United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600/1 -79-041
October 1979
Research and Development
In Vitro
Microbiological
Mutagenicity and
Unscheduled DNA
Synthesis Studies of
Eighteen Pesticides
EP 600/1
79-OUl
••'. r>\
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U S. Environmental
Protection Agency, have been grouped into nine series These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields
The nine series are
1 Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4 Environmental Monitoring
5 Socioeconomic Environmental Studies
6 Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8 "Special" Reports
9 Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-79-041
October 1979
IN VITRO MICROBIOLOGICAL MUTAGENICITY AND UNSCHEDULED
DNA SYNTHESIS STUDIES OF EIGHTEEN PESTICIDES
by
Vincent F. Simmon, Ph.D.
SRI International
Menlo Park, California 94025
Contract No. 68-01-2458
Project Officer
Dr. Michael D. Waters
Genetic Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects.
These studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory participates in the development
and revision of air quality criteria documents on pollutants for which
national ambient air quality standards exist or are proposed, provides
the data for registration of new pesticides or proposed suspension of
those already in use, conducts research on hazardous and toxic materials,
and is primarily responsible for providing the health basis for non-ioni-
zing radiation standards. Direct support to the regulatory function of
the Agency is provided in the form of expert testimony and preparation
of affidavits as well as expert advice to the Administrator to assure
the adequacy of health care and surveillance of persons having suffered
imminent and substantial endangerment of their health.
The Federal Insecticide, Fungicide, and Rodenticide Act designates
the Environmental Protection Agency 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 environment when used according
to its labeling restrictions. Thus, relative to new law as well as to
specific directives included in Public Law 93-135, 1973, EPA now is
conducting a thorough review of the implications of using alternative
chemicals, including older registered pesticides, for pest control.
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory
iii
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ABSTRACT
Eighteen pesticides being reviewed as part of the EPA Substitute
Chemical Program were tested for mutagenic activity by the following jm
vitro procedures:
o Reverse mutation in Salmonella typhimurium strains TA1535,
TA1537, TA1538, TA98, and TA100 and in Escherichia coli WP2
uvrA.
o Induction of mitotic recombination in the yeast Saccharomyces
cerevisiae D3.
o Relative toxicity assays in DNA repair-proficient and -deficient
strains of E. coli (strains W3110 and p3478, respectively) and
°f Bacillus subtil is (strains H17 and M45, respectively).
o Unscheduled DNA synthesis (UDS) in human fibroblasts (WI-38
cells).
Nine of the 18 pesticides were mutagenic in one or more of the
assays. One compound, demeton, was mutagenic in all of them. Trichlorofon
was mutagenic in all the assays except those for relative toxicity.
Acephat was mutagenic in the Salmonella typhimurium in TA100, Saccharomyces
cerevisiae D3, and UDS assays"! Dicamba, 2,4-D acid, 2,4-DB acid, and
propanil were positive only in the assay for relative toxicity. Disulfoton
was positive only in the UDS assay, and then only in the absence of the
metabolic activation system. Crotoxyphos was positive only in the S.
cerevisiae D3 assay.
iv
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CONTENTS
LIST OF ILLUSTRATIONS ..................... v
LIST OF TABLES ......................... vii
SUMMARY ............................ 1
INTRODUCTION .......................... 3
METHODS ............................ 5
Microbiological Assays .................... 5
Salmonella typhimurium Strains TA1535,
TA1537, TA1538, TA98, and TA100 .............. 5
Escherichia coll WP2
Saccharomyces cerevisiae D3 ................ 8
Escherichia coli W3110/p3478
and Bacillus subtilis H17/M45 ............... 9
Aroclor 1254-Stimulated Metabolic
Activation System ..................... 10
Unscheduled DNA Synthesis Assay ............... H
Cell Culture ........................ 12
Dilution of Compounds ...... . ............ 13
Metabolic Activation .................... 13
Controls .......................... 13
Test Procedure ....................... 13
Interpretation of Results ................. 15
RESULTS AND DISCUSSION ..................... 17
Microbiological Assays .............. , ..... 17
UDS Assay .......................... 24
CONCLUSIONS .......................... 37
REFERENCES ........................... 163
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ILLUSTRATIONS
1 Assays of Acephate With Salmonella typhimurium
Strain TA100 19
2 Assays of Demeton With Salmonella typhimurium
Strains TA1535 and TA100 20
3 Assays of Trichlorfon With Salmonella typhimurium
Strain TA100 21
4 Assays of Demeton With Escherichia coli
Strain WP2 22
5 Assays of Trichlorfon With Escherichia coli
Strain WP2 23
6 Assays of Demeton With Saccharomyces cerecisiae D3 . . . 25
7 Assays of Crotoxyphos With Saccharomyces
cerevisiae D3
26
8 Assay of Acephate With Saccharomyces cerevisiae D3
Without Metabolic Activation 27
C Assays of Trichlorfon With Saccharomyces
cerevisiae D3 28
10 Unscheduled DNA Synthesis Assays of Demeton 30
11 Unscheduled DNA Synthesis Assays of Acephate
Without Metabolic Activation 31
12 Unscheduled DNA Synthesis Assays of Disulfoton
Without Metabolic Activation 33
13 Unscheduled DNA Synthesis Assays of Trichlorfon
Without Metabolic Activation 34
vi
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28 Ethion, Experiment 1 65
29 Ethion, Experiment 2 66
30 Ethion, Experiment 3 67
31 Fensulfothion, Experiment 1 ..... 68
32 Fensulfothion, Experiment 2 69
33 Fonofos, Experiment 1 70
34 Fonofos, Experiment 2 71
35 Methoxychlor, Experiment 1 72
36 Methoxychlor, Experiment 2 73
37 Propanil, Experiment 1 74
38 Propanil, Experiment 2 75
39 Propanil, Experiment 3 76
40 Siduron, Experiment 1 77
41 Siduron, Experiment 2 78
42 Trichlorfon, Experiment 1 79
43 Trichlorfon, Experiment 2 80
44 Trichlorfon, Experiment 3 81
In Vitro Assays with Escherichia coli WP2:
45 Acephate, Aspon, Carbofuran, Crotoxyphos,
Demeton, Diaziuon, Dicamba, Disulfoton,
and Ethion 82
46 Acephate 84
47 Fensulfothion, Fonofos, Methoxychlor, Propanil,
Siduron, Trichlorfon, 2,4-D Acid, 2,4-DB Acid,
and Endrin 85
48 Trichlorfon 87
49 Differential Toxicity of Repair-Proficient
and -Deficient Microorganisms 88
J_n Vitro Assays with Saccharomyces cerevisiae D3
50 Acephate 91
51 Aspon 92
52 Carbofuran 93
53 Crotoxyphos 94
54 2,4-D Acid 95
55 2,4-DB Acid 96
vii
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TABLES
1 Eighteen Pesticides Evaluated by SRI International
for Kutagenicity 38
2 In Vitro Mutagenesis: Summary Data for EPA
Pesticides . . . . , 39
In Vitro Assays with Salmonella typhimurium:
3 Acephate, Experiment 1 40
4 Acephate, Experiment 2 41
5 Acephate, Experiments 3 and 4 42
6 Aspon, Experiment 1 43
7 Aspon, Experiment 2 44
u Carbofuran, Experiment 1 45
9 Carbofuran, Experiment 2 46
10 Crotoxyphos, Experiment 1 47
11 Crotoxyphos, Experiment 2 48
12 2,4-D Acid, Experiment 1 '. . . . 49
13 2,4-D Acid, Experiment 2 . . 50
14 2,4-DB Acid, Experiment 1 51
15 2,4-DB Acid, Experiment 2 52
16 Demeton, Experiment 1 53
17 Demeton, Experiment 2 54
18 Diazinon, Experiment I 55
19 Diazinon, Experiment 2 56
20 Dicamba, Experiment 1 57
21 Dicamba, Experiment 2 58
22 Dicamba, Experiment 3 59
23 Disulfoton, Experiment 1 60
24 Disulfoton, Experiment 2 61
25 Disulfoton, Experiment 3 62
26 Endrin, Experiment 1 63
27 Endrin, Experiment 2 64
viii
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88 Third Assay of Dicamba with Metabolic Activation .... 132
89 Assay of Fensulfothion 133
90 Repeat Assay of Fensulfothion 134
91 Assay of Fensulfothion with Metabolic Activation .... 135
92 Repeat Assay of Fensulfothion with Metabolic Activation 136
93 Assay of Fndrin 137
94 Assay of Endrin with Metabolic Activation 138
95 Repeat Assay of Endrin with Metabolic Activation .... 139
96 Third Assay of Endrin with Metabolic Activation .... 140
97 Assay of Aspon 141
98 Assay of Aspon with hetabolic Activation 142
99 Assay of Carbofuran 143
100 Assay of Carbofuran with Metabolic Activation 144
101 Assay of Crotoxyphos 145
102 Assay of Crotoxyphos with Metabolic Activation 146
103 Assay of 2,4-D Acid 147
104 Assay of 2,4-D Acid with Metabolic Activation 148
105 Assay of 2,4-DB Acid 149
106 Assay of 2,4-DB Acid with Metabolic Activation 150
107 Assay of Diazinon 151
108 Assay of Diazinon with Metabolic Activation 152
109 Assay of Fonofos 153
110 Assay of Fonofos with Metabolic Activation 154
111 Assay of Ethion 155
112 Assay of Ethion with Metabolic Activation 156
113 Assay of Methoxychlor 157
114 Assay of Methoxychlor with Metabolic Activation .... 158
115 Assay of Siduron 159
116 Assay of Siduron with Metabolic Activation 160
117 Assay of Propanil 161
118 Assay of Propanil with Metabolic Activation 162
IX
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56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
94
85
86
87
Demeton
Diazinon
Dicamba
Disulfoton
Endrin
Ethion
Fensulf othion
Fonofos
Methoxychlor
Propanil
Siduron
Trichlorfon
Imscheduled DNA Synthesis:
Assay of Demeton
Repeat Assay of Demeton
Assay of Demeton with Metabolic Activation
Repeat Assay of Demeton with Metabolic Activation . . .
Assay of Acephate
Repeat Assay of Acephate
Assay of Acephate with Metabolic Activation
Repeat Assay of Acephate with Netabolic Activation . . .
Assay of Disulfoton
Repeat Assay of Disulfoton
Assay of Disulfoton with Metabolic Activation
Repeat Assay of Disulfoton with Metabolic Activation . .
Assay of Trichlorfon
Repeat Assay of Trichlorfon
Assay of Trichlorfon with Metabolic Activation
Repeat Assay of Trichlorfon with Metabolic Activation
Assay of Dicamba
Repeat Assay of Dicamba
Assay of Dicamba with Metabolic Activation
Hepeat Assay of Dicamba with Metabolic Activation . . .
97
99
101
102
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
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SUMMARY
Eighteen pesticides being reviewed as a part of the EPA Substitute
Chemical Program were tested for mutagenic activity by the following
in vitro procedures:
• Reverse mutation in Salmonella typhimurium strains TA1535,
TA1537, TA1538, TA98, and TA100 and in Escherichia coli
WP2 uvrA.
• Induction of mitotic recombination in the yeast Saccharomyces
cerevisiae D3.
• Relative toxicity assays in DNA repair-proficient and -deficient
strains of _E. colj. (strains W3110 and p3A78, respectively) and of
Bacillus subtilis (strains H17 ans M45 respectively).
• Unscheduled DNA synthesis (UDS) in human fibroblasts (WI-38 cells)
Nine of the 18 pesticides were mutagenic in one or more of the assays.
One compound, demeton, was mutagenic in all of them. Trichlorofon was
mutagenic in all the assays except those for relative toxicity. Acephate
was mutagenic in the Salmonella typhimurium in TA100, Saccharomyces
cerevisiae D3, and UDS assays. Dicamba, 2,4-D acid, 2,4-DB acid, and
propanil were positive only in the assay for relative toxicity. Disul-
foton was positive only in the UDS assay, and then only in the absence
of the metabolic activation system. Crotoxyphos was positive only in
the J3. cerevisiae D3 assay.
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INTRODUCTION
The Federal Insecticide, Fungicide, and Rodenticide Act designates
the Environmental Protection Agency 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 environment when used according
to its labeling restrictions. Thus, relative to new law as well as to
specific directives included in Public Law 93-135, 1973, EPA now is
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 development of
scientific criteria for evaluating the safety of compounds substituted
for those pesticides found to be hazardous. In addition to reviewing
and evaluating the literature on pesticides and maintaining liaison
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 Committee recommended
that "Regulatory agencies should take steps to insure that new scientific
data raising the possibility of new or extended hazards from chemicals in
use are subject to careful process of scientific review for merit inter-
pretation. "
Development of methods for evaluating the mutagenic hazard of
chemical compounds has advanced markedly in the last few years. In
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contrast to the undefined empirical tests used a short time ago, pro-
cedures now available can detect chromosome breaks, DNA damage, and
mutational events caused by chemical stress. Mutant strains of micro-
organisms and human fibroblast cells are effective in vitro systems
for reliable detection of genotoxic agents.
Many pesticide chemicals in commercial use today have not been
adequately investigated for their potential mutagenic hazard. With
the public's increasing concern about possible pollution of our environ-
ment by chemicals, the widely used pesticides must be evaluated.
Under contract to EPA, SRI studied 18 pesticides to determine their
potential for mutagenic activitiy. We used in vitro test methods that
are appropriate for such evaluations. The 18 pesticides tested are
listed in Table 1; the common name, trade name, manufacturer, purity,
batch or lot number, and supplier are shown. (SRI had previously
reported on "In Vivo and In Vitro Studies of Selected Pesticides To
Evaluate Their Potential as Chemical Mutagens" in February 1977.1
That study of 20 pesticides was a part of this contract, No. 68-01-
2458.)
The six in vitro assay systems used were the reverse mutation in
Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and
TA100 and in Escherichia coli WP2; induction of mitotic recombination
in the yeast Saccharomyces cerevisiae D3; relative toxicity assays in
DNA repair-proficient and -deficient strains of IS. coli (strains W3110
and p3A78, respectively) and of Bacillus subtilis (strains H17 and M45,
respectively); and unscheduled DNA synthesis (UDS) in human fibroblasts
(WI-38 cells).
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METHODS
Microbiological Assays
The in vitro microbiological assay systems used to examine the 18
pesticides for mutagenicity were Salmonella typhimurium (TA1535, TA1537,
TA1538, TA98, and TA100), Escherichia coli WP2, repair-deficient and
-proficient strains of Bacillus subtilis (H17 and M45) and of J5. coli
(W3110 and p3478) , and the yeast Saccharomyces cerevisiae D3. In each
procedure except the relative toxicity assays, an Aroclor 1254-stimulated,
rat liver homogenate metabolic activation system was included to provide
metabolic steps that the microorganisms either are incapable of conducting
or do not carry out under the assay conditions.
The assay procedure with S_. typhimurium has proven to be 80 to 90%
accurate in detecting carcinogens as mutagens, and it has about the same
accuracy in identifying chemicals that are not carcinogenic.2'3 The assay
procedure with S^. cereyisiae is about 55% accurate in detecting carcino-
gens as agents that increase mitotic recombination.'' IS. coli WP2 and the
relative toxicity assays are three additional methods of detecting muta-
gens; however, the reliability of these test methods has not been ade-
quately validated yet. The combination of these five assay procedures
significantly enhances the probability of detecting potentially hazardous
chemicals.
Salmonella typhimurium Strains TA1535, TA1537, TA1538,
TA98, and TA100
The Salmonella typhimurium strains used at SRI are all histidine
auxotrophs by virtue of mutations in the histidine operon. When these
histidine-dependent cells are grown on a minimal media petri plate
containing a trace of histidine, only those cells that revert to histi-
dine independence (his ) are able to form colonies. The small amount
of histidine allows all the plated bacteria to undergo a few divisions;
in many cases, this growth is essential for mutagenesis to occur. The
his revertants are easily scored as colonies against the slight
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background growth. The spontaneous mutation frequency of each strain is
relatively constant, but when a mutagen is added to the agar, the
mutation frequency is increased 2- to 100-fold, usually in a dose-
related manner.
We obtained our ^. typhimurium strains from Dr. Bruce Ames of the
University of California at Berkeley.2'5"10 In addition to having
mutations in the histidine operon, all the indicator strains have a
mutation (rfa) that leads to a defective lipopolysaccharide coat; they
also have a deletion that covers genes involved in the synthesis of the
vitamin biotin (bio) and in the repair of ultraviolet (uv)-induced DNA
damage (uvrB). The rfa mutation makes the strains more permeable to
many large aromatic molecules, thereby increasing the mutagenic effect
of these molecules. The uvrB mutation causes decreased repair of some
types of chemically or physically damaged DNA and thereby enhances the
strains' sensitivity to some mutagenic agents. Strain TA1535 is reverted
to his by many mutagens that cause base-pair substitutions. TA100 is
derived from TA1535 by the introduction of the resistance transfer
factor, plasmid pKMLOl. This plasmid is believed to cause an increase
in error-prone DNA repair that leads to many more mutations for a given
dose of most mutagens.7 In addition, plasmid pKMlOl confers resistance
to the antibiotic ampicillin, which is a convenient marker to detect the
presence of the plasmid in the cells. We have shown that TA100 can
detect mutagens such as benzyl chloride and 2-(2-furyl)-3-(5-nitro-2-
furyl)acrylamide (AF2) that are not detected by TA1535. The presence of
this plasmid also makes strain TA100 sensitive to some frameshift muta-
gens [e.g., ICR-191, benzo(a)pyrene, aflatoxin Bj., and 7,12-dimethylben-
z(a)anthracene]. Strains TA1537 and TA1538 are reverted by many frame-
shift mutagens. Strain TA98 is derived from TA1538 by the addition of
plasmid pKMlOl, which makes it more sensitive to some mutagenic agents.
All indicator strains are kept at 4° C on minimal agar plates,
supplemented with an excess of biotin and histidine. The plates with
the plasmid-carrying strains contain, in addition, ampicillin (25 yg/ml)
to ensure stable maintenance of plasmid pKMlOl. New stock culture
plates are made every four to six weeks from single colony reisolates
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that have been checked for their genotypic characteristics (his, rfa,
uvrB, bio) and for the presence of the plasmid. For each experiment, an
inoculum from the stock culture plates is grown overnight at 37° C in
nutrient broth (Oxoid, CM67). After stationary overnight growth, the
cultures are shaken for 3 to 4 hours to ensure optimal growth.
To a sterile 13 x 100 mm test tube placed in a 43° C heating
block, we add in the following order:
(1) 2.00 ml of 0.6% agar*
(2) 0.05 ml of indicator organisms
(3) 0.50 ml of metabolic activation mixture (optional)
(4) 0.05 ml of a solution of the pesticide dissolved in DMSO.
For negative controls, we use steps (1), (2), and (3) (optional) and
0.05 ml of the solvent used for the test chemical. For positive
controls, we test each culture by specific mutagens known to revert
each strain, using steps (1), (2), (3) (optional), and (4).
This mixture is stirred gently and then poured onto minimal agar
plates.T After the top agar has set, the plates are incubated at 37° C
for 2 days. The number of his revertant colonies is counted and
recorded.
A positive response in the Salmonella/microsome assay is indicated
by a reproducible, dose-related increase in the number of revertants in
one or more of the tester strains.
Escherichia coli WP2
The .E. coli WP2 (uvrA) strain used at SRI was obtained originally from
Dr. D. McCalla. X1 It is a tryptophan auxotroph (trg) by virtue of a
*
0.6% agar contains 0.05 mM histidine, 0.05 mM biotin, and 0.6% NaCl.
See page 9.
^Minimal agar plates consist of, per liter, 15 g of agar, 20 g of
glucose, 0.2 g of MgSOi,»7H20, 2 g of citric acid monohydrate, 10 g
of K2HP04, and 3.5 g of
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base-pair substitution mutation in the tryptophan operon. In addition,
WP2 is deficient in the repair of some physically or chemically induced
DNA damage (uvrA). 12 This uyrA mutation makes the strain more sensitive
to certain mutagens.
A procedure similar to the Ames Salmonella assay is used to measure
the reversion of WP2 to tryptophan independence. However, the minimal
agar is supplemented with 1.25 g of Oxoid nutrient broth (CM67) per
liter to provide each plate with the trace of tryptophan required for
enhancement of any mutagenic effect of the test chemical.12 No addi-
tional tryptophan is added to the top agar.
Saccharomyces cerevisiae D3_
The yeast ^. cerevisiae D3 is a diploid microorganism heterozygous
for a mutation leading to a defective enzyme in the adenine-metabolizing
pathway. 13 When grown on medium containing adenine, cells homozygous
for this mutation produce a red pigment. These homozygous mutants can
be generated from the heterozygotes by mitotic recombination. The
frequency of this recombinational event may be increased by incubating
the organisms with various mutagens. The degree of mutagenicity of a
compound or of its metabolite is determined from the number of red-
pigmented colonies appearing on the plates. lh
The S_. cerevisiae tester strain is stored at -80° C. For each
experiment, the tester strain is inoculated in 1% tryptone and 0.5%
yeast extract and grown overnight at 30° C with aeration.
The in vitro yeast mitotic recombination assay in suspension is
conducted as follows. The overnight culture is centrifuged, and the
cells are resuspended at a concentration of ~10B cells/ml in a 67 mM
phosphate buffer (pH 7.4). To a sterile test tube are added:
• 1.30 ml of the resuspended culture
• 0.50 ml of either the metabolic activation mixture or buffer
• 0.20 ml of a solution of pesticide dissolved in DMSO or 0.20 ml
of DMSO alone.
Several doses of the pesticide (up to 5%, w/v or v/v) are tested in
each experiment, and appropriate controls are included.
8
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The suspension mixture is incubated at 30° C for 4 hours on a
roller drum. The sample is diluted serially in sterile physiological
saline, and 0.2-ml aliquots of the 10"3 and 10~3 dilutions are spread
on tryptone-yeast agar plates; five plates are used for the 10~3
dilution and three plates are used for the 10~s dilution. The plates are
incubated for 2 days at 30° C, followed by 2 days at 4° C to enhance
the development of the red pigment indicative of adenine-deficient
homozygosity. Plates of the 10~3 dilution are scanned with a dissecting
microscope at 10X magnification, and the number of red colonies or red
sectors (mitotic recombinants) is recorded. The surviving fraction of
organisms is determined from the number of colonies appearing on the
plates of the 10"5 dilution. The number of mitotic recombinants is
calculated per 10s survivors.
A positive response in this assay is indicated by a dose-related
increase in the absolute number of mitotic recombinants per milliliter
as well as in the relative number of mitotic recombinants per 10
survivors.
Escherichia coll W3110/p3478 and Bacillus subtilis H17/M45
The E. coll strains W3110 and p3478 that are used at SRI were
obtained from Dr. H. Rosenkranz, who devised the DNA polymerase repair
assay.1 Strain p3478 is a DNA polymerase-deficient (polA ) derivative
of W3110 and is very sensitive to the effects of some physical and
chemical agents that react with cellular DNA. The repair assay is based
on the finding that when exposed to agents that alter the DNA, bacteria
tend to protect themselves by removing the altered DNA segment and then
by resynthesizing the correct DNA sequence. Thus, their survival is
enhanced. The enzyme DNA polymerase is involved in this resynthesizing
process.16 The extent of chemically induced DNA damage can be measured
by comparing the relative toxicity (zone of growth inhibition) of the two
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strains. Therefore, if a chemical interacts with DNA, strain p3478 should
be more sensitive than strain W3110 to any toxic effect due to this
interaction.
The J3. subtilis strains H17 and M45 were obtained from Dr. T. Kada.17
Strain M45 (rec ) is derived from H17 but is deficient in the genetic
recombination mechanism necessary to repair DNA damage. Cells deficient
in this repair mechanism are killed more easily by chemical mutagens than
are wild-type cells (rec ). If the chemical is toxic to rec cells but
at the same concentration is not toxic to rec cells, the chemical is
assumed to interact with DNA.
For each experiment, an inoculum from frozen stock cultures is grown
overnight at 37° C with shaking in nutrient broth consisting of 1% tryp-
tone and 0.5% yeast extract. A 0.1-ml aliquot of this bacterial culture
(approximately 3 x 108 cells) is added to 2 ml of nutrient broth containing
0.6% agar. The suspension is mixed and poured onto the surface of a plate
containing the same ingredients as the broth plus 2% agar (25 ml). When
the top agar has solidified, a sterile filter disc impregnated with the
test substance is placed in the center of the plate. The plates are
incubated at 37° C fur 16 hours; then the width (diameter) of the zone
of inhibition of growth is measured. Several concentrations of the sub-
stance are usually tested. We routinely use DMSO as diluent and as solvent
for crystalline chemicals.
The positive control for this assay is l-phenyl-3,3-dimethyltriazine.
The negative control is chloramphenicol, which should cause equal zones
of inhibition in both strains because it is toxic to bacteria but does
not kill by interacting with DNA.
Aroclor 1254-Stimulated Metabolic Activation
Some carcinogenic chemical (e.g., of the aromatic amino type or
polycyclic hydrocarbon type) are inactive unless they are metabolized
to active forms. In animals and man, an enzyme system in the liver or
other organs (e.g., lung or kidney) is capable of metabolizing a large
number of these chemicals to carcinogens.8"10 Some of these inter-
-«?
mediate metabolites are very potent mutagens in the S^. typhimurium
10
-------
test. Ames has described the liver metabolic activation system that
we use.10 In brief, adult male Sprague-Dawley rats (250 to 300 g) are
given a single 500-mg/kg intraperitoneal injection of a polychlorinated
biphenyl, Aroclor 1254. This treatment enhances the synthesis of
enzymes involved in the metabolic conversion of chemicals. Four days
after the injection, the animals' food is removed, but drinking water
is provided ad libitum. On the fifth day, the rats are killed and the
liver homogenate is prepared as follows.
The livers are removed aseptically and placed in a preweighed
sterile glass beaker. The organ weight is determined, and all sub-
sequent operations are conducted in an ice bath. The livers are
washed in an equal volume of cold, sterile 0.15 M KC1 (1 ml/g of
wet organ), minced with sterile surgical scissors in three volumes
of 0.15 M KC1, and homogenized with a Potter-Elvehjem apparatus. The
homogenate is centrifuged for 10 minutes at 9000 x j*, and the super-
natant, referred to as the S-9 fraction, is quickly frozen in dry ice
and stored at -80° C.
The metabolic activation mixture consists of, for 10 ml:
• 1.00 ml of freshly thawed S-9 fraction
• 0.20 ml of MgCl2 (0.4 M) and KC1 (1.65 M)
• 0.05 ml of glucose-6-phosphate (1 M)
• 0.40 ml of NADP (0.1 M)
• 5.00 ml of sodium phosphate (0.2 M, pH 7.4)
• 3.35 ml of H20.
Unscheduled DNA Synthesis Assays
Many mutagenic and carcinogenic agents have been shown to induce
unscheduled DNA synthesis (UDS) in an i.n vitro tissue culture system
of mammalian cells.18 UDS is a form of mammalian repair synthesis
that involves at least two processes: first, the agent interacts with
DNA, resulting in damage to the DNA; then follows incorporation
of nucleotide(s) to repair the DNA. UDS, which occurs in a wide
variety of mammalian cell types, is considered to be a fairly universal
11
-------
system because it has been observed in all stages of the cell cycle
(Go, GI, G2 , and M) other than S, the normal synthetic phase.19'20
(UDS is not observed during S-phase because the high level of incorp-
oration of nucleotides during scheduled DNA synthesis obscures the
relatively low level of incorporation of nucleotides during UDS.)
A number of chemicals have been shown to be ineffective in
producing DNA damage in in vitro cultures of mammalian cells; yet in
the metabolically active environment of the whole animal they are
rapidly converted to mutagenic and/or carcinogenic intermediates.
Therefore, the investigator must attempt to recreate this metabolic
environment in vitro. This is usually done by adding a microsoinal
preparation from a mammalian liver homogneate to the test system.
Thus, we routinely perform a parallel series of UDS assays in the
presence and absence of a metabolically active environment to predict
the ability of an agent to induce genetic damage.
We used the UDS assay system in the previous testing of 20 substitute
pesticides.1 Reported here are the results of UDS testing, with and
without metabolic activation, of the 18 additional substitute pesticides.
Cell Culture
WI-38 cells grown in T-25 tissue culture flasks were used for the
UDS assays. Replicate cultures of these cells were initiated in
Eagle's Basal Medium containing 10% (v/v) fetal calf serum. The
cells were grown to confluency and were maintained in medium containing
0.5% serum for 5 to 6 days preceding the UDS assays.* This produced
contact-inhibited cells in synchronous cultures in the G0 phase of
the mitotic cycle. To further reduce the possibility of incorporation
of 3H-TdR by an occasional S-phase cell that might escape the contact-
* As a check against the presence of mycoplasma, which could incorporate
tritiated thymidine (3H-TdR) and thus obscure measurments of UDS, stock
cultures were periodically sent to Microbiological Associates, who
cultured them on Difco Beef Heart Infusion agar or broth for analysis
for the presence of mycoplasma. The results of these analyses were
consistently negative.
12
-------
inhibition synchrony and thus obscure measurements of UDS, the
cultures were preincubated for 1 hour with 10~2 M hydroxyurea (HU)
before each assay and 10~2 M HU was added during each subsequent
step of the assays.
Dilution of Compounds
Immediately prior to each assay, the pesticide was diluted in an
appropriate solvent (ethanol or DMSO) to form a series of concentra-
tions that, when diluted into culture medium, yielded the appropriate
set of test concentrations. To facilitate solubilization or achieve
an even suspension of the stock solutions of the compounds in solvent,
some of the compounds were sonicated for a brief period of time prior
to dilution. The final concentration of solvent was maintained at 1%
or less, which we have previously found to be not cytotoxic.
Metabolic Activation
For testing with metabolic activation, a preparation consisting
of the 9000 x j> supernatant of a liver homogenate (250 mg of liver/ml)
from adult Swiss-Webster mice was used. To this was added the following
cofactors: nicotinamide, 3.05 mg/ml; glucose-6-phosphate, 16.1 mg/ml;
MgCl2»6H20, 5.08 mg/ml; and NADP, 0.765 mg/ml.
Controls
The positive controls were 4-nitroquinoline-N-oxide (4NQO), a
compound that induces UDS in the absence of a metabolic activation
system, and dimethyInitrosamine (DMN), a compound that induces UDS
in vitro only when an exogenous metabolic activation system is in-
corporated into the treatment protocol. The negative control was
the solvent diluted in culture medium.
Test Procedure
The contact-inhibited WI-38 cells were incubated at 37° C with
dilutions of the pesticides and with 1 yCi/ml of 3H-TdR (specific activity,
6.7 Ci/mmole). For testing in the absence of metabolic activation,
the cells were exposed simultaneously to the pesticide and to 3H-TdR for
3 hours. For testing with metabolic activation, the cells were incubated
13
-------
together with pesticide, 3H-TdR, and the metabolic activation prepaira-
tion for 1 hour. (The shorter exposure time for metabolic activation
testing was used because longer exposures of WI-38 cells to the liver
homogenate preparation could be cytotoxic.) In both cases, the cells
were then incubated with 3H-TdR and HU, but without pesticide, for
an additional 3 hours.
DNA was extracted from the cells using a modification of the PCA-
hydrolysis procedure;21 one aliquot of the DNA solution was used to
measure the DNA content, after the reaction with diphenylamine, and
a second aliquot was used for scintillation-counting measurements of
the extent of incorporation of 3H-TdR. The results were expressed
as disintegrations per minute (dpm) of incorporated 3H-TdR per unit
of DNA and were compared with the rate of incorporation of 3H-TdR
into cells exposed to solvent only (negative controls).
We have defined as an acceptable assay one in which the response
of the positive control compound is predicted, within the 95% confi-
dence limits, by regressions of average dpm/yg DNA versus average
dpm/iJg for background.23 The regressions that follow are based on
data that we have acquired in previous testing:
Sample Correlation
Type of Testing Regression* Size (n) Coefficient (r)
Without metabolic
activation Yt = 629 + 16.42 (X)-j- 55 0.8066
With metabolic
activation Y2 = 212 + 2.11 (X)t 25 0.8307
If the observed average level of incorporation for the positive control
compound is outside the 95% confidence limits of the regression, we assume
that some variation has occurred in the experimental procedures and the
test is repeated.
* Regressions over a range of background dpm/yg DNA of 0 to 450.
t Yi = Average dpm/yg DNA for 10~s M 4NQO (positive control).
Y2 = Average dpm/yg DNA for 5 x 10~2 M DMN (positive control).
X = Average dpm/yg DNA for background (negative control).
14
-------
Interpretation of Results
We have tested 41 compounds of defined carcinogenic activity,
based on the results of in vivo bioassays, and have analyzed these results
using either the parametric One-Way Classification Analysis of Variance
or the nonparametric Kruskal-Wallis One-Way Analysis of Variance, depend-
ing on which was more appropriate.* Of the 16 compounds generally
recognized as being direct-acting carcinogens, 15 induced statistically
significant elevations in the incorporation of 3H-TdR into DNA, at the
99% confidence level. In all but three of these, the response was
dose-related. The assay of the sixteenth carcinogen, p>-rosaniline,
failed to suggest a positive response. Of the 13 compounds reported to
be noncarcinogenic, only one (glycidol) had a statistically significant
positive response, which was not dose-related. Thus, it appears that
the 99% confidence limits of these statistical analysis coupled with
the indication of a dose-response relationship can be used with reason-
able accuracy to predict the biological significance of the UDS
response to an ultimate carcinogen or a noncarcinogen.
The correlation between UDS response and biological significance
for testing with metabolic activation is less clear. Of the 12 pro-
carcinogens (compounds requiring chemical modifications to become
active) that we have tested with metabolic activation, seven induced
statistically significant increases in 3H-TdR uptake at the 99%
confidence level, all of which were dose-related. The remaining 5 pro-
carcinogens failed to indicate any increase in 3H-TdR incorporation.
Thus, it appears that the metabolic activation preparation presently
used for UDS testing is capable of activating only a portion of the
spectrum of procarcinogens. However, for those that can be activated,
a statistically significant and dose-related response can be observed.
Thus, if these two criteria are met, we can interpret the results as
indicating the repair of DNA damage by the cells in response to the compound
* If there is reason to believe that the variances of each of the
treatments in a test are equal (.i.e., Bartlett^s test of the variance
is negative), the parametric analysis is the appropriate one. If the
variances are not equal, the nonparametric analysis is the appropriate
one.24'25
15
-------
being evaluated. However, lack of a positive response in testing cannot
be assumed to be indicative of an absence of potential biological hazard.
16
-------
RESULTS AND DISCUSSION
The results of the in vitro microbiological and UDS assays are
summarized in Table 2. 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 observed for 9 of the 18 pesticides
tested. The 9 pesticides that had a positive response on one or more of
the assays were acephate, crotoxyphos, 2,4-D acid, 2,4-DB acid, demeton,
dicamba, disulfoton, propanil, and trichlorfon. Demeton was positive
in all the assays. Trichlorfon was positive in all but the relative
toxicity assays with 15. coli and Ji. subtilis. Acephate was weakly
mutagenic in S^. typhimurium TA100, increased mitotic recombination in
S_. cerevisiae D3, and increased unscheduled DNA synthesis in WI-38 cells.
No toxicity was observed in the relative toxicity assays for either
acephate or trichlorfon; therefore, the negative result in these assays
may indicate that the pesticide did not diffuse into the agar. Dicamba,
2,4-D acid, 2,4-DB acid, and propanil were positive in the relative
toxicity assays but were without activity in all the other assays.
Crotoxyphos increased mitotic recombination in JS. cerevisiae D3 and
disulfoton increased UDS, but no other effects were observed for these
two pesticides. Except for the relative toxicity assays, dose-response
curves are presented for pesticides that gave a positive response.
Aspon, carbofuran, diazinon, endrin, ethion, fensulfothion, fonofos,
methoxychlor, and siduron were not genotoxic or mutagenic in any of the
six assays we performed.
Microbiological Assays
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
mutagenicity was observed, the second experiment was conducted at
higher concentrations. If mutagenicity was observed, a dose response
17
-------
was determined. An assay that gave a mutagenic response was always
repeated to confirm that the results were reproducible.
Tables 3 through 44 present the results of the microbiological
assays in agar with Salmonella typhimurium. In this reverse-mutation
assay system, three pesticides were mutagenic. Acephate (Tables 3-5)
was very weakly mutagenic in assays with strain TA100 at doses above
2500 yg/plate. Although the response was marginal (the greatest increase
observed was 61 revertants above a background of 150 spontaneous revert-
ants in Experiment 3), Figure 1 shows that it was reproducible and dose-
related. Demeton (Tables 16 and 17) was strongly mutagenic in strains
TA1535 and TA100, inducing as much as a 50-fold increase in revertants
above the background in strain TA1535 and a 10-fold increase in TA100;
Figure 2 presents the dose-response curves. Demeton was slightly more
mutagenic when the metabolic activation mixture was added to the plate
than when it was not.
Trichlorfon increased reverse mutations in strain TA100 only (Tables
42-44; Figure 3). However, it was only moderately mutagenic, with a
3- to 5-fold increase in revertants above the background frequency. It
was more mutagenic without the addition of the metabolic activation mix-
ture (Experiment 3). In previous studies, trichlorfon was not found to
be mutagenic in the dominant lethal26 and Drosophila27 assays.
We observed slight increases in the number of revertants with strain
TA100 in the assays with ethion (Tables 28-30). However, because the
amount of this pesticide was limited, we were unable to test at doses
greater than 5 mg/plate. We believe additional tests using higher
doses are needed to determine whether a dose response could be obtained.
Tables 45 through 48 present the results of the assays with E_. coli
WP2. Demeton and trichlorfon were mutagenic in this reverse-mutation
assay. Figures 4 and 5 show the dose response. Their order of mutagenic
activity was the same as in the j^. typhimurium assays; i.e., demeton was
more mutagenic than trichlorfon.
Table 49 presents the results of assays for microbial inhibition in
repair-deficient and -proficient strains of B^. sub til is and .E. coli.
18
-------
to
Q.
S-
]
\
O)
01
£
(U
225
200 -
175 -
150
125 -
100
I I I I
345678
mg of Acephate per Plate
10
FIGURE 1 ASSAYS OF ACEPHATE WITH SALMONELLA TYPHIMURIUM
STRAIN TA100.
19
-------
1000
0
1000
5000
2000 3000 4000
ug Demeton per Plate
FIGURE 2 ASSAYS OF DEMETON WITH SALMONELLA TYPHIMURIUM
STRAIN TA1535 and TA100.
20
-------
345678
mg/Plate of Trichlorfon
10
FIGURE 3
ASSAYS OF TRICHLORFON WITH SALMONELLA TYPHIMURIUM
STRAIN TA100.
21
-------
UJ
200 400 600 800
ug Demeton per Plate
1000
FIGURE 4 ASSAYS OF DEMETON WITH ESCHERICHIA COLI
STRAIN WP2.
22
-------
80
n
IT 70
S-
0)
Q.
60
« 50
40
c\j
Q.
(U
CD
03
0)
30
20
10
0
I I I I I I I I \
+ S-9 mix
CH30 0
^
CH30X HC-CC13
012345 6789 10
mg/plate of Trichlorfon
FIGURE 5 ASSAYS OF TRICHLORFON WITH ESCHERICHIA
COLI STRAIN WP2.
23
-------
Toxic chemicals that do not act by damaging DNA (e.g., chloramphenicol)
should give equal zones of toxicity on both repair-proficient and repair-
deficient strains. However, a given concentration of a chemical that is
genotoxic may be toxic for a repair-deficient strain but not for a strain
that effectively repairs its DNA. Dicamba, 2,4-D acid, 2,4-DB acid,
demeton, and propanil gave positive responses in these assays; i.e., each
gave zones of inhibition that were larger in the repair-deficient strains
than in the repair-proficient strains.
Tables 50 through 68 present the results of the assays for mitotic
recombination in Saccharomyces cerevisiae D3. Four pesticides—demeton,
crotoxyphos, acephate, and trichlorfon—increased the mitotic recombina-
tion frequency in each experiment and are considered positive by these
procedures. Figures 6 to 9 show the curves. Two pesticides—diazinon
and disulfoton—gave an increased number of mitotic recombinants in one
experiment but appeared to be negative when tested a second and third
time. 2,4-D acid has been found to be positive in Saccharomyces cerevisiae
D4, D5, and RAD18 when tested at low pH (4.3),28 but it was not recombi-
nogenic in our assays with S^. cerevisiae D3.
UPS Assays
Table 69 through 119 present the results of the UDS testing, with
and without metabolic activation, of 18 substitute pesticides. For the
initial testing, cell cultures were treated with a series of 10-fold
dilutions of each compound to cover a large range of concentrations.
However, because of the spacing of the dilutions, dose-response relation-
ships were not clearly defined. Frequently, significant increases in
3H-TdR uptake were observed at only one concentration. Therefore, to
establish dose-response relationships for compounds that indicated a
significant (statistically significant at the 95% confidence level)
increase in UDS in the initial tests, additional tests were performed
using narrower concentration ranges and higher concentrations of test
chemical, if necessary.
The first test of demeton without metabolic activation (Table 68)
indicated a statistically significant elevation in 3H-TdR incorporation
24
-------
-------
in
O
LO
O
-------
% Acephate
FIGURE 8 ASSAY OF ACEPHATE WITH SACCHAROMYCES
CEREVISIAE D3 WITHOUT METABOLIC ACTIVATION.
27
-------
in
S-
o
>
i_
3
to
in
o
s-
(D
O.
V)
-M
c
to
c
JD
O
O
(U
o:
-M
o
+j
CO
o
012345
% Trichlorfon
FIGURE 9 ASSAYS OF TRICHLORFON WITH SACCHAROMYCES CEREVISIAE D3.
28
-------
at the 99% confidence level (H = 25.81 5 xl > 15.09); however, a dose-
response relationship was not observed in this test. A second test of
demeton under similar conditions, but with a narrower dose range (Table 69),
again indicated a statistically significant (99% confidence) response
(Fs,3o = 31.17 > 3.70), with a characteristic dose-response relationship.
Similarly, the initial test of demeton with metabolic activation (Table 70)
indicated an increase in UDS (F5,30 = 53.58 > 3.70) without an apparent
dose-response relationship. A repeat of this test (Table 71) also indicated
a statistically significant (99% condidence) response (F5,29 = 14.11 > 3.73).
In addition, this assay provided evidence of the existence of a dose-related
response to demeton in the presence of the metabolic activation preparation.
Figure 10 depicts the results of the testing of demeton with and without
metabolic activation. The dose-response curves are illustrative of the
characteristic differences between the testing of direct-acting compounds
in the assay with metabolic activation and the testing in the assay without
it. The two most striking features are: (1) an order of magnitude differ-
ence exists between the concentration ranges at which the dose-response
curves are observed, with the concentration range for testing with metabolic
activation being higher; and (2) the extent of UDS is diminished in the
assay with metabolic activation. Three nonexclusive hypotheses can account
for these variations. The first is that demeton is—at least in part—
enzymatically inactivated by the metabolic activation preparation. Alter-
natively, a nonspecific interaction occurs between demeton and the activa-
tion preparation that reduces the effective concentration to which the cells
are exposed. The third hypothesis is that the reduced duration of exposure
to the compound (the exposure duration with metabolic activation is one-
third that used without metabolic activation) accounts for the decreased
activity. Unfortunately, the data presented do not offer any evidence
that would support one of these hypotheses over the others.
The initial results of testing acephate without metabolic activation
(Table 73) suggested that if this compound increases UDS, the response
occurs at concentrations above 100 ug/ml and the maximum response could
be obtained at some concentration above 1000 yg/ml. An additional
test with this compound (Table 73) confirmed this. Figure 11 depicts
the results of the first and second tests of acephate. We obtained a
29
-------
160
120
DC
UJ
tu
z
Q
til
fe
80
40
0 . k
1.0
I
Initial Assay, -MA
Repeat Assay, -MA
Initial Assay, +MA _
Repeat Assay, +MA
I
10 100
CONCENTRATION OF DEMETON
1000
10,000
LSR-56
FIGURE 10 UNSCHEDULED DNA SYNTHESIS ASSAYS OF DEMETON
30
-------
ou
50
O
3
r°
UI
< 30
uu
b 20
ADJUSTED
o ? S
1 1 1 1 .
/ _
A Initial Assay •
• Repeat Assay I
I
\ \ ~
1
J -
1 1 I 1
1 1.0 10 100 1000 10,000
CONCENTRATION OF ACEPHATE — jig/ml
LSR-57
FIGURE 11 UNSCHEDULED DNA SYNTHESIS ASSAYS OF ACEPHATE WITHOUT
METABOLIC ACTIVATION
31
-------
statistically positive (99% confidence) response at 2000 yg/ml (F5,30 =
7.99 > 3.70). Because of the normal variability between the cultures used
for each assay, the increases in incorporation of 3H-TdR observed between
250 vjg/ml and 1000 yg/ml were not statistically significant at the 99%
confidence level. However, the second test clearly demonstrated a dose-
response relationship as well as the reproducibility of the initial test
results. The first and second tests of acephate with metabolic activation
(Tables 74 and 75) failed to indicate an increase in UDS under these test
conditions.
The results on disulfoton and trichlorfon were similar to those ob-
served for acephate. The first test of disulfoton without metabolic acti-
vation (Table 76) was statistically positive (F5,29 = 39.79 > 3.73), as was
the test of trichlorfon (Table 80; F5>29 = 13.42 > 3.73); yet neither
demonstrated a dose-response relationship. The second assays of these com-
pounds '^Tables 77 and 81', Figures 12 and 13) demonstrated dose-related as
well as statistically significant (99% confidence) increases in UDS (disul-
foton, F5,30 = 18.40 > 3.70; trichlorfon, F5,30 = 10.44 > 3.70). The first
and second assays of each of these compounds with metabolic activation
(Tables 78, 79, 82, and 83) failed to suggest a positive response.
The failure of acephate, disulfoton, and trichlorfon to induce UDS in
the presence of metobolic activation but not in its absence could be relat-
ed to the type of phenomenon observed in the testing of demeton. We
believe that if a response could be observed in assays with acephate, di-
sulfoton, or trichlorfon with metabolic activation, it would occur in
a concentration range roughly an order of magnitude greater than that at.
which it occurs without metabolic activation. Since the maximum concen-
trations tested, both with and without metabolic activation, for each of
these compounds were near the maximum feasible under the constraints of
the solubilities of the compound and the procedures described in "Dilution
of Compounds'", probably none of these three compound could be tested at
high enough concentrations with metabolic activation to obtain a positive
response.
The testing of dicamba and fensulfothion (Tables 84 to 92)
indicatfij that although neither was positive without metabolic
32
-------
p
C2H,0" VS-CH2-CH2-S-CH2-CH3
2 3
mg/ml of Disulfoton
FIGURE 12 UNSCHEDULED DNA SYNTHESIS ASSAYS OF DISULFOTON
WITHOUT METABOLIC ACTIVATION.
33
-------
100 1000
Trichlorfon Concentration (yg/m«,)
FIGURE 13 UNSCHEDULED DMA SYNTHESIS ASSAYS OF TRICHLORFON WITHOUT
METABOLIC ACTIVATION
10,000
34
-------
activation, both initially appeared to induce UDS in the presence of
metabolic activation. In the initial tests with metabolic activation,
no UDS effects were observed for dicamba below 100 yg/ml, although a
statistically significant response was observed at 1000 yg/ml (Table 84) .
No effects were observed for fensulfothion below 1.0 yg/ml, and a
broad plateau of positive responses was obtained at concentrations
between 1 and 1000 yg/ml (Table 89). However, the retesting of these
compounds with metabolic activation (Tables 86-88, 91 and 92), failed to
indicate either a dose-response relationship or a positive response for
either compound. Thus, the initial results were not confirmed, and we
conclude that these two compounds are unable to induce UDS.
Tests of endrin without metabolic activation gave negative results
(Table 93). The results of initial testing with activation (Table 94)
suggested an increase at the highest concentration tested (1000 yg/ml),
but it was not statistically positive. Therefore, additional testing
of endrin was performed in an attempt to show a dose-response
relationship (Tables 95 and 96). We attempted to achieve a maximum
concentration at 3000 yg/ml; however, due to the incomplete solubility
of this compound in DMSO at such a high concentration, it is doubtful
that a true solution was obtained. Although the first retest (Table 97)
again indicated an apparent, but statistically insignificant, elevation
of 3H-TdR incorporation at the highest concentration tested, it did not
resolve the question of whether a dose-response relationship can be
observed for the effects of endrin with metabolic activation. Therefore,
a third assay was conducted using modified dilution procedures that
would allow us to achieve the higher test concentrations of endrin. The
results of this test (Table 96) failed to indicate that this compound is
capable of inducing UDS even when higher concentrations are achieved.
Hence, we conclude that the results of the UDS testing of endrin are
negative.
The tests of aspon (Tables 97 and 98), carbofuran (Tables 99 and 100),
crotoxyphos (Tables 101 and 102), 2,4-D acid (Tables 103 and 104), 2,4-DB
acid (Tables 95 and 96), diazinon (Tables 107 and .108), fonofos
(Tables 109 and 110), ethion (Tables 111 and 112), methoxychlor (Tables 113
35
-------
and 114), siduron (Tables 115 and 116), and propanil (Tables 117 and 118),
both with and without metabolic activation, all failed to indicate
increased 3H-TdR incorporation. Consequently, we conclude that none of
these compounds increases UDS either in the presence or in the absence
of metabolic activation.
36
-------
CONCLUSIONS
Nine of the 18 pesticides tested in Phase II were mutagenic or
genotoxic in in vitro assays. Two of the pesticides had a broad spectrum
of activity: demeton and trichlorfon increased reverse mutation in
Salmonella and E_. coli WP2. Acephate caused a small but reproducible
increase in S_. typhimur ium TA100 revertants, increased mitotic recombin-
ation in S^ cerevisiae D3, and increased unscheduled DNA synthesis.
Demeton also was more toxic to DNA repair-deficient strains of E^. co] i
and ]3. subtilis than to repair-proficient strains. Based on these results,
substituting these pesticides for other pesticides currently in use would
be unwise unless long-term animal tests indicate that they do not pose a
carcinogenic or mutagenic risk.
Crotoxyphos, 2,4-D acid, 2,4-DB acid, dicamba, disulfoton, and
propanil exhibited a narrow range of effects. The observation of in-
creased toxicity to B.. subtilis strain M45 compared with toxicity to
strain H17 by 2,4-D acid is in contrast to the absence of such an effect
reported by Shirasu. Interestingly, none of these six pesticides
was mutagenic in Salmonella. Perhaps testing them in other in vitro
assays (cytogentics, sister chromatid exchange, transformation or
mutation of mammalian cells) would be appropriate before drawing any
conclusions about their potential effect on the environment.
Nine pesticides—aspon, carbofuran, diazinon, endrin, ethion,
fensulfothion, fonofos, methoxychlor, and siduron—were not active in
any of the assays. These results indicate that these pesticides are
good candidates to substitute for pesticides that have been found to be
harmful to the environment.
37
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Table 68
UNSCHEDULED DNA SYNTHESIS ASSAY OF DEMETON
(dpra/yg DNA)
Concentration of Compounds Tested
Demeton ()jg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
67
58
-*
78
71
57
66
9
4
0.1
32
42
42
55
50
67
48
12
5
1.0
33
37
67
35
45
48
44
13
5
10
53
50
40
60
68
73
57
12
5
100
244
159
294
206
211
173
214
49
20
1000T
-*
100
95
95
74
71
87
13
6
4NQO (M)
10~5
4367
3593
2267
3039
2960
4142
3395
791
323
Negative control and compound solvent, 0.5% EtOH.
"fPrecipitate observed at 1000 yg/ml.
^Sample lost.
112
-------
Table 69
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF DEMETON
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
68
72
75
53
49
69
64
11
5
50
161
176
190
175
146
150
166
17
7
Demeton
100
166
166
264
183
174
206
193
38
15
(yg/ml)
200t
215
231
280
202
183
179
215
37
15
400t
155
153
188
156
131
141
154
19
8
t
soot
90
72
129
76
82
69
87
22
9
iNQO (M)
lO-5
1360
1237
1564
1095
1630
1381
1378
199
81
Negative control and compound solvent, 0.5% DMSO.
"'"Precipitates observed at 200, 400, and 800 yg/ml.
113
-------
Table 70
UNSCHEDULED DNA SYNTHESIS ASSAY OF DEMETON WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
87
117
87
98
97
104
98
11
5
0.1
31
45
42
37
36
28
36
6
3
D erne ton
1.0
73
106
84
88
91
97
90
12
5
(yg/ral)
JLO
71
61
80
64
80
84
73
10
4
100
89
69
79
93
71
117
87
18
7
looot
147
169
134
149
142
137
146
13
5
DMN (M)
5 x 10~2
577
505
550
485
518
529
527.
33
14
?v
Negative control and compound solvent, 0.5% EtOH.
Precipitate observed at 1000 yg/ml.
114
-------
Table 71
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF DEMETON
WITH METABOLIC ACTIVATION
(dpm/pg DNA)
Concentration of Compounds Tested
Demeton (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
69
87
— t
69
77
113
83
18
8
250t
62
90
95
84
85
114
88
17
7
soot
122
135
86
84
90
115
106
21
9
. lOOOt
153
155
159
162
148
119
149
16
6
2000t
127
133
134
138
185
140
142
21
9
4000t
53
104
85
78
105
47
78
25
10
DMN (M)
5 x 10~2
387
306
368
326
388
362
356
34
14
Negative control and compound solvent, 0.5% DMSO.
Precipitates observed at all concentrations.
^Sample lost.
115
-------
Table 72
UNSCHEDULED DNA SYNTHESIS ASSAY OF ACEPHATE
(dpm/yg DNA)
Concentration of Compounds Tested
Acephate (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
223
243
242
259
225
226
236
14
6
0.1
116
123
208
162
129
132
145
35
14
1.0
165
173
134
121
137
150
147
20
8
10
186
233
217
181
182
208
201
22
9
100
179
230
209
177
220
254
212
30
12
1000
314
332
323
290
280
216
292
42
17
4NQO (M)
10- 5
3618
3807
2923
3397
2796
2889
3238
427
174
Negative control and compound solvent, 0.5% DMSO.
116
-------
Table 73
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF ACEPHATE
(dpm/yg DNA)
Concentration of Compounds Tested
Acephate (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
155
127
159
120
124
127
135
17
7
125
103
118
127
124
146
134
126
15
6
250
125
149
150
152
154
121
142
15
6
500
152
154
186
159
137
136
154
18
7
1000
152
154
149
213
231
199
183
36
15
2000
192
163
224
173
247
172
195
33
14
4NQO (M)
10~5
2387
2579
2657
2173
2093
2239
2355
227
93
Negative control and compound solvent, 0.5% DMSO.
117
-------
Table 74
UNSCHEDULED DNA SYNTHESIS ASSAY OF ACEPHATE WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Acephate (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
207
202
231
150
167
165
187
31
13
0.1
142
139
110
138
174
169
145
23
10
1.0
140
139
127
153
141
159
143
11
5
10
208
157
123
193
184
155
170
31
13
100
216
193
251
214
176
186
206
27
11
1000
242
156
152
224
228
247
208
43
18
DMN (M)
5 x 10-2
574
507
580
420
410
433
487
78
32
Negative control and compound solvent, 0.5% DMSO.
118
-------
Table 75
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF ACEPHATE
WITH METABOLIC ACTIVATION
(dpm/pg DNA)
Concentration of Compounds Tested
Acephate (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
— t
106
88
59
83
69
81
18
8
250
65
70
75
83
80
93
78
10
4
500
61
50
55
62
83
49
60
13
5
1000
75
87
77
81
61
73
76
9
4
2000
69
70
71
70
89
70
73
8
3
4000
89
75
64
69
81
64
74
10
4
DMN (M)
5 x ID"2
486
299
252
256
242
414
325
101
41
t
k
Negative control and compound solvent, 0.5% DMSO.
Sample lost.
119
-------
Table 76
UNSCHEDULED DNA SYNTHESIS ASSAY OF DISULFOTON
(dpm/yg DNA)
Concentration of Compounds Tested
Disulfoton (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
67
58
— f
78
71
57
66
9
4
0.1
40
32
40
37
26
27
34
6
3
1.0
33
27
38
58
30
51
39
12
5
10
23
31
43
32
36
31
33
7
3
loot
64
43
59
73
68
65
62
11
4
iooot
92
76
111
106
109
107
100
14
6
4NQO (M)
10~5
4367
3593
2267
3039
2960
4142
3395
791
323
Negative control and compound solvent, 0.5% EtOH.
^Precipitates observed at 100 and 1000 yg/ml.
*Sample lost.
120
-------
Table 77
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF DISULFOTON
(dpm/pg DNA)
Concentration of Compounds Tested
Disulfoton (ye/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
162
158
132
109
202
160
154
31
13
250t
173
142
149
163
179
202
168
22
9
500+
222
200
180
214
188
218
203
17
7
1000+
272
294
281
209
223
237
253
35
14
2000+
361
258
249
267
305
397
302
53
22
i
4000+
271
335
267
272
291
248
281
30
12
+NQO (M)
10-5
2293
2200
2604
2681
2066
2316
2360
237
97
•x
Negative control and compound solvent, 0.5% DMSO.
+Precipitates observed at all concentrations.
121
-------
Table 78
UNSCHEDULED DNA SYNTHESIS ASSAY OF DISULFOTON WITH METABOLIC ACTIVATION
(dpm/ug DNA)
Concentration of Compounds Tested
Disulfoton (pg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
223
191
209
204
181
194
200
15
6
0.1
188
190
208
209
218
166
197
19
8
1.0
163
163
175
77
165
153
149
36
15
10
146
152
202
164
165
206
172
25
10
loot
211
224
202
173
220
207
206
18
7
lOOOt
163
159
214
202
227
190
193
27
11
DMN (M)
5 x 10~2
1163
969
1023
956
1019
1008
1023
74
30
Negative control and compound solvent, 0.5% DMSO.
'Precipitates observed at 100 and 1000 pg/ml.
122
-------
Table 79
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF DISULFOTON
WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Djsulfoton (pg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
-*
187
164
137
144
143
155
21
9
250t
122
171
119
118
180
124
139
29
12
500t
127
118
110
169
183
165
146
31
13
lOOOt
113
121
62
150
110
137
115
30
12
2000t
116
110
86
106
116
111
108
11
5
4000t
127
111
108
121
93
115
112
12
5
DMN (M)
5 x 10-2
383
242
263
289
297
372
308
58
24
Negative control and compound solvent, 0.5% DMSO.
j.
Precipitates observed at all concentrations.
^Sample lost.
123
-------
Table 80
UNSCHEDULED DNA SYNTHESIS ASSAY OF TRICHLORFON
(dpm/pg DNA)
Concentration of Compounds Tested
Trichlorfon (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
36
33
54
34
38
31
37
8
3
0.1
36
42
40
26
37
47
38
7
3
1.0
39
32
26
23
42
28
32
8
3
10
32
31
38
37
36
55
38
9
3
100
42
26
29
50
46
34
38
10
4
1000
87
83
70
56
64
— t
72
13
6
4NQO (M)
10-5
2076
1748
2349
2168
1771
1814
1988
247
101
Negative control and compound solvent, 0.5% EtOH.
^Sample lost.
124
-------
Table 81
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF TRICHLORFON
(dpm/yg DNA)
Concentration of Compounds Tested
Trichlorfon (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0
155
127
159
120
124
127
135
17
7
125
162
162
162
177
169
221
175
23
9
250
188
172
194
232
186
224
199
23
9
500
210
167
237
_.t
170
208
198
30
13
1000
230
208
178
224
182
211
205
22
9
2000
341
214
232
226
258
229
250
47
19
4NQO (M)
2387
2579
2657
2173
2093
2239
2355
227
93
Negative control and compound solvent, 0.5% DMSO.
Sample lost.
125
-------
Table 82
UNSCHEDULED DNA SYNTHESIS ASSAY OF TRICHLORFON WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Trichlorfon (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
113
102
141
158
218
136
145
41
17
0.1
65
98
105
55
89
84
83
19
8
1.0
76
95
96
74
74
117
89
17
7
10
107
102
130
107
71
98
102
19
8
100
48
115
163
129
100
150
117
41
17
1000
112
168
132
159
131
158
143
22
9
DMN (M)
5 x 10~2
400
397
— t
529
645
448
484
105
50
"ft
Negative control and compound solvent, 0.5% EtOH.
1"Sample lost.
126
-------
Table 83
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF TRICHLORFON
WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Trichlorfon (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
__t
106
88
59
83
69
81
18
8
250
48
71
40
59
72
75
61
14
6
500
73
76
61
57
52
82
67
12
5
1000
70
91
64
86
97
76
81
13
5
2000
84
105
80
113
72
70
87
18
8
4000
-t
105
83
63
73
79
81
16
7
DMN (M)
5 x 10~2
486
299
252
256
242
414
325
101
41
Negative control and compound solvent, 0.5% DMSO.
Sample lost.
127
-------
Table 84
UNSCHEDULED DNA SYNTHESIS ASSAY OF DICAMBA
(dpm/wg DNA)
Concentration of Compounds Tested
Dicamba (pg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE '
0*
152
148
140
99
138
167
141
23
9
0.1
125
109
125
110
111
114
116
7
3
1.0
142
151
75
117
121
115
120
27
11
10
122
134
162
154
160
125
143
18
7
100
136
146
177
105
135
121
137
25
10
lOOOt
73
71
119
101
115
97
96
21
8
4NQO (M)
10~5
2671
2538
2660
2443
2652
1455
2458
497
203
if
Negative control and compound solvent, 0.5% DMSO.
"("Precipitate observed at 1000 yg/ml.
128
-------
Table 85
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF DICAMBA
(dpm/Mg DNA)
Concentration of Compounds Tested
Dicamba (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
61
70
70
86
55
59
67
11
4
125
59
72
56
60
70
60
63
6
3
250
52
62
77
64
52
56
60
9
4
500
59
64
80
72
57
66
66
8
3
1000
58
49
67
50
67
63
59
8
3
2000+
30
41
28
44
35
43
37
7
3
4NQO (M)
10~S
1760
1269
533
1878
1907
1929
1546
554
226
Negative control and compound solvent, 0.5% DMSO.
Precipitate observed at 2000 yg/ml.
129
-------
Table 86
UNSCHEDULED DNA SYNTHESIS ASSAY OF DICAMBA WITH METABOLIC ACTIVATION
(dpm/vg DNA)
Concentration of Compounds Tested
Dicamba (pg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0~
101
76
54
67
89
38
71
23
9
0.1
81
89
81
79
76
95
84
7
3
1.0
91
82
101
83
66
100
87
13
5
10
98
94
102
90
99
113
99
8
3
100
105
107
111
82
124
83
102
17
7
lOOOt
133
116
105
128
97
164
124
24
10
DMN (M)
5 x 10~2
468
639
842
576
739
819
681
146
60
Negative control and compound solvent, 0.5% DMSO.
'Precipitate observed at 1000 yg/ml.
130
-------
Table 87
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF DICAMBA
WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Dicamba (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
83
153
64
93
84
147
104
37
15
37
45
59
74
47
106
74
68
23
9
111
56
69
73
60
93
91
74
16
6
333
52
66
65
75
63
67
65
7
3
lOOOt
67
53
72
61
98
118
78
25
10
3000t
62
60
60
63
53
76
62
8
3
DMN (M)
5 x 10-2
269
224
229
220
235
247
237
18
7
Negative control and compound solvent, 0.5% DMSO.
^Precipitates observed at 1000 and 3000 yg/tnl.
131
-------
Table 88
THIRD UNSCHEDULED DNA SYNTHESIS ASSAY OF DICAMBA
WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Dicamba (ug/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
75
81
90
41
72
57
69
18
7
37
74
80
42
71
74
81
70
15
6
111
111
80
82
41
46
123
81
33
13
333
85
78
90
85
95
84
86
6
2
lOOO1"
66
89
55
61
114
66
75
22
9
3000+
86
65
148
87
85
136
101
33
14
DMN (M)
5 x 10~2
567
596
492
506
472
437
512
59
24
Negative control and compound solvent, 0.5% DMSO.
"''Precipitates observed at 1000 and 3000 yg/ml.
132
-------
Table 89
UNSCHEDULED DNA SYNTHESIS ASSAY OF FENSULFOTHION
(dpm/Mg DNA)
Concentration of Compounds Tested
Fensulfothion (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
f
68
54
53
45
47
53
9
4
0.1
63
— f
38
43
49
36
46
11
5
1.0
38
23
40
35
34
34
34
6
2
10
28
27
47
26
33
29
32
8
3
100
12
4
13
21
14
13
13
5
2
10001"
4
0
5
11
15
2
6
6
2
4NQO (M)
10~5
1134
1101
1076
830
1190
976
1051
129
53
* Negative control and compound solvent, 0.5% DMSO.
' Precipitate observed at 1000 yg/ml.
t Sample lost.
133
-------
Table 90
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF FENSULFOTHION
(dpm/yg DNA)
Concentration of Compounds Tested
Fensulfothion (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
61
70
70
86
55
59
67
11
4
6.25
42
60
55
56
57
52
54
6
2
1.25
58
49
52
52
48
49
51
4
2
25
36
36
53
58
40
47
45
9
4
50
30
30
35
36
30
27
31
3
1
100
18
27
22
23
17
30
23
5
2
4NQO (M)
10~5
1760
1269
533
1878
1907
1929
1546
554
226
Negative control and compound solvent, 0.5% DMSO.
134
-------
Table 91
UNSCHEDULED DNA SYNTHESIS ASSAY OF FENSULFOTHION
WITH METABOLIC ACTIVATION
(dpm/ug DNA)
Concentration of Compounds Tested
Fensulfothion (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
101
76
54
67
89
38
71
23
9
0.1
79
111
114
103
131
130
112
19
8
1.0
124
130
139
125
174
137
138
19
8
10
103
163
169
160
126
128
142
27
11
100
148
118
118
155
180
128
141
24
10
lOOOt
107
95
142
142
131
118
123
19
8
DMN (M)
5 x 10 2
468
639
842
576
739
819
681
146
60
*
Negative control and compound solvent, 0.5% DMSO.
^Precipitate observed at 1000 yg/ml.
135
-------
Table 92
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF FENSULFOTHION
WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Fensulfothion (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
(T
83
153
64
93
84
147
104
37
15
12
70
70
83
51
78
36
65
18
7
37
64
64
79
61
83
50
67
12
5
111
37
59
52
55
49
60
52
9
4
333
59
48
63
40
52
52
52
8
3
1000
31
57
54
43
43
47
46
9
4
DMN (M)
5 x 10~2
269
224
229
220
235
247
237
18
7
Negative control and compound solvent, 0.5% DMSO.
136
-------
Table 93
UNSCHEDULED DNA SYNTHESIS ASSAY OF ENDRIN
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
149
103
111
109
99
106
113
18
8
0.1
78
80
98
75
56
72
76
14
6
Endrin
1.0
103
89
74
89
84
103
90
11
5
(yg/ral)
1,0
95
94
92
95
99
110
97
7
3
loot
116
87
92
94
82
118
98
15
6
lOOOt
119
100
73
106
108
121
105
17
7
4NQO (M)
io-5
1271
1288
1421
1271
1363
1446
1343
78
32
Negative control and compound solvent, 0.5% DMSO.
^Precipitates observed at 100 and 1000 yg/ml.
137
-------
Table 94
UNSCHEDULED DNA SYNTHESIS ASSAY OF ENDRIN WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
162
138
122
182
106
166
143
28
11
0.1
76
97
109
110
99
121
102
15
6
Endr in
1.0
103
116
106
53
90
108
96
23
9
(yg/ml)
10
151
148
115
129
111
143
133
17
7
loot
156
138
181
124
165
159
154
20
8
1000 t
185
213
206
176
289
134
201
52
21
DMN (M)
5 x ID"2
690
360
448
348
388
-*
447
141
63
Negative control and compound solvent, 0.5% DMSO.
'''Precipitates observed at 100 and 1000 yg/ml.
tSample lost.
138
-------
Table 95
REPEAT UNSCHEDULED DNA SYNTHESIS ASSAY OF ENDRIN
WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
-*
187
164
137
144
143
155
21
9
188t
93
141
141
173
156
144
141
27
11
Endrln
375t
134
151
133
_-f
106
122
129
17
7
(Mg/mi;
750t
114
131
132
190
118
136
137
27
11
1500t
151
147
165
130
148
194
156
22
9
3000 1
187
135
160
163
178
219
174
29
11
DMN (M)
5 x 10~2
383
242
263
289
297
372
308
58
24
Negative control and compound solvent, 0.5% DMSO.
Precipitates observed at all concentrations.
^Sample lost.
139
-------
Table 96
THIRD UNSCHEDULED DNA SYNTHESIS ASSAY OF ENDRIN
WITH METABOLIC ACTIVATION
(dpm/pg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
72
79
87
77
77
63
76
8
3
37t
70
73
84
101
66
62
76
14
6
Endrin
111+
72
64
63
93
79
65
73
12
5
(pg/ml)
333f
67
75
69
63
64
76
69
5
2
1000f
72
80
77
82
73
71
76
4
2
3000f
62
65
62
80
74
59
67
8
3
DMN (M)
5 x 10~2
348
363
352
377
297
183
320
72
30
Negative control and compound solvent, 0.5% DMSO.
Precipitate observed at all concentrations.
140
-------
Table 97
UNSCHEDULED DNA SYNTHESIS ASSAY OF ASPON
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
223
243
242
259
225
226
236
14
6
0.1
169
190
175
163
187
-*
177
11
5
Aspon
1.0
163
183
141
145
165
184
163
18
7
(yg/ml)
10
221
234
266
243
369
208
257
58
24
loot
286
248
305
194
229
263
254
40
16
lOOOt
234
228
293
228
269
198
242
34
14
4NQO (M)
10- 5
3618
3807
2923
3397
2796
2889
3238
427
174
Negative control and compound solvent, 0.5% DMSO.
Precipitates observed at 100 and 1000 yg/ml.
Sample lost.
141
-------
Table 98
UNSCHEDULED DNA SYNTHESIS ASSAY OF ASPON WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
223
191
209
204
181
194
200
15
6
0.1
184
173
160
120
92
116
132
30
12
Aspon
1.0
199
146
174
130
150
124
154
28
12
(yg/ml)
10
170
165
222
~+
-*
154
178
30
15
loot
175
211
168
159
198
176
181
19
8
lOOOt
239
197
219
185
210
160
202
28
11
DMN (M)
5 x lO-2
1163
969
1023
956
1019
1008
1023
74
30
Negative control and compound solvent, 0.5% DMSO.
Precipitates observed at 100 and 1000 pg/ml.
t
'Sample lost.
142
-------
Table 99
UNSCHEDULED DNA SYNTHESIS ASSAY OF CARBOFURAN
(dpm/yg DNA)
Concentration of Compounds Tested
Carbofuran (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
73
100
77
112
77
74
85
17
7
0.1
63
65
61
39
59
47
56
10
4
1.0
60
58
72
48
55
63
59
8
3
10
42
56
52
82
82
59
62
16
7
100
64
74
68
64
86
76
72
9
3
1000T
55
54
51
83
69
49
60
13
5
4NQO (M)
10- 5
2694
2325
2592
2404
2223
2292
2422
184
74
*
Negative control and compound solvent, 0.5% DMSO.
Precipitate ooserved at 1000 yg/ml.
143
-------
Table 100
UNSCHEDULED DNA SYNTHESIS ASSAY OF CARBOFURAN WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Carbofuran (pg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
87
117
87
98
97
104
98
11
5
0.1
67
116
74
121
88
96
94
22
9
1.0
82
86
73
55
63
76
73
12
5
10
89
53
62
64
53
71
67
17
7
100
81
106
71
70
86
84
83
13
5
1000T
189
118
84
93
93
99
113
39
16
DMN (M)
5 x 10-2
577
505
550
485
518
529
527
33
14
Negative control and compound solvent, 0.5% DMSO.
Precipitate observed at 1000 vg/ml.
144
-------
Table 101
UNSCHEDULED DNA SYNTHESIS ASSAY OF CROTOXYPHOS
(dpm/yg DNA)
Concentration of Compounds Tested
Crotoxyphos (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
73
100
77
112
77
74
85
17
7
0.1
80
99
93
128
109
126
106
19
8
1.0
72
109
100
146
141
105
112
28
11
10
149
121
62
88
154
100
116
31
13
100
42
43
33
40
54
44
43
7
3
1000T
15
10
18
12
11
11
13
3
1
4NQO (M)
10- 5
2694
2325
2592
2404
2223
2292
2422
184
75
*
Negative control and compound solvent, 0.5% DMSO.
Precipitate observed at 1000 ug/ml.
145
-------
Table 102
UNSCHEDULED DNA SYNTHESIS ASSAY OF CROTOXYPHOS WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Crotoxyphos (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
162
138
122
182
106
166
143
28
11
0.1
144
165
221
180
176
158
174
26
11
1.0
65
115
93
63
222
84
82
55
22
10
117
77
111
124
76
90
99
21
8
100
101
83
111
143
102
99
106
20
8
lOOOt
122
139
119
115
161
139
133
17
7
DMN (M)
5 x 10-2
690
360
448
348
388
-f
447
141
63
*
Negative control and compound solvent, 0.5% DMSO.
Precipitate observed at 1000 yg/ml.
±
'Sample lost.
146
-------
Table 103
UNSCHEDULED DNA SYNTHESIS ASSAY OF 2,4-D ACID
(dpra/pg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
153
164
204
161
182
195
111
20
8
0.1
115
107
111
119
81
_t
107
15
7
2,4-D
1.0
98
132
131
119
101
169
125
25
11
(yg/tnl)
10
149
159
150
198
157
163
163
18
7
100
149
161
143
138
151
149
148
8
3
1000
206
197
196
175
173
173
186
15
6
4NQO (M)
10- 5
3589
3320
2671
2769
2494
2644
2914
436
178
t
Negative control and compound solvent, 0.5% DMSO.
Sample lost.
147
-------
Table 104
UNSCHEDULED DM SYNTHESIS ASSAY
OF 2,4-D ACID WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SD
0*
165
125
134
142
159
142
144
15
6
0.1
162
143
130
165
128
141
145
16
6
2,4-D
1.0
131
176
81
141
112
147
131
32
13
(yg/ml)
10
128
122
127
138
136
121
128
7
3
100
146
123
143
138
160
117
138
16
7
1000
81
126
128
137
139
107
120
22
9
DMN (M)
5 x 10 2
334
328
291
330
304
264
308
27
11
*Negative control and compound solvent, 0.5% DMSO.
148
-------
Table 105
UNSCHEDULED DNA SYNTHESIS ASSAY OF 2,4-DB ACID
(dpm/yg DNA)
Concentration of Compounds Tested
2,4-DB (vg/mi;
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
153
16.4
204
161
182
195
177
20
8
0.1
74
118
85
109
117
79
97
20
8
1.0
107
129
111
109
100
98
109
11
5
10
197
109
107
134
116
91
126
38
15
I
100
123
168
136
150
191
-f
154
27
12
1000
118
67
143
147
66
94
106
36
15
4NQO (M)
10
3589
3320
2671
2769
2494
2644
2914
436
178
Negative control and compound solvent, 0.5% DMSO.
^Sample lost.
149
-------
Table 106
UNSCHEDULED DNA SYNTHESIS ASSAY
OF 2,4-DB ACID WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
165
125
134
142
159
142
144
15
6
0.1
117
132
154
119
121
176
137
24
10
2,4-DB
1.0
122
117
122
126
116
133
123
6
3
(yg/ml)
10
129
181
134
133
143
164
147
21
8
100
141
106
119
146
171
145
138
23
9
1000
120
115
111
115
146
119
121
13
5
DMN (M)
5 x 10"2
334
328
291
330
304
264
308
27
11
Negative control and compound solvent, 0.5% DMSO.
150
-------
Table 107
UNSCHEDULED DNA SYNTHESIS ASSAY OF DIAZINON
(dpm/yg DNA)
Concentration of Compounds Tested
Diazinon (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
36
33
54
34
38
31
37
8
3
0.1
44
57
65
51
43
49
51
8
3
1.0
36
43
40
22
65
52
43
15
6
10
61
44
31
25
25
22
35
15
6
loot
20
14
12
23
34
40
24
11
4
lOOOt
0
0
0
26
0
3
5
10
4
4NQO (M)
10 5
2076
1748
2349
2168
1771
1814
1988
247
101
Negative control and compound solvent, 0.5% EtOH.
^Precipitates observed at 100 and 1000 yg/ml
151
-------
Table 108
UNSCHEDULED DNA SYNTHESIS ASSAY OF DIAZINON WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Diazinon (ug/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
94
107
132
118
153
120
121
20
8
0.1
77
82
72
71
72
77
75
4
2
1.0
87
78
81
99
82
107
89
11
5
10
102
100
93
52
120
98
94
23
9
loot
95
85
100
98
80
116
96
13
5
1000+
113
122
75
77
114
90
98
20
8
DMN (M)
5 x ID"2
327
399
343
429
372
455
387
50
20
Negative control and compound solvent, 0.5% EtOH.
tprecipitates observed at 100 and 1000 ijg/ml.
152
-------
Table 109
UNSCHEDULED DNA SYNTHESIS ASSAY OF FONOFOS
(dpm/pg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
61
49
53
49
42
43
49
7
3
0.1
43
44
66
66
35
46
50
13
5
Fonofos
1.0
46
50
55
67
49
33
50
11
5
(yg/ml)
10
52
59
66
92
65
63
66
14
6
loot
55
77
76
85
49
71
69
14
6
lOOOt
67
75
92
38
54
27
59
24
2
4NQO (M)
ID'5
2022
1046
2460
2090
2177
2179
1995
489
199
*
Negative control and compound solvent, 0.5% DMSO.
^Precipitate observed at 100 and 1000 pg/ml.
153
-------
Table 110.
UNSCHEDULED DNA SYNTHESIS ASSAY OF FONOFOS WITH METABOLIC ACTIVATION
(dpm/vig DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
94
107
' 132
118
153
120
121
20
8
0.1
115
111
95
131
130
134
119
15
6
Fonofos
1.0
128
112
153
85
114
135
121
23
10
(yg/ml)
10
70
110
110
131
95
110
104
20
8
loot
96
131
143
170
132
130
134
24
10
100QT
125
125
149
186
121
151
143
25
10
DMN (M)
5 x 10 2
327
399
343
429
372
455
387
50
20
Negative control and compound solvent, 0.5% DMSO.
^Precipitate observed at 100 and 1000 yg/ml.
154
-------
Table 111
UNSCHEDULED DNA SYNTHESIS ASSAY OF ETHION
(dpm/Pg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
163
173
173
196
188
219
186
20
8
0.1
196
100
128
101
255
__t
156
68
30
Ethion
1.0
171
186
188
150
165
143
167
18
8
(pg/ml)
10
160
220
102
141
186
166
162
40
16
IQOt
117
136
159
192
171
182
160
28
11
lOOOt
119
248 .
147
124
173
151
160
47
19
4NQO (M)
10- 5
3144
3221
3781
3556
3374
— f
3415
258
116
Negative control and compound solvent, 0.5% DMSO.
"^Precipitates observed at 100 and 1000 yg/ml.
*Sample lost.
155
-------
Table 112
UNSCHEDULED DNA SYNTHESIS ASSAY OF ETHION WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
142
109
67
69
93
113
99
29
12
0.1
70
82
110
104
117
103
96
17
7
Ethion
1.0
98
80
108
112
90
90
96
12
5
(y^/ml)
10
92
83
90
87
102
114
95
12
5
loot
. 91
95
105
105
113
119
105
11
4
1000+
89
72
105
100
109
109
97
14
6
DMN (M)
5 x ID"2
374
366
426
397
435
449
408
34
14
Negative control and compound solvent, 0.5% DMSO.
^Precipitates observed at 100 and 1000 pg/ml.
156
-------
Table 113
UNSCHEDULED DNA SYNTHESIS ASSAY OF METHOXYCHLOR
(dpm/pg DNA)
Concentration of Compounds Tested
Methoxychlor (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
149
103
111
109
99
106
113
18
8
0.1
75
78
88
80
87
70
79
8
3
1.0
84
73
104
74
72
68
79
13
6
10
86
84
77
85
100
163
99
32
13
loot
86
92
83
76
119
83
90
15
6
lOOOt
102
123
87
113
106
107
106
12
5
4NQO (M)
10~5
1271
1288
1421
1271
1363
1446
1343
78
32
rt
Negative control and compound solvent, 0.5% DMSO.
^Precipitates observed at 100 and 1000 yg/ml.
157
-------
Table 114
UNSCHEDULED DNA SYNTHESIS ASSAY OF METHOXYCHLOR
WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Methoxychlor (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
98
171
143
119
160
126
136
27
11
0.1
92
128
106
88
94
76
97
18
7
1.0
107
85
86
64
82
54
80
19
8
10
80
103
107
101
114
-f
101
13
5
IQOt
142
89
101
104
132
102
112
20
8
lOOOt
129
99
104
104
99
89
104
13
5
DMN (M)
5 x 10~2
595
635
508
487
670
661
593
78
32
Negative control and compound solvent, 0.5% DMSO.
^Precipitates observed at 100 and 1000 yg/ml.
^Sample lost.
158
-------
Table 115
UNSCHEDULED DNA SYNTHESIS ASSAY OF SIDURON
(dpm/yg DNA)
Concentration of Compounds Tested
Siduron (yg/tnl)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
-f
68
54
53
45
47
53
9
4
0.1
41
32
30
29
39
39
35
5
2
1.0
32
38
30
24
28
29
32
6
2
10
33
34
26
25
37
32
31
5
2
100
16
15
14
17
21
15
16
3
1
lOOOt
26
19
26
29
22
19
24
4
1
4NQO (M)
1C-5
1134
1101
1076
830
1190
976
1051
129
53
Negative control and compound solvent, 0.5% DMSO.
^Precipitate observed at 1000 yg/ml.
rSample lost.
159
-------
Table 116
UNSCHEDULED DNA SYNTHESIS ASSAY OF SIDURON WITH METABOLIC ACTIVATION
(dpm/Mg DNA)
Concentration of Compounds Tested
Siduron (yg/ml)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
207
202
231
150
167
165
187
31
13
0.1
224
162
294
196
173
206
209
47
19
1.0
64
142
132
99
148
134
120
32
13
10
137
108
130
150
151
195
145
29
12
100
176
178
103
163
132
172
154
30
12
lOOOt
175
118
203
126
136
156
152
32
13
DMN (M)
5 x 10~2
574
507
580
420
410
433
487
78
32
*
Negative control and compound solvent, 0.5% DMSO.
^Precipitate observed at 1000 yg/ml.
160
-------
Table 117
UNSCHEDULED DNA SYNTHESIS ASSAY OF PROPANIL
(dpm/yg DNA)
Concentration of Compounds Tested
Propanil
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
152
148
140
99
138
167
141
23
9
0.1
104
147
79
55
52
103
89
36
15
1.0
120
105
136
111
112
148
122
17
7
(Mg/ml)
10
117
145
117
109
105
81
112
21
9
100
24
36
51
31
39
22
34
11
4
10001"
30
28
23
101
99
0
47
43
17
4NQO (M)
ID"5
2671
2538
2660
2773
2652
1455
2458
497
203
#
Negative control and compound solvent, 0.5% DMSO.
''"Precipitate observed at 1000 yg/ml.
161
-------
Table 118
UNSCHEDULED DNA SYNTHESIS ASSAY OF PROPANIL WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Concentration of Compounds Tested
Propanil
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
98
171
143
119
160
126
136
27
11
0.1
76
67
78
64
63
67
69
6
3
1.0
75
67
73
95
64
65
73
12
5
(jag /ml)
10
76
95
111
76
91
76
87
14
6
100
113
73
97
124
104
85
100
19
8
1000 t
107
85
105
105
104
89
99
10
4
DMN (M)
5 x 10~2
595
635
508
487
670
661
593
78
32
Negative control and compound solvent, 0.5% DMSO.
"^Precipitate observed at 1000 yg/ml.
162
-------
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27. V. V. Brsheskii. Possibility of induction of mutations in
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28. IARC. 2,4-D and Esters. Jja IARC Monographs on the Evaluation of
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29. Y. Shirasu. Significance of mutagenicity testing on pesticides.
Environ. Qual. Sat. 4... 226-231 (1975).
164
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/ 1-79-041
2.
4. TITLE AND SUBTITLE
In Vitro Microbiological Mutagenicity and Unscheduled
DNA Synthesis Studies of Eighteen Pesticides
7 AUTHOR(S)
Vincent F. Simmon, Ph.D. , SRI International
Menlo Park CA 94025
9. DERFORMING ORGANIZATION NAME Af>
Microbial Genetics Program
SRI International
333 Ravenswood Avenue, Men]
JO ADDRESS
.0 Park CA 94025
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory RTP , NC
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
3. RECIPIENT'S ACCESSION NO.
5. REPORT OATH
October 1979
6. PERFORMING ORGANIZATION CODE
S. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
68-01-2458
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Eighteen pesticides being reviewed as a part of the EPA Substitute Chemical
Program were tested for mutagenic activity by the following iri vitro procedures:
Reverse mutation in Salmonella typhimuriua strains TA1535, TA1537,
TA1538, TA98, and TA100 and in Escherichia coli WP2 uvrA.
Induction of mitotic recombination in the yeast Saccharomyces
cerevisiae D3.
Relative toxicity assays in DNA repair-proficient and -deficient
strains of _E. coli (strains W3110 and p3478, respectively) and of
Bacillus subtilis (strains H17 and M45 respectively) .
Unscheduled DNA synthesis (UDS) in human fibroblasts (WT-38 cells).
Sine of the 13 pesticides were mutagenic in one or more of the assays. One
compound, demeton, was mutagenic in all of them. Trichlorofon was mutagenic in all
the assays except those for relative toxicity. Acephat was mutagenic in the
Salmonella typhimurium in TA100, Saccharomyces cerevisiae D3, and UDS assays.
Dicamba, 2,4-D acid, 2,4-DB acid, and propanil were positive only in the assay for
relative toxicity. Disulfoton was positive only in the UDS assay, and then only in
the absence of the metabolic activation system. Crotoxyphos was positive only in
the S. cerevisiae D3 assay.
17. KEY WORDS AND DOCUMENT ANALYSIS ~~
a. DESCRIPTORS
In Vitro analysis
Microbiology
Mutagens
Deoxyribonucleic acids
Pesticides
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
b. IDENTIFIERS/OPEN ENDED TERMS
19. SECURITY CLASS (This Report)
UNCLASSIFIED
20. SECURITY CLASS i This page)
UNCLASSIFIED
c. COSATI Field/Group
06C,F,M,T
21. NO. OF PAGES
173
22. PRICE
EPA Form 2220-] (Rev. 4-77) previous EDITION is OBSOLETE
165
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