United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA/600/S1 -85/006 May 1985
Project Summary
In Vitro Microbiological
Mutagenicity and Unscheduled
DNA Synthesis Studies of
Fifteen Pesticides
Vincent F. Simmon, Edward S. Riccio, Douglas E. Robinson, and
Ann D. Mitchell
Fifteen pesticides being reviewed as
part of the EPA Substitute Chemical
Program were examined by SRI Inter-
national by several In vitro test proce-
dures, for the following:
• Reverse mutation in Salmonella
typhlmurium strains TA1525,
TA1537, TA98, and TA100 and in
Escherichia coli WP2.
• Induction of mitotic recombination
in the yeast Saccharomyces cere-
visiae D3.
• Relative toxicity in DNA repair-pro-
ficient and repair-deficient strains of
E. coli (strains W311O and p3478.
respectively) and of Bacillus subtilis
(strains HI 7 and M45, respectively).
• Unscheduled DNA synthesis (UDS)
in human fibroblasts (WI-38 cells).
None of the fifteen pesticides demon-
strated genetic activity in all six of the in
vitro assays. Bioallethrin was the only
pesticide that was mutagenic in the S.
typhimurium reverse mutation assay.
Manzate-D and manzate 200 increased
both mitotic recombination in S. cere-
visiae D3 and UDS in WI-38 cells.
Dithane M-22, dithane M-45, ethylchry-
santhemate, and zineb increased mitotic
recombination in S. cerevisiae D3. DL-
cis/trans chrysanthemic acid was geno-
toxic in the relative toxicity assay, being
more toxic to the repair-deficient (roc'}
B. subtilis strain M45 than to the repair-
proficient (rec*) strain HI 7.
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
The Federal Insecticide, Fungicide, and
Rodenticide Act designates the U.S. Envi-
ronmental Protection Agency (EPA) as
the governmental body responsible for
the safety of all pesticides used in the
United States. More recently, the Federal
Environmental Pesticide Control Act (PL
92-516) strengthened EPA's regulatory
responsibilities in the area of pesticides
to include intra- as well as interstate
commerce.
To be federally registered, a pesticide
must have been determined not to be
hazardous to health or to the environ ment
when used according to its labeling
restrictions. Thus, in accordance with
new law as well as with specific directives
included in Public Law 93-135, 1973,
EPA is now conducting a thorough review
of the implications of using alternative
chemicals, including older registered
pesticides, for pest control.
In the pesticide review process, EPA
emphasizes the development of scientific
criteria for evaluating the safety of com-
pounds substituted for those pesticides
found to be hazardous. In addition to
review and evaluation of the literature on
-------
pesticides and maintenance of liaisoh
with industry and academia, the strategy
program includes laboratory studies to
obtain additional data. One of these
laboratory programs is directed toward
gathering mutagenesis data on a selected
number of compounds.
EPA's program is responsive to one of
the recommendations included in the
President's Scientific Advisory Committee
Report of September 1973, Chemicals
and Health. In that document, the Com-
mittee recommended that "Regulatory
agencies should take steps to insure that
new scientific data raising the possibility
of new or extended hazards from chem-
icals in use are subject to careful process
of scientific review for merit interpreta-
tion."
Results and Discussion
The results of the in vitro microbio-
logical and UDS assays of the fifteen
compounds are summarized in Table 1. A
positive response in these assays is
defined as a reproducible dose-related
increase in the effect being observed. A
genotoxic or mutagenic effect was ob-
served for eight of the fifteen pesticides
tested. The eight pesticides that had a
positive response in one or more of the
assays were bioallethrin, DL-cis/trans
chrysanthemic acid, dithane M22, di-
thane M-45, ethylchrysanthemate, man-
zate-D, manzate 200, and zineb. None of
the pesticides tested were positive in all
six of the in vitro assays. Bioallethrin was
the only compound mutagenic in the
Sa/mone//a/microsome assay. Dithane
M-22, dithane M-45, ethylchrysanthe-
mate, manzate-D, manzate 200, and
Zineb all increased mitotic recombination
in S. cerevisiae D3, but only manzate-D
and manzate 200 also induced unsched-
uled DNA synthesis in WI-38 cells. DL-
cis/ trans chrysanthemic acid was geno-
toxic in the B. subtilis relative toxicity
assay but was inactive in all other assays.
Dose-response curves are presented for
pesticides that give a positive response in
the assays except for ther relative toxicity
assays. Biphenyl, chlordimeform, NRDC-
149, permethrin, polyram, resmethrin,
and sumithrin were not genotoxic or
mutagenic in any of the six assays we
performed.
Microbiological A ssays
Each pesticide was tested at least twice
on separate days, using one plate per
dose. The first experiment was a test over
a wide range of doses to look for toxicity or
mutagenicity. If no toxicity or mutagenic-
ity was observed, the second experiment
was conducted at higher concentrations.
If mutagenicity was observed, a dose re-
sponse was determined. An assay that
gave a mutagenic response was always
repeated to confirm that the results were
reproducible.
Table 1. In Vitro Mutagenesis: Summary Data for EPA Pesticides
Pesticide
Salmonella
typhimurium
(His* Reversion)
-MA +MA
Escherichia
coli WP2
(Try* Reversion)
-MA +MA
Saccharomyces
cerevisiae D3
(Mitotic
Recombinants)
-MA +MA
Conclusions and
Recommendations
Our results indicate that none of th
fifteen pesticides tested in Phase III wer
mutagenic or genotoxic in all six of the/
vitro assays. Only two pesticides wer
positive in more than one assay. The
were manzate-D and manzate 200, whic
increased mitotic recombination in 6
cerevisiae D3 and induced UDS in WI-3
cells. Dithane M-22, dithane M-45, ethyl
chrysanthemate, and zineb increase
mitotic recombination. The Salmonella
microsome assay detected mutageni
activity in only one pesticide, bioallethrir
No pesticide was found to be mutageni
with E. coli WP2. DL-cis/trans chrysan
themic acid was genotoxic in the relativi
toxicity assay.
It is recommended that the eight pesti
cides that were positive in at least on<
assay be considered for further study t<
characterize more completely their poten
tial hazards to human health. Although <
mutagenic response does not mean that <
chemical is harmful to humans, th<
combination of six separate-assay sys
terns greatly enhances the probability o
detecting potentially hazardous chemi
cals. It is apparent that no one assay ii
uniquely capable of detecting the spec
trum of mutagenic events that differen
chemical structures may cause.
Escherichia Bacillus
coli subtilis UDS
(Relative (Relative (DNA Repair}
Toxicity) Toxicity) -MA +MA
Bioallethrin
Biphenyl
Chlordimeform
DL-c\s/trans
Chrysanthemic
acid
Dithane M-22
Dithane M-45
Ethylchrysan-
themate
Manzate-D
Manzate 200
NRDC-149
Permethrin
Polyram
Resmethrin
Sumithrin
Zineb
Negative response. •; positive response, +.
-------
Vincent F. Simmon, Edward S. Riccio, Douglas E. Robinson, and Ann D. Mitchell
are with SRI International, Menlo Park, CA 94025.
M. D. Waters is the EPA Project Officer (see below).
The complete report, entitled "In Vitro Microbiological Mutagenicity and
Unscheduled DNA Synthesis Studies of Fifteen Pesticides," (Order No. PB
85-193 761/AS; Cost: $17.5O, 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, NC27711
* U.S. GOVERNMENT PRINTING OFFICE: 196S-55M16/27083
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
OCOC329 PS
------- |