++ — — 15 1
5 1
5 0
--++-- + 12 6
+ +-c-d - 10 8
40
50
84
?+ 6 2
'+ 62
--++-- - 12 2
81
81
+ ++-++ - 14 10
+ + + 84
40
82
5 2
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Table 3. (continued) fle/af/ve Tol(Kity
.
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most relevant to commercial products,
it is possible that the genotoxic activity
of the technical grade chemicals may be
related to the presence of contaminants.
• The data base is incomplete; not all
chemicals were evaluated in all test
systems.
• The quantitative dose-response data that
exist for each chemical in each test have
been used only to establish whether the
test was positive or negative.
• No dosimetry studies have been per-
formed; only the quantity of chemical to
which the organism was exposed has
been recorded.
Despite these limitations, a great deal of
preliminary information has been gleaned
from this examination of the qualitative data.
Undoubtedly, much can be gained from a
careful analysis of the quantitative data.
Thirty-nine of the pesticides examined
were positive in one or more the 11 test
systems employed. Of these 35 pesticides,
21 caused point/gene mutation and 31
caused DNA damage. Nine pesticides pro-
duced chromosomal effects; however, they
also caused gene mutation and/or primary
DNA damage, and relatively few tests for
chromosomal effects-were performed.
Six pesticides evaluated in this study
displayed genotoxic acitivity in three or more
eukaryotic bioassay systems. These com-
pounds are acephate, cacodylic acid,
demeton, diallate, monocrotophos, and
trichlorfon. Cacodylic acid also caused
chromosomal damage in the in vivo mouse
micronucleus test; monocrotophos was
negative in the relatively insensitive mouse
dominant lethal test. With further testing,
other compounds may also show similar ef-
fects; nonetheless, because of their diverse
genotoxic activities, these six compounds
should be assessed carefully.
References
1. Ames, B.N., J. McCann, and E.
Yamaski. Methods for Detecting Car-
cinogens and Mutagens with the
5a//no/?e//a/Mammalian Microsome
Mutagenicity Test. Mutat. Res., 31:347,
1975.
2. Bridges, B.A. Simple Bacterial Systems
for Detecting Mutagenic Agents. Lab.
Pract., 21:413, 1976.
3. Brusick, D.J., and V.W. Mayer. New
Developments in Mutagenicity Screen-
ing Techniques with Yeast. Environ.
Health Perspect., 6:83, 1973.
4. Zimmerman, F.K. Procedures Used in
the Induction of Mitotic Recombination
and Mutation in the Yeast Sac-
charomyes cerevisiae. Mutat. Res.,
31:71, 1975.
5. Zimmerman, F.K. A Yeast Strain for
Simultaneous Detection of Induced
Mitotic Crossing Over, Mitotic Gene
Conversion, and Reverse Mutation.
Mutat. Res., 28:381, 1975.
6. Slater, E.E., M.D. Anderson, and H.S.
Rosenkranz. Rapid Detection of
Mutagens and Carcinogens. Cancer
Res., 31:970, 1971.
7. Kada, T. Mutagenecity Testing of
Chemicals in Microbial Systems. In:
New Methods in Experimental
Chemistry and Toxicology, F. Coulston,
F. Corte, and M. Coto, eds. Interna-
tional Academic Printing, Tokyo, 1973.
8. Perry, P., and H.J. Evans. Cytological
Detection of Mutagen-Carcinogen Ex-
posure by Sister Chromatid Exchange.
Nature, 158:121, 1975.
9. Stetka, D.G., and S. Wolff. Sister
Chromatid Exchanges as an Assay for
Genetic Damage Induced by
Mutagens/Carcinogens. Part II. In Vitro
Tests for Compound Requiring
Metabolic Activation. Mutat. Res.,
41:343, 1976.
10. Clive, D., K.O. Johnson, J.F.S. Spec-
tor, A.G. Batson, and M.M.M. Brown.
Validation and Characterization of the
L5178Y/TK + /-Mouse Lymphoma
Mutagen Assay System. Mutat. Res.,
59:61-108, 1979.
11. Simmon, V.F. In Vitro Microbiological
Mutagenicity and Unscheduled DNA
Synthesis Studies of Eighteen
Pesticides, Final Report. U.S. En-
vironmental Protection Agency, 1978.
12. Simmon, V.F., A.D. Mitchell, and T.A.
Jorgenson. Evaluation of Selected
Pesticides as Chemical Mutagens. In
Vitro and in Vivo Studies. Report no.
EPA-600/1-77-028, U.S. Environmental
Protection Agency, 1977.
13. Schmid, W. The Micronucleus Test for
Cytogenetic Analysis. In: Chemical
Mutagens, Vol. 4, A. Hollaender, ed.
Plenum Press, New York, 1976.
14. Wurgler, F.E., F.H. Sobels, and E.
Vogel. Drosophila as Assay System for
Detecting Genetic Changes. In: Hand-
book of Mutagenicity Test Procedures,
B.J. Kilbey, M. Legator, W. Nichols,
and C. Ramels, eds. Elsevier/North
Holland Biomedical Press, Amsterdam,
1977.
15. Simmon, V.F. In vivo and In vitro
Mutagenicity Assays of Selected
Pesticides. In: A Rational Evaluation of
Pesticidal vs. Mutagenic/Carcinogenic
Action, R.W. Hart, H.F. Kraybill, and
F.J. de Serres, eds. DHEW Publication
No. (NIH) 78-1306, U.S. Department of
Health, Education, and Welfare,
Washington, D.C. 1978.
David C. L. Jones, Vincent F. Simmon, Kristien E. Mortelmans, Ann D. Mitchell,
Elizabeth L Evans, Mary M. Jotz, Edward S. Riccio, Douglas E. Robinson, and
Barbara A. Kirkhart are with SRI International, Menlo Park, CA 94025.
Michael D. Waters is the EPA Project Officer (see below).
The complete report, entitled "In Vitro and In Vivo Mutagenicity Studies of
Environmental Chemicals,"(Order No. PB 84-138 973; Cost: $41.50, subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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