United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-83-006 Sept. 1983
4>EPA Project Summary
The Detection of Mutagenic
Properties of Pesticides Used in
Commercial Corn Production
Michael J. Plewa and James M. Gentile
This project encompassed the analy-
sis of 21 pesticides and 12 combina-
tions of herbicides for their genotoxic
properties. Three genetic assays were
employed; reverse mutation in Salmo-
nella typhimurium, gene conversion in
Saccharomyces cerevisiae and reverse
mutation in Zea mays. The use of these
genetic indicator organisms established
a comprehensive data base that re-
solved a spectrum of genetic damage
induced by single agents or by com-
binations of agents. The pesticides
were evaluated with the microbial as-
says directly, after in vitro mammalian
microsomal activation and after in vivo
plant activation protocols. Finally, all
the pesticides were tested for their
ability to induce mutations \nZeamays
under in situ (in field) conditions.
This project was divided into two
options. Option one included pesti-
cides from a wide range of chemical
classes and the entire spectrum of
genetic assays and activation proto-
cols were used. Option two was in-
cluded to gather additional informa-
tion on the genotoxic properties of s-
triazine herbicides. The range of ge-
netic assays used in option two was
abridged.
Of the insecticides assayed, cura-
cron, carbofuran, chlorpyrifos, metham
and phorate were negative in all assays.
Two insecticides, terbufos and etho-
prop were positive in one assay. Chlor-
dane, fonofos and heptachlor were
positive in two of the three genetic
assays. No insecticide was positive in
all three assays.
Of the herbicides assayed under op-
tion one of this project, eradicane and
bifenox were negative in all assays.
Alachlor, propachlor, procyazine and
SD50093 were positive in one assay.
Cyanazine, dicamba and metolachlor
were positive in two assays; no her-
bicide was found to be positive in all
three assays.
The combinations of herbicides test-
ed under option one were assayed
under the condition of in vivo plant
activation. All the herbicide combina-
tions were positive in one of the three
assays except metolachlor plus atra-
zine which was positive in two assays.
The herbicides and combinations of
herbicides tested in option two were
evaluated with the maize wx locus
assay only. Only butylate and the com-
bination of butylate plus cyanazine
were negative in the maize assay.
This report covers the period of
August 21, 1976 to March 31, 1980
and the work was completed as of May
1980.
This Project Summary was developed
by EPA's Health Effects Research Lab-
oratory, Research Triangle Park, NC, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction and Summary
This project encompassed the analysis
of 21 pesticides and 1 2 combinations of
herbicides for their genotoxic properties.
Three genetic assays were employed; re-
verse mutation in Salmonella typhimurium,
gene conversion in Saccharomyces cerevi-
siae and reverse mutation in Zea mays.
The results of this project are summarized
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in Table 1. A list of the pesticides eval-
uated for their genotoxic properties is
presented in Table 2. The common and
chemical names for the pesticides used in
this report were those indicated by the
U.S. EPA publication (EPA-600/2-81-
011) entitled, "Analytical Reference Stand-
ards and Supplemental Data for Pesti-
cides and Other Organic Compounds,"
R.R Watts, Ed. For an agent to be con-
sidered genotoxic the following criteria
were imposed. For the Salmonella assay,
a test agent with or without activation was
appraised as positive if it induced an
increase in the number of revertants per
plate that was twice the appropriate con-
trol value or if the highest dose tested
indicated a significant 6 value. In addition
the agent had to induce a reproducible
dose dependent response. For the Sac-
charomyces assay, a test agent was con-
sidered as positive if it induced an increase
in the number of convertants per 105
survivors that was twice the control value
or if the highest dose tested indicated a
significant 0 value. Additionally the agent
had to induce a reproducible dose depen-
dent response. When the microbial assays
were conducted, the agents were eval-
uated directly, with in vitro mammalian
microsome activation (S-9) or with in vivo
plant activation (1 S) protocols. For the
maize test the agent was appraised as
positive if it induced an increase in the
mutation rate among gametophytes that
was twice the control value or indicated a
significant 0 value. In addition a
significant t-test was required for each
agent. These statistical requirements
were imposed to aid m the interpretation
of the data and to reduce the type one and
type two errors that may arise when a
large number of agents are assayed with
a variety of genetic indicator organisms.
This project was divided into two op-
tions. Option one included pesticides from
a wide range of chemical classes and the
entire spectrum of genetic assays and
activation protocols were used. Option
two was included to gather additional
information on the genotoxic properties of
s-triazine herbicides. The range of genetic
assays used in option two was abridged.
Of the insecticides assayed, curacron,
carbofuran, chlorpyrifos, metham and
phorate were negative in all assays. Two
insecticides, terbufos and ethoprop were
positive in one assay. Chlordane, fonofos
and heptachlor were positive in two of the
three genetic assays. No insecticide was
positive in all three assays.
Of the herbicides assayed under option
one of this project, eradicane and bifenox
Table 1. Summary of Results
Genetic Assays
Pesticide
(Option 1)
S. typhimunum
D S-9 IS
S. cerevisiae
D S-9 IS
Alachlor (H,C)
Alachlor (H. T)
Bifenox (H,Cj
Bifenox (H, T)
Curacron (I, C)
Curacron (I, T)
Carbofuran (I, C)
Carbofuran (I, T)
Chlordane (I. C)
Chlordane (1,7)
Chlorpyrifos (1,0}
Chlorpyrifos (I, T)
Cyanazine (H,C)
Cyanazine (H, T)
Dicamba (H,C)
Dicamba (H, T}
Eradicane (H,Cj
Eradicane {H, T)
Ethoprop (1,0}
Ethoprop (I, T}
Fonofos (1,0}
Fonofos (I, T)
Heptachlor {1,0}
Heptachlor (I, T)
Metolachlor (H,C)
Metolachlor (H.T)
Phorate (I.C)
Phorate (I, T)
Procyazine (H, C)
Procyazine (H, T)
Propachlor(H,C)
Propachlor (H, T)
SD50093(H,C)
SD50093 (H, T)
Metham (iQ
Metham (IJ)
Terbufos (I, C}
Terbufos (I.T)
Alachlor + Bifenox
Alachlor + Dicamba
Cyanazine + Alachlor
Cyanazine + Propachlor
Metolachlor + Atrazine
Metolachlor + Dicamba
Procyazine + Metolachlor
Pesticide
(Option 2)
NT
NT
NT
NT
NT
NT
NT
- - NT
- + -
NT NT NT
NT NT NT
NT
+
NT
NT
- - NT
- NT
- NT
Atrazine
Butylate
Butylate + Atrazine
Butylate + Cyanazine
Eradicane + Atrazine
Eradicane + Cyanazine
Metolachlor + Atrazine
Metolachlor + Cyanazine
Simazine
Z. mays
wx
NA
NA
NA
NA
+
NA
NA
+
NA
NA
NA
+
NA
NA
+
NA
NA
NA
NA
NA
+
NA
NA
NA
/ — herbicide, /= insecticide, C= commercial grade, and 7"= Technical grade.
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were negative in all assays. Alachlor, pro-
pachlor, procyazine and SD50093 were
positive in one assay. Cyanazine, dicamba
and metolachlor were positive in two
assays; no herbicide was found to be
positive in all three assays.
The combination of herbicides tested
under option one were assayed under the
condition of in vivo plant activation. All the
herbicide combinations were positive in
one of the three assays except metolachlor
plus atrazine which was positive in two
assays.
The herbicides and combinations of
herbicides tested in option two were eval-
uated with the maize wx locus assay only.
Only butylate and the combination of
butylate plus cyanazine were negative in
the maize assay.
Recommendations
1. Since the overwhelming majority of
pesticides are applied to or on plant
species it is incumbent to incorporate
plant dependent assays in the evalua-
tion of their mutagenic properties.
2. Mutagenicity of pesticide chemical
should be evaluated in the laboratory as
well as under field conditions.
3. In vitro bioassays should employ
mammalian as well as plant-activation
systems.
4. Commercial formulations as well as
technical grade pesticides should be
evaluated for their potential mutagenic
effects.
5. A battery of prokaryotic and eukaryotic
bioassays should be applied to eval-
uate different genetic endpoints such
as primary DNA damage gene muta-
tion and chromosome aberrations. This
would provide data that are consistent
with the ways these agents are used.
Analysis under laboratory conditions
needs to be complemented with in situ
genetic analysis of pesticides used in
the agricultural environment.
Table 2. Pesticides Evaluated for Their Genotoxic Properties
Insecticides
Common Name
Trade Name
Chemical Name
CA Reg. No.
Chlordane Chlordane 1,2,4,5,6,7,8,8-Octachloro- 57-74-9
2,3,3a,4,7,7a-hexahydro-4,7-
methanoindene and related
compounds
Terbufos Counter S-{ [(1,1-Dimethylethyl) thio] 13071-79-9
methyl] 0,0-diethyl phosphoro-
dithioate
Fonofos Dyfonate 0-Ethyl S-phenyl ethyl- 944-22-9
phosphonodithioate
Heptachlor Heptachlor 1,4,5,6,7,8,8-Heptachloro- 76-48-8
3a,4,7,7a-tetrahydro-4,7-
methanoindene
Carbofuran Furadan 2,3-Dihydro-2,2-dimethylbenzo- 1563-66-2
furan-7-yl methylcarbamate
Chlorpyrifos Lorsban 0,0-Diethyl 0-f3,5,6-trichloro- 2921-88-2
2-pyridyl) phosphorothioate
Ethoprop Mocap 0-Ethyl S,S-dipropyl 13194-48-4
phosphorodithioate
Phorate Thimet 0,0-Diethyl-S-[(ethylthio) 298-02-2
methyl] phosphorodithioate
Curacron CGA 0-(4-bromo-2-ch/oro-pheny//-
0-ethyl-S-propyl
phosphorothioate
Metham SRA Sodium methyldithiocarbamate 6734-80-1
Herbicides
Cyanazine Bladex 2-[(4-Chloro-6- 21725-46-2
(ethylamino)-s-triazin-
2-yl]-2-methyl-
propionitrile
Eradicane Eradicane S-Ethyl dipropyl- 759-94-4
(EPTC) thiocarbamate
Alachlor Lasso 2-Chloro-2',6'-diethyl- 15972-60-8
N-(methoxymethyl) acetanilide
Bifenox Modown Methyl 5-(2,4-dichloro- 42576-02-3
phenoxy)-2-nitrobenzoate
Procyazine Cycle 2-{ [4-Chloro-6-(cyclopropyl- 32889-48-8
amino)-s-triazin-2-yl]
amino}-2-methylpropionitnle
Metolachlor Dual 2-Chloro-N-(2-ethyl-6- 51218-45-2
methylphenyl)-N-(2-methoxy-
1-methylethyl) acetamide
Propachlor Ramrod 2-Chloro-N-isopropyl- 1918-16-7
acetanilide
Simazine Princep 2-Chloro-4,6-bis(ethyl- 122-34-9
amino)-s-triazine
Atrazine Aatrex 2-Chloro-4(ethylamino)-6- 1912-24-9
(isopropylamino)-s-triazine
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Table 2. (Continued}
Herbicides
Common Name Trade Name
Chemical Name
CA Reg No.
Butylate
Dicamba
SD50093
Sutan S-Ethyl N,N-diisobutylthio-
carbamate
Banvel 2-Methoxy-3,6-dichloro-
benzoic acid
3,5-Dichloro-O-anisic acid
SD50093 Formulation of Atrazine
plus Cyanazine
2008-41-5
1ST 8-00-9
MichaelJ. Plewa is with the University of Illinois, Urbana, IL 61801, and James M.
Gentile is with Hope College, Holland, Ml 49423.
Michael D. Waters is the EPA Project Officer (see below).
The complete report, entitled "The Detection ofMutagenic Properties of Pesticides
Used in Commercial Corn Production," (Order No. PB 83-226 118; Cost:
$19.00, subject to change! will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
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
«US GOVERNMENT PRINTING OFFICE 1983-659-017/7196
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