EPA-600/1-77-028
May 1977
Environmental Health Effects Research Series
EVALUATION OF SELECTED PESTICIDES AS
CHEMICAL MUTAGENS
In Vitro and In Vivo Studies
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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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-600/1-77-028
May 1977
EVALUATION OF SELECTED PESTICIDES AS CHEMICAL MUTAGENS
In Vitro and In Vivo Studies
By
Vincent F. Simmon, Ann D. Mitchell, and Ted. A. Jorgenson
Stanford Research Institute
Menlo Park, California 94025
Contract No. 68-01-2458
Project Officer
Michael D. Waters
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE' PARK, N.C. 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 develops and revises 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 preparing the health basis for
non-ionizing 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.
This report describes the testing of a series of twenty technical
grade pesticide chemicals for genotoxic properties by use of a battery
of in vitro and in vivo methods. The battery includes tests for gene
and chromosomal mutations and primary damage to DNA as measured by
effects on DNA repair recombination. Since DNA is chemically similar in
all species, test results from a variety of cells and organisms are
relevant in assessing the potential genetic hazard of pesticide chemicals
in humans.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
iii
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ABSTRACT
Twenty pesticides being reviewed as a part of the EPA Substitute
Chemical Program were studied for mutagenic activity by several in vivo
and in vitro test procedures. Ten of the twenty compounds were evaluated
in vivo by the mouse dominant lethal test. All twenty compounds were
tested by the following in vitro procedures:
Unscheduled DNA synthesis (UDS) in human fibro-
blasts (WI-38 cells); reverse mutation in Salmonella
typhimurium strains TA1535, TA1537, TA1538, and
TA100 and in Escherichia coli WP2; mitotic recombi-
nation in the yeast Saccharomyces cerevisiae D3;
and preferential toxicity assays in DNA repair-
proficient and -deficient strains of IS. coli
(strains W3110 and p3478, respectively) and Bacillus
subtilis (strains H17 and M45, respectively).
None of the ten compounds tested in the mouse produced a dominant
lethal response.
Ten of the twenty compounds were mutagenic in one or more in vitro
assays. Two were mutagenic in all of the in vitro assays: captan and
folpet. In a heritable translocation study in mice, under the experimental
procedures employed, captan at 5000 ppm in the diet of male mice for 8
consecutive weeks produced a heritable mutagenic event in F^ generation
male mice.
iv
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CONTENTS
LIST OF TABLES ill
SUMMARY 1
TWENTY PESTICIDES EVALUATED BY SRI FOR MUTAGENIC ACTIVITY 3
INTRODUCTION 4
DOMINANT LETHAL TEST IN THE MOUSE 6
General 6
Experimental 6
Animals and Chemicals 6
Determination of Acute Toxicity 7
Maximum Tolerated Dose Study 7
Treatment Levels 7
Administration of the Compounds 8
Test Groups 8
Necropsy and Evaluation 9
Results and Discussion 9
MAMMALIAN IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAYS 12
General 12
Experimental 13
Cell Culture 13
Dilution of Compounds 13
Controls 13
UDS Assays 14
Interpretation of Results 15
MICROBIOLOGICAL ASSAYS 17
General 17
Experimental 18
Salmonella typhimurium Strains TA1535,
TA1537, TA1538, and TA100 18
Escherichia coli WP2 19
Escherichia coli W3100/p3478 and
Bacillus subtilis H17/M45 20
Saccharomyces cerevisiae D3 21
Aroclor 1254-Stimulated Metabolic Activation System ... 22
Results and Discussion 22
DISCUSSION 25
REFERENCES 26
APPENDIX A: Mutagenesis Studies of Pesticide Compounds - Mouse
Heritable Translocation Test - Captan 171
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TABLES
DOMINANT LETHAL TEST
1 Chi-Square Test of the Fertility Index - Monocrotophos .... 28
2 Average Implants per Pregnant Female - Monocrotophos 29
3 Average Dead Implants per Pregnant Female - Monocrotophos. . . 30
4 Chi-Square Test of the Death Index - Monocrotophos 31
5 Number of Dead Implants per Total Implants - Monocrotophos . . 32
6 Chi-Square Test of the Fertility Index - Bromacil 33
7 Average Implants per Pregnant Female - Bromacil 34
8 Average Dead Implants per Pregnant Female - Bromacil 35
9 Chi-Square Test of the Death Index - Bromacil 36
10 Number of Dead Implants per Total Implants - Bromacil 37
11 Chi-Square Test of the Fertility Index - Captan 38
12 Average Implants per Pregnant Female - Captan 39
13 Average Dead Implants per Pregnant Female - Captan . 40
14 Chi-Square Test of the Death Index - Captan 41
15 Number of Dead Implants per Total Implants - Captan 42
16 Chi-Square Test of the Fertility Index - Folpet 43
17 Average Implants per Pregnant Female - Folpet 44
18 Average Dead Implants per Pregnant Female - Folpet 45
19 Chi-Square Test Of the Death Index - Folpet 46
20 Number of Dead Implants per Total Implants - Folpet 47
21 Chi-Square Test of the Fertility Index - Azinphos-Methyl ... 48
22 Average Implants per Pregnant Female - Azinphos-Methyl .... 49
23 Average Dead Implants per Pregnant Female - Azinphos-Methyl. . 50
24 Chi-Square Test of the Death Index - Azinphos-Methyl 51
25 Number of Dead Implants per Total Implants - Azinphos-Methyl . 52
26 Chi-Square Test of the Fertility Index - Malathion 53
27 Average Implants per Pregnant Female - Malathion 54
28 Average Dead Implants per Pregnant Female - Malathion 55
29 Chi-Square Test of the Death Index - Malathion 56
30 Number of Dead Implants per Total Implants - Malathion .... 57
31 Chi-Square Test of the Fertility Index - Parathion 58
32 Average Implants per Pregnant Female - Parathion 59
33 Average Dead Implants per Pregnant Female 60
34 Chi-Square Test of the Death Index - Parathion 61
35 Number of Dead Implants per Total Implants - Parathion .... 62
36 Chi-Square Test of the Fertility Index - Parathion-Methyl. . . 63
37 Average Implants per Pregnant Female - Parathion-Methyl. ... 64
38 Average Dead Implants per Pregnant Female - Parathion-Methyl . 65
39 Chi-Square Test of the Death Index - Parathion-Methyl 66
40 Number of Dead Implants per Total Implants - Parathion-Methyl. 67
41 Chi-Square Test of the Fertility Index - Quintozene (PCNB) . . 68
42 Average Implants per Pregnant Female - Quintozene (PCNB) ... 69
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43 Average Dead Implants per Pregnant Female -
Quintozene (PCNB) 70
44 Chi-Square Test of the Death Index - Quintozene (PCNB).... 71
45 Number of Dead Implants per Total Implants -
Quintozene (PCNB) 72
46 Chi-Square Test of the Fertility Index - Phorate 73
47 Average Implants per Pregnant Female - Phorate 74
48 Average Dead Implants per Pregnant Female - Phorate 75
49 Chi-Square Test of the Death Index - Phorate 76
50 Number of Dead Implants per Total Implants - Phorate 77
UDS ASSAYS
51 DNA Repair Synthesis Assay of Monocrotophos 78
52 DNA Repair Synthesis Assay of Monocrotophos with
Metabolic Activation 79
53 DNA Repair Synthesis Assay of Bromacil 80
54 DNA Repair Synthesis Assay of Bromacil with
Metabolic Activation 81
55 DNA Repair Synthesis Assay of Cacodylic Acid 82
56 DNA Repair Synthesis Assay of Cacodylic Acid with
Metabolic Activation 83
57 DNA Repair Synthesis Assay of Captan 84
58 DNA Repair Synthesis Assay of Captan with
Metabolic Activation 85
59 DNA Repair Synthesis Assay of Chloropyrifos 86
60 DNA Repair Synthesis Assay of Chloropyrifos with
Metabolic Activation 87
61 DNA Repair Synthesis Assay of Dinoseb 88
62 DNA Repair Synthesis Assay of Dinoseb with
Metabolic Activation 89
63 DNA Repair Synthesis Assay of DSMA 90
64 DNA Repair Synthesis Assay of DSMA with
Metabolic Activation 91
65 DNA Repair Synthesis Assay of Fenthion 92
66 DNA Repair Synthesis Assay of Fenthion with
Metabolic Activation 93
67 DNA Repair Synthesis Assay of Folpet 94
68 DNA Repair Synthesis Assay of Folpet with
Metabolic Activation 95
69 DNA Repair Synthesis Assay of Azinphos-Methyl ... 96
70 DNA Repair Synthesis Assay of Azinphos-Methyl with
Metabolic Activation 97
71 DNA Repair Synthesis Assay of Malathion 98
72 DNA Repair Synthesis Assay of Malathion with
Metabolic Activation. 99
73 DNA Repair Synthesis Assay of Methomyl 100
74 DNA Repair Synthesis Assay of Methomyl with
Metabolic Activation 101
75 DNA Repair Synthesis Assay of Monuron 102
vii
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76 DNA Repair Synthesis Assay of Monuron with
Metabolic Activation 103
77 DNA Repair Synthesis Assay of MSMA 104
78 DNA Repair Synthesis Assay of MSMA with
Metabolic Activation 105
79 DNA Repair Synthesis Assay of Parathion 106
80 DNA Repair Synthesis Assay of Parathion with
Metabolic Activation 107
81 DNA Repair Synthesis Assay of Parathion-Methyl 108
82 DNA Repair Synthesis Assay of Parathion-Methyl
with Metabolic Activation 109
83 DNA Repair Synthesis Assay of Quintozene (PCNB) 110
84 DNA Repair Synthesis Assay of Quintozene (PCNB)
with Metabolic Activation Ill
85 DNA Repair Synthesis Assay of Phorate 112
86 DNA Repair Synthesis Assay of Phorate with
Metabolic Activation 113
87 DNA Repair Synthesis Assay of Simazine 114
88 DNA Repair Synthesis Assay of Simazine with
Metabolic Activation 115
89 DNA Repair Synthesis Assay of Trifluralin 116
90 DNA Repair Synthesis Assay of Trifluralin with
Metabolic Activation 117
MICROBIOLOGICAL ASSAYS
91 In Vitro Assays with Salmonella typhimurium 118
92 Results of Assays with Escerichia coli WP2 138
93 Microbial Inhibition in Escerichia coli and
Bacillus subtilis 148
94 In Vitro Assays with Saccharomyces cerevisiae D3 -
Monocrotophos . . . 150
95 In Vitro Assays with Saccharomyces cerevisiae D3 -
Bromacil 151
96 In Vitro Assays with Saccharomyces cerevisiae D3 -
Cacodylic Acid 152
97 In Vitro Assays with Saccharomyces cerevisiae D3 -
Captan 153
98 In Vitro Assays with Saccharomyces cerevisiae D3 -
Chloropyrifos 154
99 In Vitro Assays with Saccharomyces cerevisiae D3 -
Dinoseb 155
100 In Vitro Assays with Saccharomyces cerevisiae D3 - DMSA . . . 156
101 In Vitro Assays with Saccharomyces cerevisiae D3 -
Fenthion 157
102 In Vitro Assays with Saccharomyces cerevisiae D3 -
Folpet 158
103 In Vitro Assays with Sac charomyc es cerevisiae D3 ~
Azinphos-Methyl 159
104 In Vitro Assays with Saccharomyces cerevisiae D3 -
Malathion 160
viii
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105 In Vitro Assays with Saccharomyces cerevisiae D3 -
Methomyl 161
106 In Vitro Assays with Sac charomyces cerevisiae D3 -
Monuron 162
107 In Vitro Assays with Saccharomyces cerevisiae D3 - MSMA . . . 163
108 In Vitro Assays with Saccharomyces cerevisiae D3 -
Parathion 164
109 In Vitro Assays with Saccharomyces cerevisiae D3 -
Parathion-Methyl 165
110 In Vitro Assays with Saccharomyces cerevisiae D3 -
Quintozene (PCNB) 166
111 In Vitro Assays with Saccharomyces cerevisiae D3 -
Phorate 167
112 In Vitro Assays with Saccharomyces cerevisiae D3 -
Simazine 168
113 In Vitro Assays with Saccharomyces cerevisiae D3 -
Trifluralin 169
114 In Vitro Mutagenesis with Salmonella typhimurium 170
ix
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ACKNOWLEDGMENTS
Stanford Research Institute wishes to thank the EPA project officers
for their guidance and assistance during the course of this project.
Dr. Robert E. McGaughy, Office of Research and Development, Washington,
D.C., was Project Officer from the beginning of the project in June 1974
until July 1975. Project responsibility then was transferred to
Dr. Ronald L. Baron, Health Effects Research Laboratory, Research
Triangle Park, North Carolina, until January 1976. At that time,
Dr. Michael D. Waters, now Chief of the Biochemistry Branch in the
Environmental Toxicology Division, Health Effects Research Laboratory,
Research Triangle Park, North Carolina, became Project Officer.
Dr. Waters has continued in this project responsibility to the present.
x«
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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 inter-state 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 con-
ducting a thorough review of the implications of using alternate chemi-
cals, 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 timely and 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 interpretation."
Development of methods for evaluating the mutagenic hazard of chemi-
cal compounds has advanced markedly in the last few years. In contrast
to the undefined empirical tests used a short time ago, procedures now
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available can detect chromosome breaks and other genetic changes caused by
chemical stress. Mutant strains of microorganisms in cell culture and
mammalian fibroblast cells in tissue culture are effective in vitro
systems for reliable detection of presumptive gene mutations, whereas the
mammalian dominant lethal test is a recognized test for the assessment
of chromosome damage to germinal cells.
Today many pesticide chemicals in commercial use have not been
investigated adequately for their mutagenic hazard. With the public's
increasing concern about possible pollution of our environment by chemi-
cals, the widely used pesticides must be evaluated. In this project,
SRI used test methods that are appropriate for these evaluations and
that are in use by the scientific community.
Under contract to EPA, SRI examined 20 pesticides for mutagenic
activity using a combintation of in vivo and in vitro mutagenicity assay
systems. The 20 pesticides tested and their sources are listed in the '
following two tables.
The assays used were the dominant lethal test in mice (only
ten compounds); unscheduled DNA synthesis (UDS) in human fibroblasts
(WI-38 cells); reverse mutation in Salmonella typhimurium strains TA1535,
TA1537, TA1538, and TA100 and in Escherichia coli WP2; mitotic
recombination in the yeast Saccharomyces cerevisiae D3; and preferential
toxicity assays in DNA repair-proficient and -deficient strains of JE. coli
(strains W3110 and p3478, respectively) and Bacillus subtilis (strains
H17 and M45, respectively.
Based on positive responses in both Tier I (in vitro test) and
Tier II (Drosophila) mutagenic studies, it was recommended that a
heritable translocation test (Tier III) in the mouse be conducted
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to further assess the mutagenic potential of Captan. The results of
these further studies are reported as Appendix A.
The experimental procedures and results for the mammalian dominant
lethal test, the UDS assay, and the microbiological assays are described
in the separate sections that follow.
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IN VIVO AMD IN VITRO MUTAGENESIS: SUMMARY DATA FOR EPA PESTICIDES
Positive Response, +; Negative Response, -
Pesticide
Mouse
Dominant
Lethal*
Salmonella
typhimuriumt
(His*" Reversion)
Escherichia coli WP2
(Try+ Reversion)
Saccharomyces cerevisiae
(Mitotlc Recombination)
Escherichia coli
(Relative Toxicity)
Bacillus subtilis
(Relative Toxicity)
UDS
(DNA Repair)
Monocrotophos
Bromacil
Cacodylic Acid
Captan
Chlorpyrifos
Dinoseb
DSMA
Fenthion
Folpet
Azinphos-methyl
Malathion
Methomyl
Monuron
MSMA
Parathion
Parathlon-methyl
Quintozene (PCNB)
Fhorate
Simazine
Trifluralin
-MA
+MA
-MA
+MA
-MA
+MA
+MA
* Only ten pesticides were tested by the dominant lethal procedure.
t See page 170.
* Marginally positive.
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TWENTY PESTICIDES EVALUATED BY SRI FOR MUTAGENIC ACTIVITY
Ul
Common Name*
Monocrotophos
Bromacil
Cacodylic Acid
Cap tan
Chlorpyrifos
Dinoseb
DSMA
Fenthion
Folpet
Az inpho s-methy 1
Malathion
Methomyl
Monuron
MSMA
Parathion
Parathion-methyl
Quintozene (PCNB)
Phorate
Simazine
Trifluralin
Trade Name of
Compound Tested
Azodrin-5
Hyvar
Phytar
Orthoside 406
Dursban
Pr emerge
Ansar
Bay t ex
Phaltan
Guthion
Malathion
Lannate
Telvar
Ansar
Niran
Methyl Parathion
Terrachlor
Thimet
Primatol
Treflan
Manufacturer
Shell Chemical Company
E.I. DuPont de Nemours
Ansul Chemical Company
Chevron Chemical Company
Dow Chemical Company
Dow Chemical Company
Ansul Chemical Company
Chemogro
Chevron Chemical Company
Chemogro
American Cyanamid Company
E.I. DuPont de Nemours
E.I. DuPont de Nemours
Ansul Chemical Company
Monsanto Chemical Company
Monsanto Chemical Company
01 in Mathieson Chemical
Corporation
American Cyanamid Company
Ciba-Geigy Chemical Co.
Eli Lilly & Company
Batch or
Lot Number
Batch H,
9-SCL-77
T80619/40
Phyton 138
5X640
MM-1114-1
(603-D1)
MM 200554
8100
4-15-2026
SX579
411-0229
40216006.300
6602-82
T-40817-20
170 H.C.
AD 1236
AD 0659
Technical
MC85
FL-740846
X-26290
Purity (%)
55.0
95.9
65.6
Technical
98.8
97.7
80.1
96.0
Technical
Technical
Technical
99.0
97.0
58.4
99.0
80.0
99.0
97.7
97.7
Supplier
Manufacturer
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
* Common name as approved by the International Organization for Standardization.
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DOMINANT LETHAL TEST IN THE MOUSE
General
In the dominant lethal test, the ten compounds under investigation
were fed in the diet to proven male breeder mice for 7 weeks. After this
period, each male was mated with two adult virgin females for 7 days;
these females were then replaced by two others for another breeding.
The sequence was continued for 8 weeks. This procedure emphasizes
possible mutagenic effects on the male sperm, the normal female acting
as a carrier to reveal in her offspring abnormalities that may have
occurred in the male. We evaluated effects by examining the condition
and state of fetal development during the middle to latter stages of
gestation.
Experimental
Animals and Chemicals
Adult ICR/SIM mice from a closed, random-bred colony were used for
the acute toxicity and maximum tolerated dose determinations as well as
for the dominant lethal assay. These male and female mice were supplied
by Simonsen Laboratories, Gilroy, California. The males were 3- to
4-month-old proven breeders, and the females were 10- to 12-week-old
virgin stock.
At the direction of EPA, the Battelle Columbus Laboratories obtained
the pesticides from the manufacturers and subsequently provided SRI with
aliquots for the studies reported here. Each pesticide was a "technical"
grade product (or equivalent) and was provided in sufficient quantity
for us to complete all aspects of the experimental program. Excess
supplies were refrigerated or frozen, should they be needed for future
reference.
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We investigated the solubility of each compound using water,
propylene glycol, polyethylene glycol, corn oil, or carboxymethyl-
cellulose to determine the most appropriate vehicle for administration.
Compounds were administered orally, by gavage for the acute toxicity
(LD.-Q) determinations, and via the diet for the maximum tolerated dose
and dominant lethal studies.
Determination of Acute Toxicity
Although acute toxicity information on some of the compounds was
available in the literature, we conducted confirmatory tests on all to
obtain an LD,._ under our laboratory conditions and for the ICR/SIM
strain of mouse. If no data were available, we conducted a preliminary
range-finding test, followed by a determination of the oral LD^Q.
Maximum Tolerated Dose Study
Based on the acute toxicity data and available information from the
literature on dose levels known to cause adverse responses when adminis-
tered in the diet, several dose levels were selected and administered in
the diet to adult male mice for 2 weeks. Treated males then were caged with
two adult virgin females each for 7 days; these females were replaced
by two others weekly for 2 weeks. The females were examined daily for
the presence of vaginal (mating) plugs. At midterm of pregnancy, the
females were sacrificed and examined for total implants, as well as
for early and late fetal deaths. For this work, we defined a maximum
tolerated dose as that dietary level which may produce up to a 20%
weight loss, mild but transient clinical signs, no inhibition of breeding
performance, and no mortality. Thus, these initial studies provided
information on changes in body weight, acceptability of the diet,
clinical signs, mortality, and breeding performance.
Treatment Levels
For the dominant-lethal study, three dose levels were administered.
The highest was the maximum tolerated dose or 5 g/kg (a maximum level
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agreed on by EPA and SRI), whichever was lower. The intermediate and
lower dosages were one-half and one-quarter of the highest dose, respec-
tively.
Administration of the Compounds
Each pesticide was fed in the diet to adult male mice for 7 weeks.
An appropriate amount of compound initially was dissolved or suspended
in corn oil; then the compound-oil concentrate was added at a level of 3%
to a finely ground commercial diet of known composition. The use of
corn oil assured even distribution of the compound and prevented strati-
fication of the test material in an otherwise dry diet. Diets were pre-
pared at 2-week intervals and were refrigerated at 4°C until fed to the
animals. Fresh diet was placed in the feed containers every other day
to minimize the loss of compound through instability or volatility.
Test Groups
Two reference control groups were included in this project. One
was run at the beginning of each of the two dominant lethal series, five
pesticides being run concurrently. In this manner, reference breeding
and implant data were obtained at two time periods, as was information
on each shipment of research animals. Males in these groups were fed
a finely ground commercial diet supplemented with corn oil at 3%.
Control groups were treated in the same manner as the compound test
groups.
Two positive control groups were run concurrently with each of the
two series of five pesticide tests. For these groups, the known mutagen
triethylenemelamine (TEM) was administered as a single intraperitoneal
injection of 0.2 mg/kg approximately 2 hours before the first mating.
A commercial pelleted diet was available at all times.
Each control and experimental test group contained 20 adult male
mice. At the end of the 7-week compound treatment period, each male
was allowed to breed with two virgin females over a period of 7 days.
Females were replaced weekly for 8 weeks.
8
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Necropsy and Evaluation
Females were sacrificed at midterm of pregnancy. A complete
necropsy was performed to determine if an intercurrent infection was
present; such a condition can induce preimplantation loss and early
fetal deaths. At sacrifice, each female was scored for early fetal
deaths, late fetal deaths, and living fetuses (all of which provide a
total implant score).
The following parameters indicate effects in dominant lethal studies:
Total implants (live fetuses plus early and late fetal deaths), total
dead (early and late fetal deaths), and dead implants per total implants.
Total implants and dead implants were analyzed for significance by the
t-test.
The index of dead implants per total implants was analyzed statis-
tically by the t-test on arcsine- (or angular) transformed data, as
described in Experimental Design (Theory and Application). This
index was computed for each female. Other parameters analyzed were the
fertility and death indices.
Results and Discussion
Single-dose oral acute toxicity data are as follows:
Compound LD50
Monocrotophos 17 mg/kg
Bromacil 3.04 g/kg
Captan > 15 g/kg
Folpet > 10 g/kg
Azinphos-methyl 15 mg/kg
Malathion 1196 mg/kg
Parathion 17 mg/kg
Parathion-methyl 39 mg/kg
Quintozene (PCNB) > 10 g/kg
Phorate 6.59 mg/kg
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After evaluating the acute toxicity data and those from subsequent
maximum tolerated dose studies, we selected the following dosage levels
for the dominant lethal studies:
Treatment Levels
Compound (mg/kg of Diet)
Monocrotophos 15, 30, 60
Bromacil 1250, 2500, 5000
Captan 1250, 2500, 5000
Folpet 1250, 2500, 5000
Azinphos-methyl 20, 40, 80
Malathion 1250, 2500, 5000
Parathion 62.5, 125, 250
Parathion-methyl 20, 40, 80
Quintozene (PCNB) 1250, 2500, 5000
Phorate 5, 10, 20
Throughout the experiment, the biological criteria used to evaluate
mutagenic effects in the mouse showed no consistent responses that could
be attributed to treatment. Although we found occasional statistical
differences between control and compound treated groups, they were random
and did not suggest a time or dose-response effect.
Summary data on the fertility index, implantations per pregnant
female, dead implants per pregnant female, death index, and number of
dead implants per total implants are presented by compound as follows:
Tables 1 through 5, Monocrotophos; Tables 6 through 10, Bromacil;
Tables 11 through 15, Captan; Tables 16 through 20, Folpet; Tables 21
through 25, Azinphos-methyl; Tables 26 through 30, Malathion; Tables 31
through 35, Parathion; Tables 36 through 40, Parathion-Methyl; Tables 41
through 45, Quintozene (PCNB); and Tables 46 through 50, Phorate.
Two copies of a description of the statistical analysis procedures
used for dominant lethal tests and computer printouts of the raw data
and the statistical analyses are on file with the current Project Officer,
Dr. Michael D. Waters, Environmental Toxicology Division, Health Effects
Research Laboratory, EPA Environmental Research Center, Research
Triangle Park, North Carolina 27711.
10
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The following statistical procedures were used:
Chi-square test of the fertility index;
Armitage test for a linear trend in proportion for the fertility
index based on dose levels, based on logarithms of the dose levels,
and based on dose levels including the control group;
t-test of the number of implantations in pregnant females;
Regression fits of implantations on dose and log dose and with and
without control group included; ';
t-test of the (Freeman-Tukey transformed) preimplantation losses
in pregnant females;
t-test of the number of dead implants;
Chi-square test of the death index;
Armitage test for a linear trend in proportion for the death index!,
based on dose levels with and without control group included and based
on logarithms of the dose levels;
Probit analysis of the proportion of pregnant females with one or
more dead implants;
t-test of the (Freeman-Tukey transformed) number of dead implants
(dead implants/total implants);
Control group analyses of variances for number of pregnant females,
number of implantations per pregnant female, preimplantation loss per
pregnant female, number of dead implants per pregnant female, ratio of
dead implants to total implants per pregnant female; and
t-test of the number of corpora lutea in pregnant females.
Careful review arid statistical evaluation of the data show that
folpet, captan, parathion-methyl, parathion, phorate, malathion,
bromacil, monocrotophos, quintozene (PCNB), and azinphos-methyl are
not mutagenic in the mouse by the dominant lethal test.
11
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MAMMALIAN IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAYS
General
Many mutagenic and carcinogenic agents have been shown to induce un-
scheduled DNA synthesis (UDS) in an in vitro tissue culture system of mam-
malian cells. UDS is a form of mammalian repair synthesis that involves
at least two processes. The first is interaction of the agent with DNA,
resulting in damage of the DNA. The second, which follows, is incopor-
ation of nucleotides to repair the DNA.
UDS may be considered a fairly universal system because it occurs in
a wide variety of mammalian cell types and because it has been observed
in all stages of the cell cycle (G0, G\, G2, and M) other than S_, the
normal DNA synthetic phase.2'3 (UDS'is not observed during S_-phase be-
cause the high level of incorporation of nucleotides during the scheduled
DNA synthesis obscures the relatively low level of incorporation of
nucleotides during unscheduled DNA synthesis.)
An additional feature of UDS is that it may detect a level of DNA
damage higher than that revealed by examination of chromosomeal aberra-
tions'4 because some DNA repair results in little or no detectable change
in chromosome morphology. For each compound tested, an in vitro metabolic
activation system should be incorporated for a parallel series of UDS
assays since some compounds may be ineffective in producing DNA damage
unless they are first activated by a microsomal preparation from a mam-
malian liver homogenate.
The UDS system we have developed is unique in that, at the end of
each assay, DNA is extracted from human diploid fibroblasts (WI-38 cells)
so that the extent of repair may be expressed per unit of DNA. We have
found that this UDS assay system affords sensitivity and precision with-
out sacrificing efficiency or economy. Under separate contact, NCI
approved our use and validation of this system for the prescreening of
chemical carcinogens. With the approval of the EPA project officer, we
used this system for testing the 20 substitute pesticides, with and
without metabolic activation.
12
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Experimental
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 (BME) containing 10% (v/v) fetal calf serum and aureomycin,
an antibiotic specific for PPLO*. For 1 to 2 weeks preceding the UDS
assays, the cells were grown in medium containing 0.5% serum. This pro-
duced contact-inhibited cells in synchronous cultures in the GI phase of
the mitotic cycle. To reduce further the possibility of incorporation
of 3H-TdR by an occasional S-phase cell that might escape the contact-
inhibition synchrony and thus obscure measurements of UDS, the cultures
were preincubated for 1 hour with 10~^ M hydroxyurea (HU) before each
assay, and 10~^ M HU was added during each subsequent step of the assays.
Dilution of Compounds
Chemicals to be tested were made up immediately before use and were
diluted in appropriate solvents (water, ethanol, or DMSO), the final
concentration of solvent being one that did not produce a cytotoxic
effect after repeated testing. Sonification and pH adjustments were
used to ensure maximum solubility or even suspension of the stock solu-
tions of the compounds. The highest concentration was diluted further
in solvent and then in culture medium to give several log dilutions of
each compound. All compounds were in apparent solution and within the
physiological pH range when tested, except as otherwise noted in the
tables.
Controls
The positive controls were 4-nitroquinoline-N-oxide (4NQO), a com-
pound that induces UDS in the absence of a metabolic activation system,
and dimethylnitrosamine (DMN), a compound that induces UDS only with
metabolic activation. The negative controls were the solvents diluted
in culture medium.
As an additional check against the presence of PPLO, which could incor-
porate tritiated thymidine (3H-TdR) and thus obscure measurements of UDS,
stock cultures were analyzed monthly for the presence of PPLO. The
results of these analyses were consistently negative.
13
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UPS Assays
The contact-inhibited WI-38 cells were incubated at 37°C with log
dilutions of the substitute pesticides and with 1 yCi/ml of 3H-TdR (sp act,
6.7 Ci/mmole). For testing in the absence of metabolic activation, the
cells were exposed simultaneously to the substitute pesticide and to 3H-TdR
for 3 hours. For testing with metabolic activation, the cells were exposed
to the substitute pesticide, to 3H-TdR, and to 500 mg/ml of the 9000 x £
supernatant fraction of a liver homogenate from adult male Swiss-Webster
mice, with appropriate cofactors,* for 1 hour; then the cells were incu-
bated with only 3H-TdR for an additional 4 hours. The shorter exposure
time for metabolic activation testing was used to preclude cytotoxic effects
of the liver homogenate preparation. Both approaches included a postincorp-
oration incubation with unlabeled thymidine. DNA was extracted from the
cells by a modification of the PCA-hydrolysis procedure;5 one aliquot of
the DNA solution was used to measure the DNA content, after reaction with
diphenylamine,6 and a second aliquot was used for scintillation counting
measurements of the extent of incorporation of 3H-TdR. Results were expres-
sed as incorporated per unit of DNA and were compared with the background
rate of incorporation.
We have defined as an acceptable assay one in which the response of
the positive control compound is predicted, within the 95% confidence
limits, by regressions of average dpm/yg DNA versus average dpm/pg for
background. 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 Y: = 696 + 17.45 (X)* 48 0.7668
activation
With Metabolic Y2 = 263 + 1.83 (X)t 13 0.9639
activation
*Nicotinamide, 3.05 mg/ml; glucose-6-phosphate, 16.1 mg/ml;
5.08 mg/ml; NADP, 0.765 mg/ml.
"^Regressions over a range of background dpm/yg DNA of 0 to 450.
*Yj = Average dpm/Mg DNA for 10~5 M 4NQO (positive control).
Y£ = Average dpm/yg DNA for 5 x 10~2 M DMN (positive control).
X = Average dpm/ g DNA for background (negative control).
14
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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 occured in the experimental procedures
and repeat the test.
Interpretation of Results
In a report to the National Cancer Institute,7 we presented the
results of tests performed without metabolic activation on 40 compounds
of known carcinogenicity. We have analyzed these results using either
the parametric One-Way Classifiction Analysis of Variance or the non-
parametric Kruskal-Wallis One-Way Analysis of Variance, depending on which
was more appropriate.* At the 99% confidence limits, all the ultimate
carcinogens significantly elevate the incorporation of 3H-TdR into the
DNA. The noncarcinogenic compounds, with one exception, fail to elevate
significantly the incorporation of 3H-TdR at this level of confidence.
Thus, the 99% confidence limits of these statistical analyses apparently
can be used with reasonable accuracy to predict the biological signifi-
cance of the response to a chemical.
The number of compounds we have tested with metabolic activation is
insufficient to establish a correlation between statistical significance
and biological significance. Therefore, we assumed that the 99% confid-
ence levels of the analyses of variance used without metabolic activation
also apply for testing with metabolic activation.
Results and Discussion
Tables 51 through 90 present the results of the UDS testing, with
and without metabolic activation, of the 20 substitute pesticides. Tables
51 and 52, the DNA repair synthesis assays of monocrotophos, include
detailed summaries of the cell culture and experimental conditions for
these assays. The assays presented in the following tables (53 through
90) were conducted under similar conditions. In routine testing in the
*If there is reason to believe that the variances of each of the treat-
ments 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.
15
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absence of metabolic activation, six samples each are used for five log
concentrations of each test compound and for the negative and positive
controls. However, because of the expense of the metabolic activation
preparations, for all compounds except bromacil we tested three replicate
samples in the presence of metabolic activation and used three concentra-
tions of the test compound (selected on the basis of the testing without
metabolic activation).
Based on the criteria for positive responses, we observed significant
increases in unscheduled DNA synthesis in the absence of metabolic activa-
tion after exposure of the cells to only two substitute pesticides, raono-
crotophos and parathion. In the presence of metabolic activation enzymes,
significantly increased UDS was detected for five substitute pesticides:
monocrotophos, captan, folpet, azinphos-methyl, and monuron.
Compared with those of negative controls, the levels of 3H-TdR incor-
poration were greatly reduced in the absence of metabolic activation at the
highest concentrations tested for captan, folpet, azinphos-methyl, and
monuron, the same four compounds that induced UDS only in the presence of
metabolic activation. The reduced levels of incorporation may be inter-
preted as cytotoxic effects or as inhibition of repair caused by the highest
concentration of the test compounds. A similar effect was observed in
the presence of metabolic activation for only one compound, captan, and this
was observed at a higher concentration than had been tested without
metabolic activation. Stich et al.8 have discussed the problem of cyto-
toxicity and possible inhibition of DNA repair systems by some chemicals
and have stressed that, whereas such factors may obscure measurements of
UDS, often a close relationship exists between concentrations that induce
UDS and concentrations that are cytotoxic or that inhibit repair.
Because of the cytotoxic or inhibitory effects of the substitute
pesticides, it should not be assumed without further testing that monocro-
tophos and parathion would be carcinogenic without metabolic activation
or that the other four substitute pesticides that induced UDS in the
presence of metabolic activation are procarcinogens. The positive UDS
results indicate that these six substitute pesticides should be tested
more extensively, with the testing to include evaluations of the effects
of these chemicals in jLn vivo bioassays.
16
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MICROBIOLOGICAL ASSAYS
General
SRI examined twenty pesticides for mutagenicity by in vitro micro-
biological assays with Salmonella typhimurium (TA1535, TA1537, TA1538,
TA100), Escherichia coli WP2, repair-deficient and -proficient strains
of Bacillus subtilis and 13. coli, and with the yeast Saccharomyces
cerevisiae D3. An Aroclor 1254-stimulated, rat-liver-homogenate meta-
bolic activation system was included in each procedure, except the rela-
tive toxicity assays, to provide metabolic steps that the bacteria are
either incapable of conducting or that they do not carry out under the
assay conditions. The purpose of this study was to determine whether
the compounds elicited a mutagenic response in microorganisms.
The assay procedure with S_. typhimurium has been proven to be 85 to
90% accurate in detecting carcinogens as mutagens, and it has about the
o
same accuracy in identifying chemicals that are not carcinogenic. The
assay procedure with ^. cerevisiae is about 50% accurate in detecting
carcinogens as agents that increase mitotic recombination. J5. coli WP2
and the microbial sensitivity assay are two additional methods of detecting
mutagens. The combination of these four assay procedures significantly
enhances the probability of detecting potentially hazardous chemicals.
17
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Experimental
Salmonella typhimurium Strains TA1535, TA1537, TA1538, and TA100
The S^. typhimurium strains used at SRI were obtained from Dr. Bruce
Ames of the University of California at Berkeley.10~12 ^-Q are histidine
auxotrophs (his") by virtue of mutations in the histidine operon. In
addition to the mutations in the histidine operon, the indicator strains
have mutations in the lipopolysaccharide coat (rfa ) and deletions that
cover a gene involved in the repair of uv damage (uvrB~). The rfa~
mutation makes the strains more permeable to large molecules, thereby
increasing their sensitivity to these molecules. The uvrB~ mutation
decreases repair of some types of chemically damaged DNA and thereby
enhances sensitivity to some mutagenic chemicals. Strain TA1535 is
reverted to histidine prototrophy (his"*") by many mutagens that cause base-
pair substitutions. Strains TA1537 and TA1538 are reverted by many
frameshift mutagens. TA1537 is more sensitive than TA1538 to mutation
by some acridine and benzanthracenes, but the difference is quantitative
rather than qualitative. TA100 is derived from TA1535 by the intro-
duction of the R factor plasmid pKMlOl.13 The introduction of this
plasmid, which confers ampicillin resistance to the strain, greatly
enhances the sensitivity of the strain to some base-pair substitution
mutagens. We have shown that mutagens such as benzyl chloride and
2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (known as AF2) can be detected
in plate assays by TA100 but not by TA1535. The presence of this plasmid
also makes strain TA100 sensitive to some frameshift mutagens—e.g.,
ICR-191, benzo(a)pyrene, aflatoxin Bj, and 7,12-dimethylbenz(a) anthracene.
All the indicator strains are stored at -80°C. For each experiment,
an inoculum from frozen stock cultures is grown overnight at 37°C in a
nutrient broth consisting of 1% tryptone and 0.5% yeast extract. After
stationary overnight growth, the cultures are shaken for 3 to 4 hours to
ensure optimal growth. Each culture is checked for sensitivity to
crystal violet. The presence of the rfa~ mutation makes the indicator
strains sensitive to this dye, whereas the parent strain, rfa+, is not
sensitive to the dye. However, the mutation is reversible, leading to
18
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the accumulation of rfa cells in the culture. Therefore, the cells
must be tested routinely to ensure their sensitivity to crystal violet.
Each culture also is tested by specific mutagens known to revert each
test strain (positive controls).
To a sterile 13 x 100 mm test tube placed in a 43°C heating block,
we add in the following order:
Assays in agar
(1) 2 ml of 0.6% agar*
(2) 0.1 ml of indicator organisms
(3) 0.5 ml of metabolic activation mixture (optional)
(4) Up to 100 yl of a solution of the test chemical.**
For negative controls, we use steps (1), (2), and (3) (optional)
and 100 yl of the solvent used for the test chemical.
This mixture is stirred gently and then poured onto minimal agar
plates.t After the soft agar has set, the plates are incubated at 37°C
for 2 days. The number of his revertants (colonies that grow on plates
lacking a sufficient amount of histidine to support colony formation) are
counted and recorded. Some of the revertants are routinely tested to
confirm that they are his+, require biotin, and are sensitive to crystal
violet (rfa~).
Escherichia coli WP2
The E. coli WP2 (uvrA ) used in this project was given to us by
14 15
Dr. D. McCalla. ' A procedure similar to the one used with Salmonella
is used to measure the reversion of WP2 to tryptophan independence. How-
ever, instead of containing a trace of tryptophan in the top agar, the
minimal agar plates contain 1.25 g of oxoid broth per liter to provide
* 0.6% agar contains 0.05 mM histidine and 0.05 mM biotin.
t Minimal agar plates consist of 15 g of agar, 20 g of glucose, 0.2 g
of MgSO/.7 H20, 2 g of citric acid monohydrate, 10 g of K2HP04, and
3.5 g of NaHNH4P04.H20 per liter.
**Solvents used as appropriate include: water, dimethyl sulfoxide,
ethanol, and benzene.
19
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the trace of tryptophan required for enhancement of any mutagenic effect
of the test chemical.
Alternatively, reversion of the mutated tryptophan gene, WP2
may undergo a forward mutation in a tryptophan tRNA gene to obtain
tryptophan independence. We do not distinguish experimentally between
the true revertants and the phenotypic revertants (although the latter
tend to form smaller colonies).
Escherichia coli W3110/p3478 and Bacillus subtilis H17/M45
The E. coli strains W3110 and p3478 were obtained from Dr. H.
Rosenkranz.16 Strain p3478 is a polA~ derivative of strain W3110. It
carries a single, revertable mutation in a gene for a DNA polymerase;
Gross and Gross showed that this mutation is involved in DNA repair
synthesis. This mutation increases the sensitivity of strain p3478 to
chemicals that lead to alterations (damage) of the DNA. Therefore, we
can assay for chemicals that damage DNA by comparing the relative sensi-
tivity of the two strains (p3478 and W3110) to the test chemical.
18
The Ii. subtilus strains H17 and M45 were obtained from Dr. Kada.
Strain H17 (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
probably is a mutagen.
Inoculums from frozen stocks are grown overnight in nutrient broth*
at 37°C with shaking. To 2 ml of nutrient broth containing 0.6% agar
is added 0.1 ml of the test culture. The suspension is mixed and poured
onto plates containing nutrient broth and 2% agar.
After the soft agar has solidified, a sterile filter disc impreg-
nated with the test chemical is placed in the center of the plate. The
plates are incubated at 37°C for 16 hours, and the width of the zone of
Tryptone, 1%, and 0.5% yeast extract, supplemented with 5 ug of
thymine/ml to prevent selection of thy+ revertants.
20
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toxicity or inhibition of growth is then measured. We usually must test
several concentrations of chemical to detect accurately differences in
the zones of growth inhibition because higher initial concentrations lead
to steep concentration gradients that may reduce the differences in
growth inhibition of the two strains.
The positive control for this assay is 1 ml of l-phenyl-3,3-dimethyl-
triazene placed on the disc. A zone of approximately 40-mm width is
observed (52 and 61 mm, respectively). An additional control is 30 yg
of chloramphenicol placed on a disc. Equal zones of inhibition are
expected in all four strains (approximately 30 mm) since the toxicity of
this chemical does not depend on a mechanism that leads to DNA damage.
All assays are performed at least three times.
Saccharomyces cerevisiae D3
The yeast j^. cerevisiae D3 is a diploid heterozygous for a mutation
in an adenine-metabolizing enzymes.** Cells homozygous for this mutation
produce a red dye when grown on medium containing adenine. Adenine-
requiring homozygotes can be generated from the heterozygotes by mitotic
recombination. Many mutagens increase the ,frequency of mitotic recombi-
nation. Mitotic recombination is indicated by the development of
colonies with red pigmentation, and the degree of conversion to this
pigmented colony indicates the mutagenicity of a compound or its
?0
metabolite.
The Saccharomyces test strain from the liquid nitrogen is grown over-
night at 30°C with aeration in 1.0% tryptone and 0.5% yeast extract.
The cells are washed twice in 0.067M PO^ buffer (pH 7.4) and resuspended
in the same buffer at a concentration of 10° cells/ml.
The in vitro yeast mitotic recombination assay in suspension con-
sists of 5 x 10 washed, stationary-phase yeast cells in 1 ml of 0.067M
PCU buffer (pH 7.4) and 50 mg/ml of the test chemical (or a fraction of
the concentration required to give 50% killing). The suspension is
incubated at 30° for 4 hours. After incubation, the sample is diluted
serially in sterile saline and plated on tryptone-yeast-agar plates.
21
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_^
Plates of a 10 -'"dilution 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-negative homozygosity. To detect red colonies or red sectors,
we scan the plates with a dissecting microscope at 10 x magnification.
Plates of a 10 dilution are incubated for 2 days at 30°C for determi-
nation of the total number of colony-forming units.
The in vitro yeast itotic recombination assay in suspension with
metabolic activation is conducted as above with the addition of the meta-
bolic activation system to the incubation mixture.
Aroclor 1254-Stimulated Metabolic Activation System
Some carcinogenic mutagens (e.g., dimethylnitrosamine) are inactive
unless they are converted to their active form by being metabolized.
Ames et al.^1 have described the metabolic activation systems we use.
Adult male mice are given a single 500-mg/kg intraperitoneal injection
of a polychlorinated biphenyl (Aroclor 1254). Four days after the
injection, the animals' food is removed. On the fifth day, the mice
are killed.
The liver are removed aseptically and placed in preweighed, sterile
glass beakers. The organ weight is determined, and all subsequent
operations to the metabolic activation step are conducted in an ice
bath. The organ is 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
volume of 0.15 KC1, and homogenized with a Potter-Elvehjem apparatus.
The homogenate is centrifuged for 10 minutes at 9000 x £, and the super-
natant is removed and stored in liquid nitrogen. To .the postmito-
chondrial supernate are added MgCl2, KC1, glucose-6-phosphate, TPN, and
sodium phosphate (pH 7.4).
Results and Discussion
All the pesticides submitted to SRI for examination were tested
at least three times in the microbiological assays. The results pre-
sented here are an average of those experiments.
22
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Table 91 presents the results of the microbiological assays in
agar with Salmonella typhimurium. In this histidine reverse-mutation
assay system, two pesticides—captan and folpet—were mutagenic. For
each chemical, we observed an increase in the number of histidine-
independcnt revertants on strains TA1535 and TA100 but not on strains
TA98, TA1537, or TA1538. These results suggest that these pesticides
can alkylate DNA, causing mutations of the base-pair substitution type.
This conclusion is consistent with the mutagenic activity of these
compounds in assays with E_. coli WP2 (Table 92), which is sensitive to
base-pair substitution mutagens. Although liver homogenate activation
was not required for mutagenic activity, the mutagenic activity was
enhanced somwhat with activation at some doses. A toxic effect
(reduction of the number of mutants) was observed at doses of 100 yg
of each compound.
Table 92 presents the results of assays with j;. coli WP2.
Essentially, the results were identical to those obtained with j>.
typhinmrium TA1535 and TA100; captan and folpet were mutagenic,
but none of the other pesticides was mutagenic.
Table 93 presents the r'esults of the assays for microbial inhibition
in repair-deficient and-proficient strains of _B. subtilis and JE. coli.
Folpet, captan, chloropyrifos, and dinoseb all gave toxic zones that
were larger on the repair-deficient strains than on the repair-proficient
strains, indicating a mutagenic response. Toxic chemicals that do not
act by damaging DNA (e.g., chloramphenicol) should give equivalent zones
of toxicity. However, many if not all mutagens damage DNA and, if the
damage is not repaired, can result in cell death. Thus, a given
concentration of mutagen may be toxic for a repair deficient strain
but not for a strain the effectively repairs its DNA.
Tables 94 through 113 present the results of the assays for mitotic
recombination in Saccharomyces cerevisiae D3. A positive response in
this assay is indicated by an increase of more than threefold in the
absolute number of mitotic recombinants per milliliter as well as in
the relative number of mitotic recombinants per 10 survivors. Folpet,
23
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captan, monocrotophos, cacodylic acid, and azinphos-methyl increased
mitotic recombination significantly and are considered positive by
these procedures. Methyl parathion gave a marginally positive response.
Our results indicate that 7 of the 20 pesticides examined give
positive responses in one or more of the four microbiological assay
procedures. Although a mutagenic response in a microorganisms does
not mean that a chemical is a mutagen in humans, the combination of
four separate assay system greatly enhances the probability of detecting
potentially hazardous chemicals. Folpet and captan are mutagenic in
all four assay procedures. Chloropyrifos and dinoseb are positive in
the microbial sensitivity. Monocrotophos, cacodylic acid, and azinphos-
methyl are positive in the yeast assays.
24
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DISCUSSION
Of the 20 pesticides tested for mutagenic activity, 9 were clearly
mutagenic in one or more in vitro assays. Of these 9, 2 were mutagenic
in all the in vitro assays, but none of them produced a dominant lethal
response in the mouse. In the Salmonella assays, these chemicals caused
base-pair substitution mutations but not frameshift mutations. The '
i
absence of activity in the dominant lethal assay may be due to a lack
of sensitivity of the mouse to these types of compounds; for example,
N-methyl-N'-nitro-N-nitrosoguanidine and other alkylating agents that
cause base-pair substitution mutations do not all cause dominant lethality.
Another explanation for the absence of activity may be that these pesti-
cides did not reach the gonadal tissues in sufficient amounts to cause a
mutagenic event. None of the other 6 pesticides was mutagenic in all
the in vitro assays.
The combination of assays used in this program is one means of
identifying those pesticides that may present a mutagenic health hazard.
Those that show positive responses in several experimental systems
should be evaluated more thoroughly before they are substituted for
other pesticides already considered as a risk to the environment. Also
apparent is that no one assay system is uniquely capable of detecting
the spectrum of mutagenic events that different chemical structures may
cause.
25
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26
-------
12. B. N. Ames, E. W. Durston, E. Yamasaki, and F. D. Lee. Carcinogens
are mutagens: A simple test system combining liver homogenates
for activation and bacteria for detection. Proc. Nat. Acad. Sci.
USA 70i, 2281-2285 (1973).
13. J. McCann, N. E. Spingarn, J. Kobori, and B. N. Ames. The detection
of carcinogens as mutagens: Bacterial tester strains with R factor
plasmids. Proc. Nat. Acad. Sci. USA 7i2, 979-983 (1975).
14. B. A. Bridges. Simple bacterial systems for detecting mutagenic
agents. Lab. Pract. 21_, 413-416 (1972).
15. D. R. McCalla and D. Voutsinos. On mutagenicity of nitrofurans.
Mutation Res. 26, 3-16 (1974).
16. M. D. Anderson, E. E. Slater, and H. S. Rosenkranz. Rapid detection
of mutagens and carcinogens. Cancer Res. 31, 970-973 (1971).
17. J. Gross and M. Gross. Genetic analysis of an E^. coli strain with
a mutation affecting DNA polymerase. Nature 224, 1166-1168 (1969).
18. T. Kada. Mutagenicity testing of chemicals in microbial systems.
New Methods in Environ. Chem. Toxicology 127-133 (November 1973).
19. F. K. Zimmermann and R. Schwaier. Induction of mitotic gene con-
version with nitrous acid, l-methyl-3-nitro-l-nitrosoguanidine and
other alkylating agents in Saccharomyces cerevisiae. Mol. Gen.
Genet. 100, 63-69 (1967).
20. D. J. Brusick and V. W. Mayer. New developments in mutagenicity
screening techniques with yeast. Environ. Health Perspectives 6^,
83-96 (1973).
21. L. D. Kier, E. Yamasaki, and B. N. Ames. Detection of mutagenic
activity in cigarette smoke condensates. Proc. Nat. Acad. Sci.
USA 71_, 4159-4163 (1974).
22. B. N. Ames, J. McCann, and E. Yamasaki. Methods for Detecting
Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome
Mutagenicity Test. Mut. Res. ^1_, 347-364 (1975).
27
-------
Table 1
CHI-SQUARE TEST OF THE FERTILITY INDEX - MONOCROTOPHOS
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-10 15 MG/K6
74-10 30 MG/KG
74-10 60 MG/KG
TFM
.2 MG/KG
N
PRG
N
MTO
PERT.
INDEX
CHISQ
N
PRG
N
MTD
PERT.
INDEX
CHISQ
N
PRG
N
MTD
PERT.
INDEX
CHISQ
N
PRG
N
MTD
PERT.
INDEX
CHISQ
N
PRG
N
MTD
PERT.
INDEX
CHISQ
MULTIPLE TREATMENT
1
NJ 2
oo
3
4
5
6
7
8
28
26
23
27
24
24
30
27
40
40
40
40
40
38
38
38
.70
.65
.57
.67
.60
.63
.79
.71
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
23
31
30
25
33
27
25
28
40
40
40
40
39
40
40
40
.57
.77
.75
.63
.85
.67
.63
.70
.07
.98
2.01
.05
4.79*1
.03
1.81
.02
18
20
19
22
25
23
21
20
40
40
40
40
40
40
40
40
.45
.50
.47
.55
.63
.57
.52
.50
4.14
1.28
.45
.84
0.00
.08
4.91*
2.78
16
16
22
15
21
25
28
24
40
40
40
40
40
38
38
38
.40
.40
.55
.38
.52
.66
.74
.63
6.11 *
4.06*
0.00
6.07*
.20
o.oo
.OT
.24
29
27
32
27
30
27
27
26
40
39
40
40
40
38
36
36
.72
.69
.80
.67
.75
.71
.75
.72
0.00
.03
3.72
.06
1.42
.24
.02
• 02
* SIGNIFICANT AT P LT 0.05
I INCREASED ABOVE CONTROL
-------
Table 2
AVERAGE IMPLANTS PER PREGNANT FEMALE - MONOCROTOPHOS
WEEK
CONTROL
74-10 15 MG/KG
74-10 30 MG/KG
74-10 60
MG/KG
TEM
.2 MG/KG
NJ
MULTIPLE TREATMENT
1 319/
2 303/
3 245/
4 309/
5 274/
6 302/
7 346/
8 292/
28=11.39
26=11.65
23=10.65
27=11.44
24=11.42
24=12.58
30=11.53
27=10.61
249/
332/
356/
314/
372/
327/
28S/
313/
23=10
31 = 10
30=11
25=12
33=11
.83
.71
.87
.56
.27
27=12.11
25=11
28=11
.40
.18
189/
242/
239/
274/
270/
275/
2S5/
228/
18*10.50
20=12.10
19=12.58**!
22=12.45
25=10.80
23=11.96
21=12.14
20=11.40
180/
180/
267/
166/
248/
255/
316/
291/
16=11.
16=11.
22=12.
15=11.
21=11.
25=10.
28=11.
24=12.
25
25
14 *I
07
81
20 **
29
12
316/
293/
348/
266/
356/
273/
306/
322/
29=10.90
27=10.85
32=10.87
27= 9.85*
30=11.87
27 = 10.11**
27=11.33
26 = 12.38**!
* SIGNIFICANT AT P LT 0.05
** SIGNIFICANT AT P LT 0.01
I INCREASED ABOVE CONTROL
-------
Table 3
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - MONOCROTOPHOS
WEEK
CONTROL
74-10 15
MG/KS
74.10 30
MG/KG
74-10 60
MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1 13/
2 8/
3 9/
4 2/
5 ll/
6 21/
7 30/
8 19/
28=
26=
23=
27=
24=
24=
30=
27=
.46
.31
.39
.07
.46
.88
1.00
.70
28/
3/
16/
7/
22/
16/
ll/
24/
23=
31 =
30=
25=
33=
27=
25=
28=
1.22
.10*D
.53
.28 *
.67
.59
.44
.86
10/
10/
9/
26/
12/
14/
4/
7/
18=
20=
19=
22=
25=
23=
21 =
20=
.56
.50
.47
1.18*
.48
'61
.19
.35
10/
2/
9/
9/
9/
6/
8/
9/
16 =
16 =
22=
15=
21 =
25-
28=
24=
.63
.13
.*!
.60**
.43
,24**D
.29
.38
62/
77/
87/
ll/
22/
17/
ll/
ll/
29=
27=
32=
27=
30=
27=
27=
26=
2.14**
2.85**
2.72**
.41*
.73
.63
.41
.42
* SIGNIFICANT AT P LT 0.05
•• SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 4
CHI-SQUARE TEST OF THE DEATH INDEX - MONOCROTOPHOS
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-10 15 MG/KG
74-10 30
MG/KG
74-10 60
MG/KG
TEM
.2 MG/KG
U>
N
WDI
N DEATH
PRG INDEX
CHISQ
N
MDI
N DEATH
PRG INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
N
CHISQ WOI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PUG
DEATH
INDEX
CHI SO
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
10
7
9
2
9
15
8
12
28
26
23
27
24
24
30
27
.36
.27
.39
.07
.38
.63
.27
.44
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
11
3
11
7
11
13
8
15
23
31
30
25
33
27
25
28
.48
.10
.37
.28
.33
.48
.32
.54
.35
1.84
.01
2.54
.00
.56
.02
.17
7
7
7
11
12
11
4
7
18
20
19
22
25
23
21
20
.39
.35
.37
.50
.48
.48
.19
.35
.01
.07
.03
9.EO**
.21
.52
.09
.12
6
2
6
9
a
5
7
8
16
16
22
15
21
25
28
24
.38
.13
.27
.6Q
.38
• 20
.25
.33
.04
.52
.28
11.21 **
.07
7.48
.02
.27
25
26
25
8
10
10
9
10
29
27
32
27
30
27~
27
26
.86
.96
.78
.30
.33
.37
.33
.38
13.27 **
24.26 **
7.05 **
3.07
.00
2.36
.07
.03
** SIGNIFICANT AT P LT 0.01
-------
Table 5
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - MONOCROTOPHOS
WEEK
u>
CONTROL
74-10 15 MG/KG
74-10 30
M6/KG
74-10 60
Mb/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1 13/ 319=
2 8/ 303=
3 9/ 245=
4 2/ 309=
5 ll/ 274=
6 21/ 302=
7 30/ 346=
8 19/ 292*
.04
.03
.04
.01
.04
.07
.09
.07
28/
3/
16/
7/
22/
16/
ll/
24/
249=
332=
356=
314=
372=
327=
285=
313=
.11
.01
.04
.02
.06
.05
.04
.08
10/
10/
9/
26/
12/
14/
4/
7/
189=
242=
239=
274=
270=
275=
255=
228=
.05
.04
.04
.09**
.04
.05
.02
.03
10/
2/
9/
9/
9/
6/
8/
9/
180 =
180 =
267=
166=
248 =
255=
316=
291 =
.06
.01
.03
.05**
.04
.0?**D
.03
.03
MX
77/
87/
ll/
22/
17/
ll/
ll/
316 =
293=
348=
266 =
356 =
273=
306=
322=
.20**
.26**
.25**
.04*
.06
.06
.04
.03
• SIGNIFICANT AT f LT 0.05
•* SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
CHI-SQUARE TEST OF THE FERTILITY INDEX - BROMACIL
1 DEGREE OF FREEDOM
MEEK
VEHICLE CONTROL
74-06 1250 MG/KG
74-06 2500 MG/KG
74-06 5000 MG/KG
TEM
.2 MG/KG
N
PRG
LO
Co
1 28
2 26
3 23
4 27
5 24
6 24
7 30
8 27
N PERT.
MTO INDEX
CHISQ
N
PRG
N
MTD
PERT.
INDEX
CHISQ
N
PRG
N
MTO
PERT.
INDEX
CHISQ
N
PRG
N
MTD
PERT.
INDEX
CHISQ
H
PRG
N
MTD
PERT.
INDEX
CHISQ
MULTIPLE TREATMENT
40
40
40
40
40
38
38
38
.70
.65
.57
.67
.60
.63
.79
.71
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
21
24
29
25
31
31
29
26
40
40
40
40
40
40
40
39
.52
.60
.72
.63
.77
.77
.72
.72
1.90
.05
1.37
.05
2.09
1.30
.16
.03
23
26
19
22
22
25
22
24
40
38
38
38
38
38
38
38
.57
.68
.50
.58
.58
.66
.58
.63
.87
.01
.19
.41
• 00
0.00
2.98
.24
29
25
25
28
27
27
29
27
40
40
40
40
40
40
40
40
.72
.63
.63
.70
.67
.67
.72
.67
0.00
0.00
.05
0.00
.22
.03
.16
.01
29
27
32
27
30
27
27
26
40
39
40
40
40
38
36
36
.72
.69
.80
.67
.75
.71
.75
.72
0.00
.03
3.72
.06
1.42
.24
.02
.02
-------
MEEK
Table 7
AVERAGE IMPLANTS PER PREGNANT FEMALE - BROMACIL
CONTROL
74-06 1250 MG/KG
74-06 2500 MG/K6
74-06 5000 MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1 319/
2 303/
3 245/
4 309/
5 274/
6 302/
7 346/
8 292/
28=11.39
26=11.65
23=10.65
27=11.44
24=11.42
24=12.58
30=11.53
27=10.81
235/
253/
335/
290/
3fel/
368/
3S5/
318/
21=11.19
24=10.54
29=11.55
25=11.60
31=11.65
31=11.87
29=12.24
28=11.36
27B/
284/
225/
268X
26l/
294/
253/
277/
23=12.09
26=10
19=11
22=12
22=11
25=11
22=11
24=11
.92
.84
.18
.86
.76
.50
.54
328/
304/
319/
346/
323/
278/
357/
307/
29=11.
25=12.
25=12.
28=12.
27=11.
27=10.
29=12.
27=11.
31
16
76**I
36
96
30**
31
37
316/
293X
348/
266/
35&/
273/
306/
322/
29=10.90
27=10.85
32=10.87
27= 9.85 *
30=11.87
27=10.11 **
27=11.33
26=12.38**!
• SIGNIFICANT AT P LT 6.05
** SIGNIFICANT At P LT 0.01
I INCREASED ABOVE CONTROL
-------
WEEK
Table 8
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - BROMACIL
CONTROL
74-06 1250 MG/KG
74-06 2500 MG/KG
74-06 5000 MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
13/
8/
9/
2/
ll/
21/
30/
19/
28=
26 =
23 =
27 =
24 =
24=
30 =
27=
.46
.31
.39
.07
.46
.88
1.00
.70
8/
12/
6/
7/
12/
7/
ll/
IS/
21 =
24 =
29 =
25=
31 =
31 =
29=
28=
.38
.50
.21
.28
.39
,23**D
.38
.54
10/
a/
12/
16/
15/
16/
14/
a/
23=
26=
19=
22s
22=
25=
22=
24 =
.43
.31
.63
.73**
.68
.64
.64
.33
17/
5/
10/
IS/
8/
21/
14/
9/
29=
25=
25=
28=
27=
27=
29=
27=
.59
.20
.40
.54**
.30
.78
.48
.33
62/
77/
87/
ll/
22/
17/
ll/
ll/
29 =
27=
32=
27=
30 =
27=
27=
26=
2.14**
2.85**
2.72**
.41*
.73
.63
.41
.42
« SIGNIFICANT AT P LT 0.05
«« SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 9
CHI-SQUARE TEST OF THE DEATH INDEX - BROMACIL
1 DEGREE OF FREEDOM
MEEK
VEHICLE CONTROL
74-06 1250 MG/KG
74-06 2500 MG/KG
74-06 5000 MG/KG
TEH
.2 MG/KG
u>
N
WOI
N
PR6
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
N
CHISQ WDI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PKG
DEATH
INDEX
CHISQ
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
10
7
9
2
9
15
8
12
28
26
23
27
2*
24
30
27
.36
.27
.39
• 07
.38
.63
.27
.44
0.00
0.00
0.00
0.00
0.00
0*00
0.00
0.00
7
10
6
6
12
5
7
11
21
24
29
25
31
31
29
28
.33
.42
.21
.24
.39
• 16
.24
.39
.02
.64
1.32
1.62
.04
j0.65**D
.01
.01
7
a
9
11
12
13
10
7
23
26
19
22
22
25
22
24
.30
.31
.47
.50
.55
.52
.45
.29
.01
0.00
.05
9.20**
.74
.21
1.24
.70
14
5
6
14
8
10
12
7
29
25
25
28
27
27
29
27
.48
.20
.24
.50
.30
.37
.*!
.26
.48
.06
.67
10.11**
.09
2.36
.84
1.30
25
26
25
a
10
10
9
10
29
27
32
27
30
27
27
26
.86
.96
.78
.30
.33
.37
.33
.38
13.27**
24.26**
7.05**
3.07
.00
2.36
.07
.03
** SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
WEEK
Table 10
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - BROMACIL
CONTROL
74-06 5000 MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
3
u>
5
6
7
8
13/
8/
9/
2/
ll/
21/
30/
19/
319=
303=
245=
309=
274=
302 =
346=
292=
.04
.03
.04
.01
.04
.07
.09
.07
8/
12/
6/
7/
12/
7/
ll/
15/
235=
253=
335=
290 =
361 =
368=
355=
318=
.03
.05
,02*D
.02
.03
.02 **D
.03
.05
10/
8/
12/
16/
15/
16/
14/
8/
278=
284 =
225=
268=
261 =
294=
253=
277=
.04
.03
.05
.06**
.06
.05
.06
.03
17/
5/
10/
15/
a/
21/
14/
9/
328 =
304=
319=
346=
323=
278=
357 =
307 =
.05
.02
.03
.04**
.02
.08
.04
.03
62/
77/
87/
ll/
22/
17/
ll/
ll/
316=
293=
348 =
266 =
356=
273=
306=
322=
.20**
.26**
.25**
.04*
.06
.06
.04
.03
• SIGNIFICANT AT P LT 0.05
«« SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 11
CHI-SQUARE TEST OF THE FERTILITY INDEX - CAPTAN
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-02 1250 MG/KG
74-02 2500 MG/Kti
74-02 5000 MG/KG
TEM
.2 MG/KG
N
PRG
N
MTD
PERT.
INDEX
CHISQ
N
PRG
N
MTD
PERT.
INDEX
CHISO
N
PRG
N
MTD
FtRT.
INDEX
CHiSO
N
PRG
N
MTD
FEHT.
INDEX
CHISO
N
PPG
N
MTO
PERT.
INDEX
CHISO
MULTIPLE TREATMENT
CO 1 23
oo
2 27
3 26
4 27
5 29
6 29
7 29
8 32
40
39
40
40
40
40
40
40
.57
.69
.65
.67
.72
.72
.72
.80
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
34
30
23
31
26
27
27
33
40
40
40
38
38
38
38
38
.85
.75
.57
•82
.68
.71
.71
.87
6.10*1
.10
.21
1.35
.02
.01
.01
.26
31
36
30
33
30
34
30
34
40
40
40
40
40
40
40
40
.77
.90
.75
.62
.75
.85
.75
.85
2.79
4.07*!
.54
1.67
0.00
1.20
0*00
.09
29
30
31
32
26
30
26
27
40
40
40
40
36
36
38
36
.72
.75
.77
.60
.68
.79
.68
.71
1.37
.10
.98
1.03
.02
.16
.02
,43
29
27
32
27
30
27
27
26
40
39
40
40
40
aa
36
36
.7?
.69
.80
.67
,7S
.71
.75
.7?
1.37
.06
1.57
.0*
0.00
.01
.00
.26
* SIGNIFICANT AT PLT 0.05
I INCREASED ABOVE CONTROL
-------
Table 12
AVERAGE IMPLANTS PER PREGNANT FEMALE - CAPTAN
MEEK
CONTROL
74-02 1250 MG/KS
74-02 2500 HG/KG
74-02 5000 MG/KG
w
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
3
4
5
6
7
6
265/
305/
268/
288/
334/
323/
323/
38l/
23=11.52
27=11.30
26=10.31
27=10.67
29 = 11. "52
29=11.14
29=11.14
32=11.91
3a7/
328/
252/
324/
2H2/
312/
soy/
382/
34=11.38
30=10.93
23=10.96
31=10.45
26=10.85
27=11.56
27=11.44
33=11.58
339/
375/
313/
359/
348/
361/
329/
383/
31=10. v4
36=10.42
30=10.43
33=10-88
30=11.60
34=10.62
30=10.97
34=11.26
310/
342X
345/
333/
298/
340/
309/
301/
29=10
30=11
31 = 11
32=10
26=11
30=11
26=11
27 = 11
.69
.40
.13
.41
.46
.33
. aa
.15
316/
293/
34fi/
266/
35ft/
273/
306/
322/
29=10.90
27=10.85
32=10.87
27= 9.85
30=11.87
27=10.11
27=11.33
26=12.38
« SIGNIFICANT AT P LT 0.05
»« SIGNIFICANT AT P LT 0.01
-------
WEEK
Table 13
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - CAPTAN
CONTROL
74-02 1250 MG/KG
74-02 2500 MG/KG
74-02 5000 MG/KG
TEM
.2 MS/KG
MULTIPLE TREATMENT
1 8/
2 ll/
• 3 16/
4 16/
5 15/
6 9/
7 21/
8 13/
23=
27 =
?6=
27=
29=
29=
29=
32=
.35
.41
.62
.59
.52
.31
.72
.*!
17/
15/
16/
12/
?4/
13/
15/
12/
34 =
30 =
23=
31 =
26=
27=
27=
33=
.50
.50
.to
.39
.92
.48
.56
.36
19/
17/
16/
15/
9/
21/
14/
ll/
31 =
36=
30 =
33=
30=
34=
30 =
34=
.61
.*T
.53
.45
.30
.62
.47
.32
IS/
14/
IS/
ll/
ll/
18/
8/
7/
29 =
30 =
31 =
32=
26=
30 =
2fi =
27=
.52
.*7
.*B
.3*
.42
.60
.31
.26
62/
77/
87/
ll/
22/
17/
ll/
I!/
29=
27 =
32 =
27=
30S
27=
27=
26=
2.14**
2.85**
2.72**
.*!
.73
.63
.41
.+2
• SIGNIFICANT AT P LT 0.05
«• SIGNIFICANT AT P LT 0.01
-------
Table 14
CHI-SQUARE TEST OF THE DEATH INDEX - CAPTAN
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-02 1250 MG/KG
74-02 2500 MG/KG
74-02 5000 MG/KG
TEM
.? MG/KG
N N DEATH
WDI PRO INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHISQ
N
WOI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHISQ
MULTIPLE TREATMENT
i a
2 9
3 10
* 11
5 11
6 8
7 12
8 11
23
27
26
27
29
29
29
32
.35
.33
.38
•41
.38
.2«
.41
• 34
0.00
0.00
0.00
0-00
0.00
0.00
0.00
0.00
10
8
12
8
10
9
12
8
34
30
23
31
26
27
27
33
.29
.27
.52
.26
.38
.33
.44
«24
.02
.07
.46
.86
.06
.03
.00
.39
12
12
11
7
7
15
11
9
31
36
30
33
30
34
30
34
.39
.33
.37
.21
.23
.44
.37
.26
.00
.07
.02
1.85
.87
1*20
.01
.19
12
10
13
8
11
10
7
5
29
30
31
32
26
30
26
27
.41
.33
.42
.25
.*2
.33
.27
.19
.04
.08
.00
1.02
.00
.0*
.71
1.15
25
26
35
8
10
10
9
10
29
27
32
27
30
27
27
26
.B6
.96
.78
.30
.33
.37
.33
.38
12.49 **
20.79**
7.85 **
.32
.01
.22
.12
.00
** SIGNIFICANT AT PLT 0.01
-------
WEEK
Table 15
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - CAPTAN
CONTROL
74-02 1250 MG/KG
74-02 2500 MG/KG
74-02 5000 MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
•0 3
(S3
4
5
6
7
a
B/ 265=
ll/ 305=
16/ 268=
16/ 288=
15/ 334=
9/ 323=
21/ 323=
13/ 381=
.03
.04
.06
.06
.04
.03
.07
.03
17/ 387=
\5/ 328=
16/ 252=
12/ 324=
24/ 282=
13/ 312=
\^/ 309=
12/ 382=
.04
.05
.06
.04
.09
.04
.05
.03
19/
17/
16/
15/
9/
21/
14/
ll/
339*
375 =
313=
359=
348=
361 =
329=
383=
.06
.05
.05
• 04
.03
.06*
.04
.03
15/
14/
15/
ll/
ll/
IB/
B/
7/
310=
342=
345=
333=
298=
340 =
309=
30i =
.05
.04
.04
.03
.04
.05
.03
.02
62/
77/
87/
ll/
22/
17/
ll/
ll/
316=
293=
348=
266=
356=
273=
306=
322=
.20**
.26**
.25**
.04
.06
.06
.04
.03
• SIGNIFICANT AT P LT 0.05
*« SIGNIFICANT AT P LT 0.01
-------
Table 16
CHI-SQUARE TEST OF THE FERTILITY INDEX - FOLPET
1 DEGREE OF FREEDOM
MEEK
VEHICLE CONTROL
74-03 1250 MG/KG
74-03 2500 M6/Kb
74-03 5000 MO/KG
TEM
.2MG/KG
.p-
CO
N
PRG
N
MTO
FERT.
INDEX
CHISU
N
PRG
N
MTO
KERT.
INDEX
CHIbQ
N
PRG
N
MTD
FERT.
INDEX
CH15Q
N
PRG
N
MTO
FERT.
INDEX
CHISQ
N
PRG
N
MTD
F-ERT.
INDEX
CHISU
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
23
27
26
27
29
29
29
32
40
39
40
40
40
40
40
40
.57
.69
.65
.67
.72
.72
.72
.80
0.00
0.00
0.00
0.00
u.oo
0.00
0.00
0.00
27
28
25
30
30
30
28
27
40
40
40
40
40
40
40
40
.67
.70
.63
.75
.75
.75
.70
.67
.48
.03
0.00
.24
0.00
O.UO
O.UO
1.03
29
23
31
18
26
28
27
29
40
40
40
40
40
4u
40
40
.72
,b7
.77
.45
.65
.70
.67
.72
1.37
.72
.98
3.25
.£3
0.00
.66
.28
30
35
32
30
3*
28
31
30
40
40
40
40
40
40
40
40
.75
.88
.80
.75
.88
.70
.77
.75
2.01
2.90
1.57
.24
1.9b
o.ou
.07
.07
29
27
32
27
30
27
27
26
40
39
40
40
40
38
J6
36
.72
.69
.80
.67
.75
.71
.75
.72
1.37
.06
1.57
.06
0.00
.01
• 00
.28
-------
Table 17
AVERAGE IMPLANTS PER PREGNANT FEMALE - FOLPET
WEEK
CONTROL
74-03 125U M6/K6
74-03 2500 MG/KG
74-03 "5000 Mto/Nti
I EM
MULTIPLE TREATMENT
1 265/
2 305/
3 268/
4 288'
5 334/
6 323/
7 323/
8 3«1'
23=11.52
27=11.30
26=10.31
27=10.67
29=11.52
29=11.14
29=11.14
32=11.91
308'
304/
271/
299/
333/
334/
307/
301'
27=11.41
28=10.86
25=10.84
30= 9.97
30=11.10
30=11.13
28=10.96
27=11.15
324/
257'
334/
183'
318'
294/
311'
34b/
29=11
23=11
31 = 10
18=10
26=12
28=10
27=11
29=11
.17
.17
.n
.17
.23
.bO
.S2
.90
328'
390'
327/
320'
399'
312/
356/
346/
30 = 10
35=11
32=10
30=10
35=11
28=11
31 = 11
30 = 11
.93
.1*
.22
.67
.40
.1*
.48
.53
316/
293'
34B/
266/
356'
273'
306'
322'
29=10.90
27=10.85
32=10.87
27= 9.85
30=11.87
27=10.11
27=11.33
26=12.36
* SIGNIFICANT AT P LT 0.05
** SIGNIFICANT AT H LT 0.01
-------
HEEK
Table IS
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - FOLPET
CONTROL
74-03 1250 MG/KG
74-03 2500 MG/KG
7«t-03 5000 M(j/K,G
TEM
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
8/
ll/
16/
16/
15/
9/
21/
13/
23=
27 =
26=
27 =
29=
29 =
29 =
32 =
.35
.41
.62
.59
.52
.31
.72
.41
10/
18/
13/
16/
12/
\Tf
12/
ll/
27 =
28=
25=
30 =
30 =
30 =
28 =
27=
.37
.64
.52
.53
.40
.57
.43
.41
25/
20/
19/
8/
V/
16/
10/
20/
29=
23=
31 =
18 =
26=
28=
27 =
29=
.86
.J>7*
.61
.44
.35
.57
.37
.69
13/
21/
ll/
7/
16/
13/
IB/
13/
30 =
35=
32=
30 =
35 =
2b =
31 =
30*
.43
.60
.34
.23*0
.46
.46
.58
.43
62/
77/
87/
ll/
22/
17/
ll/
ll/
29=
27=
32=
27=
30=
27=
27=
26=
2.14**
2.85**
2.72**
.41
.73
.63
.41
.42
* SIGNIFICANT AT P LT 0.05
•« SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 19
CHI-SQUARE TEST OF THE DEATH INDEX - FOLPET
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-03 1250 MG/KG
74-03 2500 Mb/KU
74-03 5000 M(j/K6
TEM
.2MG/KG
N
nOI
N
PRG
DEATH
INDEX
CHISU
N
WDI
N
HK6
DEATH
INDEX CHIMJ
N N
WDI PRG
DEATH
INDEX CHlbQ
N
NU1
N
PRG
DEATH
INDEX
CHISU
N N
woi PKG
DEATH
INDEX
CHISO
MULTIPLE TREATMENT
1
2
3
4
5
6
7
e
8
9
10
11
11
e
12
11
23
27
26
27
29
29
29
32
.35
.33
.38
.41
.38
• 28
• M
• 34
0.00
0.00
0.00
0.00
0.00
o.oo
o.oo
0.00
u
13
9
11
10
11
a
9
27
28
25
30
30
30
28
27
.30 .01
.46 .bl
.36 .01
.37 .00
.33 .01
•37 tCZ
.29 .54
.33 .04
13 29
12 23
9 31
7 lb
8 26
11 28
9 27
16 29
.45 .10
.52 1.12
.29 .22
.39 .03
.31 -07
.39 .43
.33 .12
.55 1.89
10
15
tt
7
12
9
13
11
30
35
32
30
35
28
31
30
.33
.43
.25
.23
• 34
• 32
• 42
.37
.03
.25
.67
1.27
.00
.01
• 05
.01
25
.86
.96
.78
.30
.33
.37
.33
.38
12.49**
20.79**
7.85**
.32
• 01
.22
.12
.00
** SIGNIFICANT AT PLT 0.01
-------
WEEK
Table 20
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - FOLPET
CONTROL
74-03
MG/KG
/4-03 iibOO M6/Kfa
74-03- 5000 MG/KG
TtM
.2MG/K6
MULTIPLE TREATMENT
1 8/
2 ll/
3 16/
4 16/
5 15/
6 9/
7 21/
8 13/
265=
305=
268=
288=
334=
323 =
323 =
381 =
.03
.04
.06
.06
.04
.03
.07
.03
10/
la/
13/
16/
12/
17/
lit
ll/
308=
304=
271 =
29V=
333=
334 =
307=
301 =
.03
.06
.05
.05
.04
.05
.04
.04
2b/
20/
19/
8/
9/
16/
10/
20/
324=
257=
334=
1«3>
318=
294=
311 =
345=
.118
,0«*
.06
•U4
.03
.Ob
.03
.06
13/
21/
ll/
7/
16/
13/
18/
13/
328 =
390 =
327=
320 =
399=
312 =
356=
346 =
.04
.05
.03
.02
.04
.04
.05
.04
62/
77/
87/
ll/
22/
17/
ll/
ll/
316=
293=
348=
266=
356=
273=
306=
322=
.20**
.26**
.25**
• 04
.06
.06
.04
.03
• SIGNIFICANT AT P LT 0.05
*« SIGNIFICANT AT P LT 0.01
-------
Table 21
CHI-SQUARE TEST OF THE FERTILITY INDEX - AZINPHOS-METHYL
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-09 20 MG/KS
74-09 40 M6/K6
74-09 RO MS/KG
TEM
.? MG/KG
oo
N
PRG
N
MTO
FERT.
INDEX
CHISQ
N
PRG
N
MTD
FERT.
INDEX
CHISQ
N
PRG
N
MTD
FERT.
INDEX
CHISQ
N
PRG
N
MTO
FERT.
INDEX
CHISQ
N
PRG
N
MTD
FERT.
INDEX
CHISQ
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
28
26
23
27
24
24
30
27
40
40
40
40
40
38
38
38
.70
.65
.57
.67
.60
.63
.79
.71
0.00
0.00
0.00
0*00
0.00
0.00
0.00
0.00
34
29
33
30
29
30
27
26
40
40
40
40
40
40
40
40
.85
.72
.82
.75
.72
.75
.67
.65
1.79
.23
4.82*1
.24
.89
.79
.78
.11
28
27
29
29
26
27
25
26
40
40
40
40
40
40
40
40
.70
.67
.72
.72
.65
.67
.63
.65
.06
0.00
1.37
.06
.05
.03
1.81
.11
27
27
24
21
20
28
24
22
40
40
38
36
34
34
34
34
.67
.67
.63
.58
.59
• 82
.71
.65
0.00
0.00
.08
.35
.02
2.41
.30
.10
29
27
32
27
30
27
27
26
40
39
40
-40
40
3«
36
36
.72
.69
.80
.67
.75
.71
.75
.72
0.00
.03
3.72
.06
1.42
.24
.02
.02
* SIGNIFICANT AT P LT 0.05
I INCREASED ABOVE CONTROL
-------
Table 22
AVERAGE IMPLANTS PER PREGNANT FEMALE - AZINPHOS-METHYL
WEEK CONTROL
74-09 20 MG/KG
74-09 40 MG/KG
74-09 80 MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
3
VO
4
5
6
7
8
319/
303/
245/
309/
274/
302/
346/
292/
28=11.39
26=11.65
23=10.65
27=11.44
24=11.42
24=12.58
30=11.53
27=10.81
375/
335/
379/
367/
337/
345/
288/
306/
34=11
29=11
33=11
30=12
29=11
30=11
27=10
26=11
.03
.55
.*8
.23
.62
.50 *
.67
.77
300/
312/
33S/
369/
310/
276/
278/
293/
28=10.71
27=11.56
29=11.66
29=12.72**!
26=11.92
27=10.22**
25=11.12
26=11.27
306/
310/
272/
247/
228/
323/
262/
250/
27=11.33
27=11.48
24=11.33
21=11.76
20=11.40
28=11.5**
24=10.92
22=11.36
316/
293/
348/
266/
356/
273/
306/
322/
29=10.90
27=10.85
32=10.87
27= 9.85 *
30=11.87
27=10.11 **
27=11.33
26=12.38 **!
* SIGNIFICANT AT P LT 0.05
•• SIGNIFICANT AT P LT 0.01
I INCREASED ABOVE CONTROL
-------
WEEK
Table 23
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - AZINPHOS-METHYL
CONTROL
74-09 20 MG/K3
74-09 40 MG/KG
74-09 80
MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
Z
o, 3
° 4
5
6
7
a
13/
8/
9/
2/
ll/
21/
30/
19/
28=
26=
23=
27=
24=
24=
30=
27=
.46
.31
.39
.07
.46
.88
1.00
.70
13/
16/
31/
z\f
15/
10/
8/
13/
34*
29=
33=
30 =
29=
30 =
27=
26=
.38
.55
.94
.70**
.52
,33**D
.30
.50
34/
15/
a/
31/
6/
17/
a/
10/
28=
27*
29=
29=
26=
27=
25=
26=
1.21 *
.56
.28
1.Q7 **
.23
.63
.32
.38
19/
19/
ll/
7/
ll/
9/
19/
a/
27=
27=
24=
21 =
20 =
28=
24=
22=
.70
.70**
.46
.33*
.55
,32**D
.79
.36
62/
77/
87/
11'
22/
17/
ll/
ll/
29=
27=
32=
27=
30=
27=
27=
26=
2.14**
2.85**
2.72**
.41*
.73
.63
.41
.42
* SIGNIFICANT AT P LT 0.05
** SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 24
CHI-SQUARE TEST OF THE DEATH INDEX - AZINPHOS-METHYL
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-09 20 HO/KG
74-09 40 MG/KG
74-09 80 MG/KG
TEM
.2 MG/KS
N
WDI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
N
CHISQ WDI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHISQ
MULTIPLE TREATMENT
1 10
(Ji g 7
(-" * '
3 9
4 2
5 9
6 15
7 8
8 12
28
26
23
27
24
24
30
27
.36
.27
.39
.07
.38
.63
.27
.44
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
12
11
16
13
13
8
7
10
34
29
33
30
29
30
27
26
.35
.38
.48
.43
.45
.27
.26
.38
.05
.34
.18
7.70**
.07
5.61**D
.06
.03
14
8
5
14
6
9
7
8
28
27
29
29
26
27
25
26
.50
.30
.17
.48
.23
.33
.28
.31
.66
.01
2.11
9.53**
.64
3.25
.04
.55
11
12
8
7
7
8
10
8
27
27
24
21
20
28
24
22
.41
.44
.33
.33
.35
.29
.42
.36
.01
1.09
.01
3.65
.02
4.73 *D
.76
.08
25
26
25
8
10
10
9
10
29
27
32
27
30
27
27
26
.86
.96
.78
.30
.33
.37
.33
.38
13.27**
24.26**
7.05**
3.07
.00
2.36
.07
.03
* SIGNIFICANT AT P LT 0.05
** SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
WEEK
Table 25
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - AZINPHOS-METHYL
CONTROL
74-09 20 MG/KG
74-09 40 MG/KG
74-09 80
MO/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
13/ 319=
8/ 303=
9/ 245=
2/ 309=
ll/ 274=
21/ 302=
30/ 346=
19/ 292=
.04
.03
.04
.01
.04
.07
.09
.07
13/
16/
31/
21/
15/
10/
a/
13/
375=
335=
379=
367=
337=
345=
288=
306=
.03
.05
.08
.06 **
.04
.03 **D
.03
.04
34/
15/
a/
31/
6/
17/
a/
10/
300=
312=
338=
369s
310=
276=
278=
293=
.11
.05
.02
.08**
.02
.06
.03
.03
19/
19/
ll/
7/
ll/
9/
19/
8/
306=
310=
272=
247=
228=
323=
262=
25o=
.06
.06
.04
.03*
.05
,03**D
.07
.03
62/
77/
87/
ll/
22/
17/
ll/
ll/
316=
293=
348=
266=
356=
273=
306=
322=
.20**
.26**
.25**
.04*
.06
.06
.04
.03
* SIGNIFICANT AT P LT 0.05
•* SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 26
CHI-SQUARE TEST OF THE FERTILITY INDEX - MALATHION
1 DEGREE OF FREEDOM
MEEK
VEHICLE CONTROL
74-07 1250 MG/KG
74-07 2500 MG/KG
74-07 5000 MG/KG
TEM
MG/KG
N
PRG
N FERT.
MTD INDEX
CHISQ
N
PRG
N
MTD
FERT.
INDEX
CHISO
N
PRG
N
MTD
FERT.
INDEX
CHISQ
N
PRG
N
MTD
FERT.
INDEX
CHISO
N
PRG
N
MTD
FERT.
INDEX
CHISO
MULTIPLE TREATMENT
W l 28
2 26
3 23
4 27
5 24
6 2*
7 30
8 27
40
40
40
40
«0
38
38
38
.70
.65
.57
.67
.60
.63
.79
.71
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
31
30
23
25
21
28
30
30
40
40
38
38
36
36
36
36
.77
.75
.61
.66
.58
.78
.83
.83
.26
.54
.00
.01
.01
1.26
.03
.96
26
24
24
22
22
19
19
16
40
40
40
38
36
34
34
34
.65
.60
.60
.58
.61
.56
.56
.47
.06
.05
0.00
.41
• 02
.15
3.39
3.36
29
31
33
27
34
35
32
37
40
39
40
40
*0
*0
*0
40
.72
.79
.82
.67
.85
.88
.80
.92
0.00
1.40
4.82*1
.06
5.o8*I
5.02*1
.03
4.72*1
29
27
32
27
30
27
27
2*
40
39
40
40
40
38
36
36
.7?
.69
.SO
.67
.75
.71
.75
.72
0.00
.03
3.72
.06
l.*2
.24
.02
.02
** SIGNIFICANT AT P LT 0.05
I INCREASED ABOVE CONTROL
-------
MEEK
Table 27
AVERAGE IMPLANTS PER PREGNANT FEMALE - MALATHION
CONTROL
74-07 5000 MG/KG
TEM
.2 M6XKG
MULTIPLE TREATMENT
1 319X
2 303X
3 245X
n
4 309X
5 274X
6 302X
7 346X
8 292X
28=11.39
26=11,65
23=10.65
27=11.44
24=11.42
24=12.58
30=11.53
27=10.81
346X
363X
281X
304X
238X
318X
337X
323X
31 = 11
30=12
23=12
25=12
21=11
28=11
30=11
30=10
.16
.10
.22*1
.16
.33
.36*
.23
.77
298X
267X
305X
256X
243X
226X
235X
187X
26=11.46
24=11.12
24=12.71*1
22=11.64
22=11.05
19=11.89
19.12.37*1
16.11.69
321X
356X
402X
300X
386X
391X
352X
397X
29=11
31 = 11
33=12
27=11
34=11
35=11
32=11
37=10
.07
.48
.18*1
.11
.35
.17**
.00
.73
316X
293X
348X
266X
356X
273X
306X
322X
29=10.90
27=10.85
32=10.87
27= 9.85*
30=11.87
27=10.11**
27=11.33
26=12.38**!
• SIGNIFICANT AT f LT 0.05
** SIGNIFICANT AT f> LT 0.01
I INCREASED ABOVE CONTROL
-------
MEEK
Table 28
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - MALATHION
CONTROL
74-07 1250 MG/KG
74-07 2500 MG/KG
74-07 5000 MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
U. 3
Ln
4
5
6
7
8
13/
8/
9/
2/
ll/
21/
30/
19/
38=
26=
23=
27=
24s
24x
30s
27*
.46
.31
.39
.07
.46
.88
1.00
.70
IB/
12/
14/
14/
4/
21/
10/
19/
31 =
30=
23=
25>
21 =
28=
30 =
30=
.58
.*0
.61
.56**
.19
.75
.33
.63
8/
ll/
14/
8/
a/
20/
6/
7/
26=
24 =
24=
22=
22=
19=
19s
16=
.31
.46
.58
.36*
.36
1.05
.32
.44
13/
ll/
14/
7/
8/
23/
IS/
32/
29=
31 =
33=
27=
34=
35=
32=
37=
.45
.35
.*2
.26
.24
.66
.*7
.86
62/
77/
87/
ll/
22/
17/
ll/
ll/
29=
27=
32=
27=
30=
27 =
27=
26=
2.14**
2.85**
2.72**
.41*
.73
.63
.*!
.*2
• SIGNIFICANT AT P LT 0.05
«• SIGNIFICANT AT f LT 0.01
-------
Table 29
CHI-SQUARE TEST OF THE DEATH INDEX - MALATHION
1 DEGREE OF FREEDOM
MEEK
VEHICLE CONTROL
74*07 1250 MG/KG
74-07 2500 MG/KG
74-07 5000 MG/KG
TEM
.2 MG/KG
N
MDI
N
PRG
DEATH
INDEX
CHI SO
N
WDI
N
PRG
DEATH
INDEX
CHI SO
N
UDI
N
PRG
DEATH
INDEX
CHISO
N
WDI
N
PRG
DEATH
INDEX
CHISO
N
WDI
N
PUG
DEATH
INDEX
CHISQ
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
10
7
9
2
9
15
8
12
28
26
23
27
24
24
30
27
.36
.27
.39
.07
.38
.63
.27
.**
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
13
8
7
10
3
11
7
14
31
30
23
25
21
28
30
30
.42
.27
.30
.*o
.1*
.39
.23
.*7
.05
.08
.10
6.04*
2.01
1.93
0.00
.01
7
9
11
5
6
6
4
7
26
24
24
22
22
19
19
16
.27
.38
.*<•
.23
.27
.32
.21
.**
.16
.25
.03
1.2*
.IB
2.91
.01
.07
10
8
9
5
7
16
12
16
29
31
33
27
34
35
32
37
.34
.26
.27
.19
.21
.46
.38
.*3
.03
.04
.*!
.66
1.26
1.01
.*!
.03
25
26
25
a
10
10
9
10
29
27
32
27
30
27
27
26
.86
.96
.78
.30
.33
.37
.33
.38
13.27 **
24.26 **
7.05 **
3.07
.00
2.36
.07
.03
* SIGNIFICANT AT P LT 0.05
** SIGNIFICANT AT P LT 0.01
-------
MEEK
Table 30
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - MALATHION
CONTROL
74-07 1250 MG/KG
74-07 2500 MG/KG
74-07 5000 MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
13/
8/
9/
2/
ll/
21/
30/
19/
319=
303=
245=
309 =
274 =
302 =
346=
292=
.04
.03
.04
.01
.04
.07
.09
.07
18/
12/
14/
14/
4/
21/
10/
19/
346=
363=
281 =
304 =
238=
318=
337 =
323=
.05
.03
.05
.05**
,02*D
.07
.03
.06
a/
ll/
14/
8/
a/
ao/
6/
7/
298=
267 =
305=
256=
243=
226=
235=
187=
.03
.04
.05
.03
.03
.09
.03
.04
13/
ll/
14/
7/
a/
23/
15/
32/
321 =
356=
402=
300=
386=
391 =
352=
397=
.04
.03
.03
.02
.02
.06
.04
.08
62/
77/
87/
ll/
22/
17/
ll/
ll/
316=
293=
348 =
266=
356=
273=
306=
322=
.20**
.26**
.25**
.04*
.06
.06
.04
.03
* SIGNIFICANT AT P LT 0.05
•« SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 31
CHI-SQUARE TEST OF THE FERTILITY INDEX - PARATHION
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-01 62.5 MG/KG
74-01 125. M6/K6
74-01 250. MG/KG
TFM
.? MG/KG
Ui
oo
N
PRO
N
MTO
PERT.
INDEX
CMISQ
N
PR6
N
MTO
PERT.
INDEX
CHISQ
N
PRG
N
MTO
PERT.
INDEX
CMISQ
N
PRG
N
MTD
PERT.
INDEX
CHISU
N
PUG
N
MTD
PERT.
INDEX
CHISQ
MULTIPLE TREATMENT
1
2
3
4
5
6
7
•
23
27
26
27
29
2»
29
32
40
39
40
40
40
40
40
41
.57
.69
.65
.67
.72
.72
.72
.•0
0.00
0.00
0.00
0.00
0.00
1.00
• .00
• .ol
25
32
23
25
25
26
29
33
40
40
40
40
40
40
40
40
.63
.80
.57
.63
.63
.70
.72
.«2
.05
.n
.21
.05
.51
o.oo
.06
• .00
27
23
20
23
25
23
21
29
40
40
40
40
40
40
40
40
.67
.57
.50
.57
.63
.57
.52
.72
.48
.72
1.28
.48
.51
1.37
2.61
.28
22
23
26
2t)
30
32
2*
30
40
40
40
40
40
40
40
40
.55
.57
.65
.70
.75
.80
.72
.75
0.00
.72
.05
0.00
0.00
.28
.06
.07
29
27
32
27
30
27
27
26
*o
39
40
40
40
3H
36
36
.72
.(S9
.80
.67
.T5
.71
.75
.72
1.37
.06
1.57
.06
0.00
.01
.no
.?8
-------
Table 32
AVERAGE IMPLANTS PER PREGNANT FEMALE - PARATHION
WEEK
CONTROL
74-01 62.5 H6/K3
74-01 125. M6/l<6
74-01 250. MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1 265/
2 305/
0, 3 268/
VO
4 288/
5 33*/
6 323/
7 323/
8 38l/
23=11.52
27=11.30
26=10.31
27=10.67
29=11.52
29=11.14
29=11.14
32=11.91
266/
337/
257/
277/
299/
322/
322/
393/
25=10.64
32=10.53
23=11.17
25=11.08
25=11.96
28=11.50
29=11.10
33=11.91
314/
250/
246/
2S3/
2B5/
259/
236/
336/
27=11
23=10
.63
.87
20 = 12.30 **I
23=11.00
25=11
23=11
21 = 11
29=u
.40
.26
.24
.59
224/
238/
277/
3H/
339/
331/
339/
355/
22 = 10
23=10
26 = 10
28=11
30=11
32=10
29=11
30 = 11
.18*
.35
.65
.11
.30
.34
.69
.83
316/
293/
348/
266/
356/
273/
30
-------
Table 33
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - PARATHION
WEEK CONTROL 74-01 62.5 MG/KG 7*-01 125. MG/KG 74-01 250. MG/KG TEM .2
• SIGNIFICANT AT P LT 0.05
•• SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
a/
ll/
16/
16/
15/
9/
21/
13/
23=
27=
26=
27=
29=
29=
29=
32=
.35
.41
.62
.59
.52
.31
.72
.41
IS/
14/
7/
9/
13/
10/
15/
13/
25=
32=
23=
25=
25=
28=
29=
33=
.72
.44
.30
.36
.52
.36
.52
.39
ll/
10/
10/
5/
14/
9/
23/
9/
27=
23=
20=
23=
25=
23=
21 =
29=
.41
.43
.50
.22*0
.56
.39
1.10
.31
B/
17/
8/
42/
ll/
14/
22/
6/
22=
23=
26=
28=
30=
32=
29=
30 =
.36
.74
.31
1.50
.37
.44
.76
.20
6?/
77/
87/
ll/
22/
17/
ll/
ll/
29=
27 =
32 =
27 =
30 =
27 =
27=
26=
2.14**
2.85**
2.72**
.41
.73
.63
.41
.42
-------
Table 34
CHI-SQUARE TEST OF THE DEATH INDEX - PARATHION
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-01 62.5 MG/KG
74-01 125. MG/KG
74-01 250. Mb/KG
.? MG/KG
N N
WDI PRG
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHI so
N
WUI
N
PRG
DEATH
INDEX
ChlSU
N N
noi PHG
DEATH
INDEX CHISU
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
8
9
10
11
11
8
12
11
23
27
26
27
29
29
29
32
.35
.33
.38
.41
.38
.28
• 41
.34
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
6
11
7
8
7
9
12
11
25
32
23
25
25
28
29
33
.24
.34
.30
.32
.28
.32
.41
.33
.25
.04
.08
.13
.23
.01
.07
.03
9
8
8
4
9
7
12
9
27
23
20
23
25
23
21
29
.33
.35
.40
.17
.36
.30
.57
.31
.04
.04
.04
2.21
.02
.01
.66
.00
5
7
7
10
e
10
11
5
22
23
26
20
30
32
29
30
.23
.30
.27
.36
.27
.31
.38
.17
.32
.01
.35
.01
.42
.00
0.00
1.70
25 29
26 27
25 32
a ^7
10 30
10 27
9 27
10 26
,«ft 12.49**
,9f, 20.79**
,7R 7.85**
.30 .32
.13 .01
.37 .22
.13 .12
..IB .no
** SIGNIFICANT AT PLT 0.01
-------
WEEK
Table 35
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - PARATHION
CONTROL
74-01 62.5 M6/KS
74-01 125. MS/KG
74-01 250. MS/KG
1EM
.2 MG/KG
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
8/ 265=
ll/ 305=
16/ 268=
16/ 288=
15/ 334=
9/ 323=
21/ 323=
13/ 381=
.03
.04
.06
.06
.04
.03
.07
.03
18/
14/
7/
9/
13/
10/
15/
13/
266=
337=
257=
277=
299=
322=
322=
393=
.07
.04
.03
.03
.04
.03
.05
.03
ll/
10/
10/
5/
14/
9/
23/
9/
314=
250=
246 =
253=
285=
259=
236=
336=
.04
.04
.04
,02*D
.n5
.03
.10
.03
8/
17/
8/
42/
ll/
14/
22/
6/
224=
238=
?77=
311 =
339=
331 =
339=
355=
.04
.07
.03
.14
.03
.04
.06
.02*0
62/
77/
R7/
ll/
2?/
17/
ll/
ll/
316 =
293 =
348 =
266 =
356 =
273=
306 =
322=
.20**
.26**
.25**
.04
.06
.06
.04
.03
* SIGNIFICANT AT f- LT o.os
*• SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 36
CHI-SQUARE TEST OF THE FERTILITY INDEX - PARATHION-METHYL
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-05 20 M6/K6
74-05 40
74-05 80 MO/KG
TEW
.2 MG/KG
N
PRG
N
MTU
PERT.
INDEX
CHISQ
N
PRG
N
MTO
PERT.
INDEX
N
CHlbO PRG
N
MTU
PERT.
INDEX
CHISQ
N
PrtG
N
MTD
PERT.
INDEX
CHISQ
N
PKG
i\
MTD
fERT.
INDEX
CHISQ
MULTIPLE TREATMENT
1
LJ z
3
4
5
6
7
8
23
27
26
27
29
29
29
32
40
39
40
40
40
40
40
40
.57
.69
.65
.67
.72
.72
.72
.80
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
29
33
30
38
32
33
36
32
40
40
40
40
40
40
40
40
.72
.82
.75
.95
.80
.82
.90
.80
1.J7
1.25
.34
8.^1 **I
.18
.65
2.95
.06
24
23
32
26
24
30
32
35
40
40
40
37
40
40
40
40
.60
.57
.80
.70
.60
.75
.80
.88
0.00
.72
1.57
.00
.69
0.00
.28
.37
29
29
2«
26
30
24
33
34
40
40
40
39
40
40
40
40
.72
.72
.70
.67
.75
.60
.82
.85
1.37
.01
.06
.03
0.00
.89
.65
.09
29
27
32
27
30
27
27
26
40
39
40
40
40
38
36
36
.72
.69
.80
.67
.75
.71
.75
.72
1.37
.06
1.57
.06
o.oo
.01
.00
.28
** SIGNIFICANT AT P LT 0.01
I INCREASED ABOVE CONTROL
-------
Table 37
AVERAGE IMPLANTS PER PREGNANT FEMALE - PARATHION-METHYL
MEEK CONTROL
74-05 20 MS/KG
74-05 80 Mb/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1 26S/
2 305/
3 268/
-P-
4 288/
5 334/
6 323/
7 323/
8 381/
23=11.52
27=11.30
26=10.31
27=10.67
29=11.52
29=11.14
29=11.14
32=11.91
301/
347/
306/
417/
389/
366/
401/
347/
29=10
33=10
30=10
38=10
32=12
33=11
36=11
32=10
.38*
.52
.20
.97
.16
.09
.1*
.a**
268X
241/
331/
275/
300/
334/
357/
407/
24=11.17
23=10.48
32=10.34
26=10.58
24=l?.bO
30= 11 ..13
32=11.16
35=11.63
303/
323/
297/
281/
368/
279/
399/
402/
29=10
29=11
28=10
26=10
30=12
24=11
33=12
34=11
.45*
.1*
.61
.81
.27
.62
.09
.82
316/
293/
348/
266/
356/
273/
306/
322/
29=10.90
27=10.85
32=10.87
27=' 9.85
30=11.87
27=10.11
27=11.33
26=12.38
* SIGNIFICANT AT P LT O.OS
** SIGNIFICANT AT f LT 0.01
-------
WEEK
Table 38
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - PARATHION-METHYL
CONTROL
74-ob 20 MG/KG
74-05 40 MG/KU
74-05 80 MG/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
3
Ul
4
5
6
7
8
8/
ll/
16/
16/
is/
9/
21/
13/
23=
27 =
26=
27=
29 =
29 =
29=
32=
.35
.41
.62
.59
.52
.31
.72
.41
9/
10/
B/
IB/
23/
20/
7/
ll/
29 =
33=
30s
38 =
32 =
33=
36s
32=
.31
.30
.27 *D
.47
.72
.61
.19 *D
.34
25/
8/
22/
5/
17/
10/
ll/
18/
24= 1.04 *
23= .J5
32= .bS
26= . j9 *D
24= ./I
30= .J3
32= .34
35= .51
16/
9/
34/
9X
12/
6/
16"/
12/
29 =
29=
28 =
26 =
30 =
24=
33=
34 =
.55
.31
1.21
.35
.40
.25
.48
.35
62/
77/
87/
ll/
22/
17/
ll/
ll/
29 =
27 =
32=
27=
30 =
27=
27=
26=
£.14**
2.85**
2.72**
.41
.73
.63
.41
.42
• SIGNIFICANT AT P LT 0.05
•• SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 39
CHI-SQUARE TEST OF THE DEATH INDEX - PARATHION-METHYL
WEEK
VEHICLE CONTROL
74-05 20
MG/K6
74-Ob 40
MG/Ko
74-05 80
Mb/KG
TEM
.? MG/KG
N
MDI
N
PR6
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHIbQ
N
MDI
N
PRG
DEATH
INDEX .
CrUSQ
N
MDI
N
PRG
DEATH
INDEX
CHISQ
N
MDI
N
PKG
DEATH
INDEX
CHISU
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
8
9
10
11
11
8
12
11
23
27
26
27
29
29
29
32
.35
.33
.38
.41
.38
• 28
.41
.34
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0*00
8
9
8
14
11
11
6
8
29
33
30
38
32
33
36
32
.28
.27
.27
.37
.34
• 33
.17
.25
.07
.05
• «3
.00
.uo
.05
3. '4
.JO
12
6
14
5
10
8
8
7
24
23
32
26
24
30
32
35
.50
.26
.44
.19
.42
.27
.25
.20
.58
.06
.02
1.98
.00
• 05
1-18
1«10
9
a
9
7
10
6
13
a
29
29
28
26
30
24
33
34
.31
.28
.32
.27
.33
• 25
.39
.24
.00
.03
.04
.60
.01
• 01
• 01
.49
25
26
25
a
10
10
4
10
29
27
32
27
30
27
27
26
.86
.96
.78
.30
.33
.37
.33
.38
12.49**
20.79**
7.85**
.32
.01
• 22
• 12
• 00
** SIGNIFICANT AT P LT 0.01
-------
WEEK
Table 40
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - PARATHION-METHYL
CONTROL
74-05 20 MG/KG
74-05 40 MG/KG
74-Ob 80
M(j/KG
TEM
.2 MG/KG
MULTIPLE TREATMENT
1
2
3
O^
4
5
6
7
8
a/
ll/
16/
16/
15/
9/
21/
13/
265 =
305=
268 =
288=
334 =
323=
323 =
381 =
.03
.04
.06
.06
.04
.03
.07
.03
9/
10/
a/
is/
23/
20/
7/
ll/
301 =
347=
306=
417=
389=
366=
401 =
347=
.03
.03
.03
.04
.06
.05
.02*D
.03
25/
8/
22/
5/
17/
10/
ll/
18/
268 =
241 =
331 =
275=
300 =
334 =
357=
407 =
.09*
.03
.07
• l)2*D
.06
.03
.03
• 0*
16/
9/
34/
9/
12/
6/
16/
12/
303=
323=
297 =
281 =
368=
279 =
399=
402=
.05
.03
.11
• 03
.03
.02
.04
.03
62/
77/
87/
ll/
22/
17/
ll/
ll/
316 =
293 =
348 =
266=
356=
273 =
306=
322=
.20**
.26**
.25**
.04
.06
.06
.04
.03
» SIGNIFICANT AT P LT 0.05
•• SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 41
CHI-SQUARE TEST OF THE FERTILITY INDEX - QU1NTOZENE (PCNB)
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-08 1250 MG/KG
74-08 2500 MG/KG
74-08 5000 MG/KG
T€M
.? MB/KG
oo
N
PRG
N
MTO
PERT.
INDEX
CHISQ
N
PRG
N
MTD
PERT.
INDEX
CHISQ
N
PRG
N
MTD
PERT.
INDEX
CHISQ
N
PRS
N
MTO
PERT.
INDEX
CHISQ
N
PRG
N
MTO
PERT.
INDF.X
CHISQ
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
28
26
23
27
24
24
30
27
40
40
40
40
40
38
38
38
.70
.65
.57
.67
.60
.63
.79
.71
0.00
0.00
OiOO
0.00
0.00
0.00
0.00
0.00
29
21
25
33
19
Z3
27
30
40
34
38
38
38
38
38
38
.72
.62
.66
.87
.50
.61
.71
.79
0.00
.00
.27
3.09
.44
0.00
.28
.28
30
22
26
30
28
26
27
31
40
40
38
40
40
40
40
40
.75
.55
.68
.75
.70
.65
.67
.77
.06
.47
.58
.24
.49
.00
.78
.15
31
26
29
28
33
36
28
30
40
40
40
40
40
40
40
40
.77
.65
.72
.70
.82
.90
.70
,75
.26
.05
1.37
0.00
3.91*1
6.47*1
.42
.02
29
27
32
27
30
27
27
26
40
39
40
40
40
38
36
36
.72
.69
.80
.67
• 75
.71
.75
.72
0.00
.03
3.72
.06
1.42
.24
.02
.02
* SIGNIFICANT AT P LT 0.05
I INCREASED ABOVE CONTROL
-------
WEEK
Table 42
AVERAGE IMPLANTS PER PREGNANT FEMALE - QUINTOZENE (PCNB)
CONTROL
74-08 1250 MG/K6
74-08 2500 MG/KG
74-08 5000 MG/KG
1EM
.2 MG/KG
MULTIPLE TREATMENT
1 319/
2 303/
3 245/
4 309/
5 274/
6 302/
7 346/
8 292/
28=11.39
26=11.65
23=10.65
27=11.44
24=11.42
24=12.58
30=11.53
27=10.81
349/
222/
296/
392/
214/
267/
287/
354/
29=12.
21=10.
25=11.
33=11.
19=11.
23=11.
27=10.
30=11.
03
57
84
88
26
61
63*
80
338/
235/
326/
349/
342/
291/
292/
336/
30=11.27
22=10.68
26=12.54**!
30=11.63
28=12.21
26=11.19 **
27=10. «1
31=10.84
361/
259/
353/
317/
376/
400/
302/
333/
31=11.
26= 9.
65
96*
29=12.17*1
28=11.
33=11.
36=11.
28=10.
30=11.
32
39
11*
79
10
316/
293/
34fl/
266/
356/
273/
306/
322/
29=10.90
27=10.85
32=10.8''
27= 9.85*
30=11.87
27 = 10.11 **
27=11.33
26=12.38**!
* SIGNIFICANT AT P LT 0.05
»• SIGNIFICANT AT f LT 0.01
I INCREASED ABOVE CONTROL
-------
WEEK
Table 43
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - QUINTOZENE (PCNB)
CONTHOL
74-08 1250 MG/KG
74-08 2500 MG/KG
74-OB SOOO MG/KG
.2 MO/KG
MULTIPLE TREATMENT
1 13/
2 8/
3 9X
4 2/
5 ll/
6 21/
7 30/
8 19/
28 =
26 =
23=
27=
24 =
24=
30=
27=
.46
.31
.39
.07
.46
.88
1.00
.70
ll/
12/
13/
IS/
10/
e/
ll/
17/
29=
21 =
25=
33=
19=
23=
27=
30=
.38
.57
.52
.45**
.53
.35*D
.41
.67
IZ/
15/
7/
7/
21/
9/
16/
10/
30=
22=
26=
30 =
28=
26=
27=
31 =
.40
.68
.£7
,i3
.75
.35 **D
.59
• 32
ll/
6/
IS/
10/
16/
16/
20/
13/
31 =
26=
29=
38=
33=
36=
28=
30=
.35
.23
.52
.36*
,48
.44*D
.71
.43
62/
77/
87/
ll/
22/
17/
ll/
ll/
29=
27=
32=
27=
30=
27=
27=
36=
2.14 **
2.85 **
2.7? **
.41 *
.73
.63
• 41
.42
• SIGNIFICANT AT f LT 0.05
•• SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 44
CHI-SQUARE TEST OF THE DEATH INDEX - QUIKTOZENE (PCNB)
1 DEGREE OF FREEDOM
WEEK
VEHICLE CONTROL
74-08 1250 MG/KG
74-08 2500 MG/KG
74-08 5000 MG/KG
Mfi/KG
N
WOI
N DEATH
PRG INDEX
CHISQ
N
WOI
N
PRG
DEATH
INDEX
N
CHISQ WOI
N
PRG
DEATH
INDEX
CHISQ
N
WDI
N
PRG
DEATH
INDEX
CHISQ
N N
WDI PrtG
DEATH
INDEX CHISQ
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
10
7
9
2
9
15
8
12
28
26
23
27
24
24
30
27
.36
.27
.39
.07
.38
.63
.27
.44
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
7
10
11
12
9
6
10
13
29
21
25
33
19
23
27
30
.24
.48
.44
.36
.47
.26
.37
.43
.44
1.35
.00
5.44 *
.12
4.91 *D
.31
.03
10
9
6
6
13
8
14
9
30
22
26
30
28
26
27
31
.33
.*!
.23
.20
.46
.31
.52
.29
.01
.51
.82
.97
.14
3.86*D
2.b]
.89
,
2
11
7
13
12
10
12
31
26
29
28
33
36
28
30
.2S
.08
.38
.25
.39
.33
.36
.40
.07
2.15
.04
1.96
.02
3.84*D
.21
.00
25 2t»
26 27
25 32
8 27
10 30
10 27
9 27
10 26
.8ft 13.27**
."6 24.26**
.7B 7.05**
.3" 3.07
.33 .00
.37 2.36
.33 .17
.3fl .03
* SIGNIFICANT AT P LT 0.05
** SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
WEEK
Table 45
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - QUINTOZENE (PCNB)
CONTROL
74-08 1250 MS/K6
74-08 2500 MG/KG
74-08 5000 MG/KG
TEW
.2 MG/KG
MULTIPLE TREATMENT
1 13/
2 8/
3 9/
4 2/
5 ll/
6 21/
7 30/
8 19/
319=
303=
245=
309=
274=
302=
346=
292=
.04
.03
.04
.01
.04
.07
.09
.07
ll/
12/
13/
15/
10/
8/
ll/
17/
349=
222=
296=
392=
214=
267=
287=
354=
.03
.05
.04
.04**
.05
.03*0
.04
.05
12/
IS/
7/
7/
21/
9/
16/
1Q/
338=
235=
326=
349=
342=
291 =
292=
336=
.04
.06
• 02 *D
.02
.06
.03 *D
.05
• 03
ll/
6/
15/
10/
16/
16/
2P/
13/
361 =
259=
353=
317"
376=
400 =
302=
333=
.03
.02
.04
.03*
.04
.04
.07
•04
6?/
77/
B7/
ll/
22/
17/
ll/
ll/
316 =
293=
348 =
266=
356=
273=
306=
322=
.20**
.20*
.25**
.04*
.06
.06
.04
• 03
« SIGNIFICANT AT P LT 0.05
«« SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 46
CHI-SQUARE TEST OF THE FERTILITY INDEX - PHORATE
1 DEGREE OF FREEDOM
KEEK
VEHICLE CONTROL
74-04 5
MQ/KG
74-04 10 MG/KG
74-0* 20 MG/KG
TEM
.? MG/K6
N
PRG
N FERT.
MTD INDEX
CHISU
HHG
N
MTD
FERT.
INDEX CHlbU
N
PRG
N
MTD
FEHT.
INDEX CHlbQ
N
PRG
N
MTD
FERT.
INDEX
N N
CHI5U PRG MTD
PERT.
INDEX
CHISU
MULTIPLE TREATMENT
1
2
3
4
5
6
7
B
23
27
26
27
29
29
29
32
40
39
40
40
40
40
40
40
.57
.69
.65
.67
.72
.72
.72
.60
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
23
30
26
26
25
25
30
26
40
40
40
40
40
40
40
40
.57 .Ub
.75 .10
.65 .05
.65 0.00
.63 .SI
.63 .51
.75 0.00
.65 1.37
21
23
24
26
27
26
29
29
40
40
40
40
40
40
40
40
.52 .05
.57 .72
.60 .05
.65 0.00
.67 .06
.65 .23
.72 .06
.72 .*a
24
29
31
32
31
33
35
30
40
40
40
40
40
40
40
40
.60
.72
.77
.80
.77
.82
.88
.75
0.00 29 40
.01 27 39
.98 32 40
1.03 27 40
.07 30 40
.65 27 38
1.95 27 36
«fl7 ?b 36
.72
.69
.80
.67
.75
.71
.75
.7?
1.37
.06
1.57
.06
0.00
.01
.00
.28
-------
Table 47
AVERAGE IMPLANTS PER PREGNANT FEMALE - PHORATE
HEEK
CONTROL
74-04 5
MG/KG
74-04 10
74-04 20
MO/KG
.2 MG/KG
MULTIPLE TREATMENT
1 265/
2 305/
3 268/
4 288/
5 334/
6 323/
7 323/
8 38l/
23=11.52
27=11.30
26=10.31
27=10.67
29=11.52
29=11.14
29=11.14
32=11.91
241/
326/
299/
291/
278/
305/
336/
31b/
23=10.48*
30=10.87
26=11.50*1
26=11.19
25=11.12
25=12.20*1
30=11.20
26=12*12
221/
255X
257/
277/
312/
295X
317/
335X
21 = 10
23*11
24=10
26=10
27*11
26=11
29= ID
29=11
.52 *
.09
.n
.65
.S6
.35
.93
.55
269/
307/
341/
353/
3t>9/
389/
407/
J24/
24=11
29=10
31 = 11
32=11
31 = 11
33=11
35=11
30=10
.21
.59
.00
.03
.90
."
.63
.80*
316/
293/
34B/
266/
356/
273/
306/
322/
29=10.90
27=10.85
32=10.87
27= 9.85
30=11.87
27=10.11
27=11.33
26=12*38
* SIGNIFICANT AT P LT 0.05
•* SIGNIFICANT AT P LT 0.01
I INCREASED ABOVE CONTROL
-------
Table 48
AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - PRORATE
KEEK CONTROL
74-04 b MG/KG
10
?4-04 20
Mti/KG
TEM
.2 MG/Kt3
MULTIPLE THEATMtNT
1
2
3
4
5
6
7
8
a/
ll/
16/
16/
15/
9/
21/
13/
23=
27 =
26 =
27=,
29 =
29=
29 =
32=
.35
.41
.62
.59
.52
.31
.72
.41
9/
16/
U/
IS/
13/
13/
13/
10/
23=
30 =
26=
26=
25 =
25=
30 =
26=
.39
.53
.50
.58
.«
.52
.43
.38
10/
4/
12/
26/
ll/
7/
22/
26/
21= .48
23= .17
24= .bO
26= 1.00
27= ,»1
26= .27
29= . r 6
29= .90
3/
17/
15/
12/
13/
7/
7/
ll/
24 =
29 =
31 =
32=
31 =
33=
35 =
30=
.13
.59
,4B
.38
.42
.21
.20*0
.37
6?/
77/
87/
ll/
22/
17/
ll/
ll/
29=
27 =
32=
27=
30 =
27=
27=
26=
2.14 **
2.85 **
2.72**
.«!
.73
.63
.*!
.*2
• SIGNIFICANT AT V LT 0.05
«« SIGNIFICANT AT f LT 0.01
D DECREASED BELOW CONTROL
-------
Table 49
CHI-SQUARE TEST OF THE DEATH INDEX - PHORATE
1 DEGREE OF FREEDOM
WEEK
VEHICLE COMTHOL
74-04 5
MS/KG
10
MG/Ko
74-04 20
Mb/HG
TEM
.2 MG/KG
N
WDI
N
PRU
DEATH
INDEX
CHISO
M
N
PMG
DEATH
INDEX CHIiU
N
WDI
N
PRG
DEATH
INDEX
CH1SU
N
401
N
PRG
DEATH
INDEX
CHISU
N N
MOI PNG
DEATH
INDEX
CHISQ
MULTIPLE THEATHENT
1
2
3
4
5
6
7
8
a
9
10
11
11
8
12
11
23
27
26
27
29
29
29
32
.35
.33
.38
.41
.38
.28
.41
.34
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
d
13
7
11
12
10
9
9
23
30
26
26
25
25
30
26
.J5 .10
.43 .«!5
.27 .J5
.42 .03
.48 .22
.40 .46
.30 .41
.35 .07
7
2
12
15
7
7
15
14
21
23
24
26
27
26
29
29
.33
.09
.50
.5B
.26
.27
.52
.48
.05
3.07
.29
.92
.46
.06
.2B
.71
2
13
10
11
11
7
5
B
24
29
31
32
31
33
35
30
• OB
.45
.32
.34
.35
.21
.14
.27
3.45
.37
.04
.05
.01
.OB
4.66*D
.15
25 29
26 27
25 32
8 £7
10 30
10 27
9 til
10 16
.86
.96
.78
.30
.33
.37
.33
.38
12.49 **
20.79**
7.B5 **
.32
.01
.22
.12
.00
* SIGNIFICANT AT P LT 0.05
** SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
KEEK
Table 50
NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - PHORATE
CONTROL
74-0* 5
MG/K6
/4-04 10
74-04 20
MG/KG
.1 MG/NG
MULTIPLE TREATMENT
1
2
3
4
5
6
7
8
8/ 265=
ll/ 305=
16/ 268=
!&/ 288=
IS/ 334=
9/ 323=
21/ 323=
13/ 381=
.03
.04
.06
.06
.04
.03
.07
.03
9/
lb/
13/
15/
13/
13/
13/
10/
241 =
326 =
299=
291 =
278=
30S =
336=
31b =
.04
.05
.04
.05
.05
.04
.04
.03
10/
4/
12/
26/
ll/
7/
22/
26/
221 =
25>5=
257=
*77 =
312=
295=
317 =
33b=
.Ob
.02
.05
.u«
.04
.02
.07
.08
3/
17/
15/
12/
13/
7/
7/
ll/
269=
307=
341 =
3b3 =
369=
3B9 =
407 =
324 =
.01*D
.06
.04
.03
.04
.02
,02*D
.03
62/
77/
87/
ll/
22/
17/
ll/
ll/
316 =
293=
348=
266 =
356=
273 =
306 =
322=
.20 **
.26 **
.25 **
.04
.06
.06
.04
.03
* SIGNIFICANT AT P LT 0.05
•• SIGNIFICANT AT P LT 0.01
D DECREASED BELOW CONTROL
-------
Table 51
DNA REPAIR SYNTHESIS ASSAY OF Monocrotophos
(dpm/^g DNA)
Mono cr o tophb s
Sample
Mean
SD
SE
1
2
3
4
5
6
0
65*
35
39
34
30
20
32
7
6
io-7
46
42
39
34
26
— f
38
7
3
io-6
31
9*
25
25
46
— f
32
10
5
(M)
io-5
54
46
40
40
53
— *
47
7
3
10- 4
70
46
36
40
64
37
50
14
6
io-3
64
86
69
51
64
92
71
15
6
4NQO (M)
10- s
1354
1273
1308
975
972
1135
1169
168
69
Sample deleted from calculations because of low DNA value.
Only five samples used.
Cell culture and experimental conditions
T-25 flask cultures of passage 24 WI-38 cells were initiated in medium con-
taining 10% serum. The medium was replaced with medium containing 0.5$
serum on day 5 following initiation and subsequently on days 11 and 15.
The assay was conducted on day 22.
Hydroxyurea (10"2M) preincubation = 1 hour.
Compound exposure time = 3 hours.
3H-TdR added with compound.
3H-TdR incorporation = 1 ^Ci/ml (S.A. =6.7 Ci/mmole), 3 hours.
Postincorporation incubation = medium containing TdR, 3/4 hour.
Cells were removed with IN NaOH, 1 minute, 70°C.
DNA was extracted by the PCA-hydrolysis procedure and measured following
reaction with diphenylamine.
Negative control and compound solvent =0.5$ EtOH.
78
-------
Table 52
DNA REPAIR SYNTHESIS ASSAY OF Monocrotophos
WITH METABOLIC ACTIVATION
(dpm/p,g DNA)
Monocrotophos (M) DMN (M)
0^_ IP"4 10-3 10-2 5 x 10"2
Sample 1 55 67 43 87 206
2 54 66 48 84 220
3 52 43 52 82 223
Mean 54 59 48 84 216
SD 2 14 4 2 9
SE 1821 5
Cell culture and experimental conditions
T-25 flask cultures of passage 24 WI-38 cells were initiated in medium
containing 10% serum. The medium was replaced with medium contain-
ing 0-5% serum on day 4 following initiation and subsequently on
day 10. The assay was conducted on day 16.
Hydroxyurea (10~2M) preincubation = 1 hour.
Compound exposure time = 1 hour, with the 9,000 g fraction of a mouse
liver homogenate.
3H-TdR added with compound.
3H-TdR incorporation = 1 ^Ci/ml (S.A. =6.7 Ci/mmole), 4 hours.
Postincorporation incubation = medium containing TdR, £ hour.
Cells were removed with IN NaOH, 10 minutes, 22°C.
DNA was extracted by the PCA-hydrolysis procedure and measured follow-
ing reaction with diphenylamine.
Negative control and compound solvent =0.5% EtOH.
79
-------
Table 53
DNA REPAIR SYNTHESIS TESTING
OF BROMACIL
(dpm/yg DNA)
Bromacil (M)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
195
153
212
230
217
298
218
48
19
10-7
207
212
156
187
182
251
199
32
13
10-6
201
215
121
218
184
220
193
38
15
T0~ **
248
178
179
231
240
178
209
34
14
1(r«, t
148
144
152
204
144
200
165
28
12
ID' 3 f
129
137
105
107
80
74
87
50
20
4NQO (M)
ID'5
2670
2850
2688
2702
2438
2662
2668
134
55
Negative control and compound solvent = 0.5% DMSO.
Slight precipitate observed at 10"3 M and 10"1* M
80
-------
Table 54
DNA REPAIR SYNTHESIS ASSAY OF BROMACIL
WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Sample
1
2
3
4
5
6
Mean
SD
SE
Bromacil (M)
0
113
102
141
158
218
136
145
41
17
io-7
191
112
174
189
152
170
165
30
12
10- 6
91
117
110
82
104
96
100
13
5
io-5
102
102
102
91
126
190
119
37
15
10-"
120
110
149
135
167
165
141
23
9
10~ 3
161
178
131
185
141
133
155
24
10
DMN (M)
5 X'10-2
400
397
_*
529
645
448
484
105
50
* Sample lost.
81
-------
Table 55
DNA REPAIR SYNTHESIS ASSAY
OF CACODYLIC ACID
DMA)
Cacodylic Acid (M)
4NQO (M)
Sample
Mean
SD
SE
1
2
3
4
5
6
0*
61
31
32
18
25
35
34
15
6
io-7
41
48
59
32
39
22
40
13
5
io-6
47
27
25
38
— *
__*
34
10
5
io-5
__*
__*
63
68
23
29
46
23
11
10- 4
31
27
30
29
22
28
28
3
1
io-3*
36
19
45
19
38
36
32
11
4
10- 5
1891
1681
2418
2245
1430
2275
1990
387
158
•si-
Negative control and compound solvent = 0.5% DMSO.
t Slight lowering of pH at 10~3 M.
+ Sample lost.
82
-------
Table 56
DNA REPAIR SYNTHESIS ASSAY OF CACODYLIC ACID
WITH METABOLIC ACTIVATION
DNA)
Cacodylic Acid (M)
Sample 1
2
3
Mean
SD
SE
0*
44
30
23
33
11
6
io-5
25
33
39
32
7
4
io-4
21
22
28
24
4
2
io-3
29
25
21
25
4
2
DMN (M)
5 X 10~2
381
384
339
368
25
15
Negative control and compound solvent =0.5% EtOH.
83
-------
Table 57
DNA REPAIR SYNTHESIS ASSAY OF CAPTAN
(dpm/^g DNA)
Sample 1
2
3
4
5
6
Mean
SD
SE
0*
37
64
76
76
63
66
64
14
6
io-8
43
58
50
60
61
44
51
8
3
Captan
io-7
41
53
68
73
56
66
59
12
5
(M)
io-6
74
60
52
37
50
82
59
16
7
io-5
81
81
57-
51
72
65
68
12
5
io-4
8
6
7
5
5
6
6
1
0.4
4NQO(M)
10'5
924
947
1106
801
760
884
904
122
50
Negative control and compound solvent =0.5$ DMSO.
84 ,
-------
Table 58
DNA REPAIR SYNTHESIS ASSAY OF CAPTAN
WITH METABOLIC ACTIVATION
DNA)
Captan (M)
Sample 1
2
3
Mean
SD
SE
0*
30
40
__t
35
7
5
io-5
53
50
48
50
2
1
10- 4
89
73
71
77
9
5
io-3
7
5
5
6
1
0.6
DMN (M)
5 X IO-2
-t
323
384
353
43
31
•si-
Negative control and compound solvent =0.5% DMSO.
Sample lost.
85
-------
Table 59
DNA REPAIR SYNTHESIS ASSAY OF CHLOROPYRIFOS
(dprn/yig/DNA)
Chloropyrifos
Sample
Mean
SD
SE
1
2
3
4
5
6
DMSO.
Sample deleted from calculations because of low DNA value.
Sample lost.
86
-------
Table 60
DNA REPAIR SYNTHESIS ASSAY OF CHLOROPYRIFOS
WITH METABOLIC ACTIVATION
DNA)
Chloropyrifos (M)
Sample 1
2
3
Mean
SD
SE
0*
72
75
55
67
10
6
10~5
79
65
67
70
8
5
io-4
70
75
63
69
6
4
io-3
52
71
79
67
14
8
DMN (M)
5 X IO-2
355
349
384
363
18
11
Negative control and compound solvent = 0.5$> DMSO.
87
-------
Table 61
DEN REPAIR SYNTHESIS ASSAY
OF DINOSEB
(dprn/^g DNA)
Dinoseb (M)
Sample
Mean
SD
SE
1
2
3
4
5
6
JL*
115
143*
96
72
97
78
92
17
7
10-7
101
101
112
61
57
58
82
26
11
10-9
67
68
54
58
63
73
64
7
3
10-5
103
100
116
62
60
60
84
25
10
ip-'t
106
100
79
66
67
76
82
17
7
4NQO (M)
10" 5
1337
__ §
1721
1220
1208
1209
1339
220
98
Negative control and compound solvent =0.5$ DMSO.
Suggestion of precipitate at lO"4 M.
Sample deleted from calculations because of low DNA value.
§ Sample lost.
88
-------
Table 62
DNA REPAIR SYNTHESIS ASSAY OF DINOSEB
WITH METABOLIC ACTIVATION
DNA)
Sample 1
2
3
Mean
SD
SE
0*
72
75
55
67
10
6
Dinoseb
10~5
93
81
51
75
22
12
(M)
10- 4
76
80
58
71
12
7
10-3
71
64
89
74
13
8
DMN (M)
5 X ID"2
355
349
384
363
18
11
Negative control and compound solvent =0.5$ DMSO.
89
-------
Table 63
DNA REPAIR SYNTHESIS ASSAY OF DSMA
(dprn/jig DNA)
Sample 1
2
3
4
5
6
Mean
SD
SE
0*
67
58
44
55
79
105
68
22
9
io-7
51
54
36
36
62
32
45
12
5
DSMA
IO"6
79
67
99
400 t
74
75
79
12
5
io-5
86
64
64
45
53
107
70
23
9
(M)
io-4
100
155
35
44
55
89
80
45
18
10- 3
62
49
58
59
68
69
61
7
3
Negative control and compound solvent = H20.
Sample deleted from calculations because of low DNA value.
4NQO (M)
1 O™
902
1241
1380
990
1087
971
1095
182
74
90
-------
Table 64
DNA REPAIR SYNTHESIS ASSAY OF DSMA
WITH METABOLIC ACTIVATION
DNA)
Sample 1
2
3
Mean
SD
SE
0*
44
30
23
33
11
6
DSMA
io-5
28
36
21
29
8
4
io-4
25
29
32
29
4
2
(M)
io-3
38
36
28
34
5
3
DMN (M)
5 X 10" 2
381
384
339
368
25
15
Negative control and compound solvent =0.5% EtOH.
91
-------
Table 65
DM REPAIR SYNTHESIS ASSAY
OF FENTHION
DNA)
Fenthion (M)
Sample
Mean
SD
SE
1
2
3
4
5
6
0*
89
43
107
62
61
94
76
24
10
io-6
65
105
83
46
34
51
64
26
11
io-5
154
337*
85
54
102
44
88
44
18
io-4
37
34
40
63
31
— §
41
13
5
10-3t
64
67
36
63
44
33
51
15
6
4NQO (M)
io-5
2983
2272
2552
4059
1728
1893
2583
857
350
Negative control and compound solvent =0.5$ EtOH.
Precipitate observed at 10~3 M.
v Sample deleted from calculations because of low DNA value.
§ Sample lost.
92
-------
Table 66
DMA REPAIR SYNTHESIS ASSAY OF FENTHION
WITH METABOLIC ACTIVATION
DNA)
Fenthion (M)
Sample 1
2
3
Mean
SD
SE
0*
55
54
52
54
2
1
io-5
54
50
42
48
6
4
10-*
60
46
64
57
10
6
io-3
51
64
63
60
7
4
DMN (M)
5 X 10~2
206
220
223
216
9
5
Negative control and compound solvent = 0.5$> EtOH.
93
-------
Table 67
DMA REPAIR SYNTHESIS ASSAY OF FOLPET
DNA)
Sample
Mean
SD
SE
1
2
3
4
5
6
0*
37
64
76
76
63
66
64
14
6
10-8
43
58
91
83
107
60
73
24
10
Folpet
io-7.
63
62
108
91
72
84
80
18
7
(M)
10~6
45
54
52
92
70
104
71
25
10
10- 5
82
58
92
65
85
107
82
18
7
.10-*
29
25
31
26
31
29
28
2
1
4NQO(M)
10" 5
924
947
1106
801
760
884
904
122
50
Negative control and compound solvent =0.5$ DMSO.
94
-------
Table 68
DNA REPAIR SYNTHESIS ASSAY OF FOLPET
WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Folpet (M) DMN (M)
0* 10"5 10" ** 10"3 5 X 10~2
Sample 1 30 49 63 49 —f
2 40 54 82 40 323
3 —f 54 98 58 384
Mean 35 52 81 49 353
SD 7 3 18 9 43
SE 5 2 10 5 31
Negative control and compound solvent = 0.5% DMSO
T Sample lost.
95
-------
Table 69
DNA REPAIR SYNTHESIS ASSAY
OF AZINPHOS-METHYL
DNA)
Azinophos-methyl (M)
Sample
Mean
SD
SE
1
2
3
4
5
6
0*
102
99
77
97
85
111
95
12
5
io-7
145
115
96
125
93
77
108
25
10
io-e
99
103
71
100
__§
72
89
16
7
io-5
264*
99
82
80
79
56
79
15
7
io-4
51
105*
55
85
57
68
63
14
6
10-st
55
39
33
34
97
35
49
25
10
4NQO (M)
io-5
804
924
629
856
761
897
822
87
36
Negative control and compound solvent = 0.5$> DMSO.
t Precipitate observed at 10~3 M.
t Sample deleted from calculations because of low DNA value.
§ Sample lost.
96
-------
Table 70
DNA REPAIR SYNTHESIS ASSAY
OF AZINPHOS-METHYL WITH METABOLIC ACTIVATION
(dpm/yg DNA)
Sample 1
2
3
Mean
SD
SE
0*
30
40
— f
35
7
5
io-5
70
66
78
71
6
3
io-4
75
65
41
60
18
10
io-3
42
55
54
50
8
4
DMN (M)
5 X }0-a
~t
323
384
353
43
31
*
Negative control and compound solvent = 0.5% DMSO.
Sample lost.
97
-------
Table 71
DNA REPAIR SYNTHESIS ASSAY OF MALATHION
(dpm/yg DNA)
Malathion (M)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
123
125
106
100
114
138
118
14
6
io-7
110
111
86
133
116
110
111
15
6
10~6
128
124
130
116
127
143
128
9
4
10- 5
144
78
74
119
132
127
112
29
12
io-"
90
126
67
113
91
156
107
31
13
10-3
33
34
23
44
39
40
35
7
3
4NQO (M)
1943
1626
1538
1264
1737
1651
1626
225
92
Negative control and compound solvent =0.5% EtOH.
98
-------
Table 72
DNA REPAIR SYNTHESIS ASSAY OF MALATHION
WITH METABOLIC ACTIVATION
(dprn/jjig-DNA)
Ma lathi on (M)
Sample 1
2
3
Mean
SD
SE
*
0
55
54
52
54
2
1
10-5
48
49
62
53
8
5
10-4
46
52
55
51
5
3
io-3
38
48
37
41
6
4
DMN (M)
5 X IO-2
206
220
223
216
9
5
*
Negative control and compound solvent =0.5$ EtOH.
99
-------
Table 73
DNA REPAIR SYNTHESIS ASSAY
OF METHOMYL
DNA)
Methomyl (M)
Sample
Mean
SD
SE
1
2
3
4
5
6
*
0
135
133
165
72
104
103
118
32
13
io-7
95
— f
100
98
85
95
94
6
3
io-6
116
133
129
97
103
93
112
17
7
10~5
117
108
97
115
116
139
115
14
6
*o-<
126
116
122
109
109
130
118
9
4
io-a
88
69
69
69
70
61
71
9
4
4NQO (M)
10- 5
1442
1544
1518
1385
1423
— f
1462
67
30
Negative control and compound solvent =0.5$ DMSO.
Sample lost.
100
-------
Table 74
DMA REPAIR SYNTHESIS ASSAY OF METHOMYL
WITH METABOLIC ACTIVATION
DNA)
Sample 1
2
3
Mean
SD
SE
.2!
44
30
23
33
11
6
10~s
26
25
22
25
2
1
io-4
22
20
26
23
3
2
io-3
24
25
30
26
3
2
Methomyl (M) _ DMN(M)
5 X IP"2
381
384
339
368
25
15
*
Negative control and compound solvent =0.5$ EtCH.
101
-------
Table 75
DNA REPAIR SYNTHESIS ASSAY
OF MONURON
DNA)
Monuron
4NQO (M)
Sample 1
2
3
4
5
6
Mean
SD
SE
0*
135
133
165
72
104
103
118
32
13
io-7
113
__*
129
__*
118
131
123
8
4
io-6
86
93
89
83
92
110
92
9
4
io-5
97
88
77
75
77
109
87
14
6
io-4
83
72
81
82
84
85
81
5
2
10- 3"
48
47
46
49
45
38
46
4
1
,-s
10
1442
1544
1518
1385
1423
—••
1462
67
30
Negative control and compound solvent =0.5% DMSO.
Precipitate observed at 10"3 M.
t Sample lost.
102
-------
Table 76
DNA REPAIR SYNTHESIS ASSAY OF MONURON
WITH METABOLIC ACTIVATION
DNA)
Monuron (M)
Sample 1
2
3
Mean
SD
SE
0*
30
40
~'r
35
7
5
10~s
78
88
92
86
7
4
io-4
81
68
63
71
9
5
io-3
74
74
88
79
8
5
DMN (M)
5 x 10~2
-t
323
384
353
43
31
-it-
Negative control and compound solvent = 0.5$> DMSO.
Sample lost.
103
-------
Table 77
DNA REPAIR SYNTHESIS ASSAY OF MSMA
DNA)
MSMA (M)
Sample
Mean
SD
SE
1
2
3
4
5
6
0*
67
58
44
55
79
105
68
22
9
10-'
38
84
114
235t
75
53
73
29
13
io-6
108
93
68
50
66
__*
77
23
9
io-5
60
53
61
63
75
50
60
9
4
io-4
93
52
66
66
48
59
64
16
7
io-3
68
39
61
67
60
75
62
12
5
4NQO (M)
10~5
902
1241
1380
990
1087
971
1095
182
74
Negative control and compound solvent = H2O.
Sample deleted from calculations because of low DNA value.
Sample lost.
104
4
-------
Table 78
DNA REPAIR SYNTHESIS ASSAY OF MSMA
WITH METABOLIC ACTIVATION
DNA)
MSMA (M)
Sample 1
2
3
Mean
SD
SE
0*
44
30
23
33
11
6
io-5
27
25
21
24
3
2
io-4
21
35
34
30
8
5
io-3
32
25
24
27
4
3
DMN (M)
5 X NT"
381
384
339
368
25
15
*
Negative control and compound solvent = 0.5% EtOH.
105
-------
Table 79
DNA REPAIR SYNTHESIS ASSAY
OF PARATHION
(dpm/yg DNA)
Parathion (M)
Sample
1
2
3
4
5
6
Mean
SD
SE
0*
123
125
106
100
114
138
118
14
6
io-7
127
129
135
124
137
145
133
7
3
10- 6
151
155
135
200
160
210
169
29
12
IO-5
221
129
157
137
155
126
154
35
14
10- •»
102
94
83
72
93
71
86
13
5
10- 3
90
104
93
116
102
93
100
10
4
4NQO (M)
10- 5
1943
1626
1538
1264
1737
1651
1626
225
92
* Negative control and compound solvent =0.5% EtOH.
106
-------
Table 80
DNA REPAIR SYNTHESIS ASSAY OF PARATHION
WITH METABOLIC ACTIVATION
(dpm/|j,g DNA)
Parathion (M)
Sample 1
2
3
Mean
SD
SE
0*
44
30
23
33
11
6
io-5
33
33
35
34
1
1
10- 4
42
32
30
35
6
4
10- 3
20
23
30
24
5
3
DMN (M)
5 X ID"2
381
384
339
368
25
15
•it-
Negative control and compound solvent =0.5$ EtOH.
107
-------
Table 81
DM REPAIR SYNTHESIS ASSAY
OF PARATHION-METHYL
(dpm/jig DNA)
Parathion-Methyl (M)
Sample
Mean
SD
SE
1
2
3
4
5
6
0*
36
34
86
94
53
112*
61
28
13
io-7
36
38
49
56
49
46
46
8
3
10~6
33
32
28
27
35
41
33
5
2
ID"5
34
55
31
52
43
85
50
20
8
10- 4
44
44
53
41
40
44
44
5
2
io-3t
40
23
28
29
29
27
28
6
3
4NQO (M)
10- 5
411*
781
782
858
1296
1103
964
227
102
Negative control and compound solvent = 0.5% EtOH.
t a
Precipitate observed at 1Q~* M.
* Sample deleted from calculations because of low DNA value.
108
-------
Table 82
DNA REPAIR SYNTHESIS ASSAY OF PARATHION-METHYL
WITH METABOLIC ACTIVATION
DNA)
Parathion-Methyl (MJ
Sample 1
2
3
Mean
SD
SE
0*
55
54
52
54
2
1
io-5
44
54
57
52
6
4
10-"
65
52
37
51
14
8
io-3
51
48
45
48
3
2
DMN (M)
5 X 10~a
206
220
223
216
9
5
Negative control and compound solvent = 0.5$ EtOH.
109
-------
Table 83
DNA REPAIR SYNTHESIS ASSAY OF QUINTOZENE (PCNB)
(dpm/^g DNA)
PCNB (M)
Sample 1
2
3
4
5
6
Mean
SD
SE
_£
61
31
32
18
25
35
34
15
6
io-7
35
43
44
30
38
20
35
9
4
10-e
33
22
31
30
27
20
27
5
2
io-5
21
21
23
16
32
39
25
8
3
io-4
27
18
18
18
22
20
20
4
2
. io-3t
23
37
19
21
27
27
26
6
3
•K-
Negative control and compound solvent =0.5$ DMSO.
Precipitate observed at 10~3 M.
4NQO (M)
1891
1681
2418
2245
1430
2275
1990
387
158
110
-------
Table 84
DNA REPAIR SYNTHESIS ASSAY OF QUINTOZENE (PCNB)
WITH METABOLIC ACTIVATION
DNA)
PCNB (M)
Sample 1
2
3
Mean
SD
SE
0*
72
75
55
67
10
6
io-5
75
79
84
79
5
3
10- 4
95
71
73
80
13
8
1Q-3
106
72
49
76
29
17
DMN (M)
5 X 10-2
355
349
384
363
18
11
Negative control and compound solvent =0.5% DMSO.
Ill
-------
Table 85
DNA REPAIR SYNTHESIS ASSAY OF PHORATE
(dpm/^g DNA)
Phorate. (M)
Sample 1
2
3
4
5
6
Mean
SD
SE
•if.
0
36
34
86
94
53
112*
61
28
13
io-7
58
45
31
26
40
56
43
13
5
io-6
38
44
107
__§
44
26
52
32
14
1Q-5
25
17
22
43
52
77
39
23
9
io-4
27
50
42
43
37
39
40
8
3
io-3
56
45
55
50
61
59
55
6
2
4NQO (M)
10~5
411*
781
782
858
1295
1103
964
227
102
Negative control and compound solvent = 0.5$> EtOH.
Precipitate observed at 10~3 M.
* Sample deleted from calculations because of low DNA value.
§ Sample lost.
112
-------
Table 86
DNA REPAIR SYNTHESIS ASSAY OF PRORATE
WITH METABOLIC ACTIVATION
DNA)
Phorate. (M)
Sample 1
2
3
Mean
SD
SE
0*
55
54
52
54
2
1
io-5
45
59
63
55
10
6
io-4
43
41
39
41
2
1
io-3
37
35
38
37
1.4
0.8
DMN (M)
5 X 10~8
206
220
223
216
9
5
it-
Negative control and compound solvent = 0.5^> EtOH.
113
-------
Table 87
DNA REPAIR SYNTHESIS ASSAY
OF SIMAZINE
(dpm/ug DNA)
Simazine (M) 4NQO (M)
Q* 10" 7 10"6 10"5 10"^ 10"3 10"5
Sample
1 195 165 151 195 177 369 2670
2 153 91 171 190 193 208 2850
3 212 131 152 312 253 233 2688
4 230 138 146 290 281 165 2702
5 217 152 179 237 166 205 2435
6 298 113 213 305 161 f 2662
Mean 218 132 169 255 205 236 2668
SD 48 27 25 55 50 78 134
SE 19 11 10 22 20 32 55
* Media control and compound solvent =0.5% DMSO.
t Sample lost.
114
-------
Table 88
DNA REPAIR SYNTHESIS ASSAY OF SIMAZINE
WITH METABOLIC ACTIVATION
DNA)
Simazine (M) DMN (M)
Sample 1
2
3
Mean
SD
SE
*
0
72
75
55
67
10
6
io-»
57
58
64
60
4
2
io-»
64
60
61
62
2
1
10- 3
59
58
76
64
10
6
5 X 10"a
355
349
384
363
18
11
Negative control and compound solvent = 0.5^> DMSO.
115
-------
Table 89
DNA REPAIR SYNTHESIS ASSAY
OF TRIFLURALIN
(dpm/^g OKA)
Trifluralin (M)
Sample
Mean
SD
SE
1
2
3
4
5
6
0*
56
68
51
45
50
29
50
13
5
io-7
42
29
78
51
47
41
48
17
7
io-6
26
—t
48
76
79
59
58
22
10
io-'
53
152*
68
89
97
57
73
19
9
10-*
51
97
158*
78
56
43
62
28
12
io-3
71
123
112
83
80
53
87
26
11
4NQO (M)
1 0""
639
1125
570
894
663
986
812
222
91
Negative control and compound solvent = 0.5$ EtCH.
Sample lost.
$
Sample deleted from calculations because of low DNA value.
t
116
-------
Table 90
DNA REPAIR SYNTHESIS ASSAY OF TRIFLURALIN
WITH METABOLIC ACTIVATION
DNA)
Trifluralin (M)
Sample 1
2
3
Mean
SD
SE
0*
72
75
55
67
10
6
io-B
67
58
74
66
8
5
io-4
79
64
79
74
9
5
io-3
51
61
-J
56
7
5
DMN (M)
5 X 10~a
355
349
384
363
18
11
*
Negative control and compound solvent =0.556 DMSO.
Sample lost.
117
-------
Table 91
IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM
Compound
Negative control
Positive control, 4-o-tolylazo-l-toluidine
£ Monocrotophos
00
Metabolic
Activation
+
-
+
-
-
-
-
-
-
-
+
+
+
+
+
+
+
pg Compound
Added/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revert ants /Plate
TA100
95
113
87
101
93
107
97
95
126
101
89
75
79
71
78
113
TA1535
13
13
9
22
14
23
13
17
14
16
20
16
14
16
19
18
TA1537
8
10
10
9
9
7
11
10
10
14
13
12
10
15
9
10
TA1538
7
10
6
183
11
10
13
10
9
7
6
12
12
11
10
11
15
10
Monocrol
1 T
O
•a
o
CD
-------
Table 91 (continued)
Average Number of
Metabolic p,g Compound Histidine-Positive Revertants/Plate
Compound Activation Added/Plate
Negative control
Positive controls
g-Propiolactone - 50 ^1
AF-2 - 0.05
2-Anthramine - 50
j. 50
Bromacil - 1
5
10
50
100
500
1000
+ 1
+ 5
+ 10
+ 50
+ 100
4. 500
TA100
145
154
372
120
129
123
117
136
140
101
118
131
157
136
138
145
162
TA1535
22
25
756
23
17
31
29
40
30
14
35
28
33
26
30
21
8
TA1537
25
24
24
13
22
16
18
15
6
21
16
21
14
12
20
5
TA1538
16
30
63
338
26
14
13
15
19
15
11
16
20
19
13
15
20
16
1000 162 8 5 16 Cd
H
§
»
o
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Cacodylic Acid
to
o
pg Compound
Added/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
56
72
48
42
42
39
43
44
44
69
53
64
50
64
54
59
TA1535
15
14
17
15
12
18
22
15
16
17
15
16
15
18
21
14
TA1537
12
9
15
15
15
10
11
11
8
14
15
12
11
15
14
13
TA1538
7
15
10
150
5
11
8
8
9
9
8
18
8
18
12
19
15
13
Cacod
H
O
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Captan
Hg Compound
Added/Plate
2
2
1
5
10
15
25
50
1
5
10
15
25
50
Average Number of
Histidine-Positive Revertants/Plate
TA100
72
98
211
532
822
820
720
Killing
141
210
285
340
330
704
TA1535
18
14
3
100
29
80
76
104
80
Killing
20
60
113
55
71
143
TA1537
7
3
2
5
0
0
0
0
2
2
2
0
0
1
TA1538
8
25
7
14
16
26
6
22
19
22
26
21
46
44
£>
•o
ft
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Chloropyrifos
Mg Compound
Added/Plate
N3
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
92
80
66
92
65
88
67
87
79
67
71
87
77
77
72
77
TA1535
18
14
20
22
14
26
• 20
17
13
11
9
11
16
11
13
14
TA1537
12
16
12
15
21 -
15
17
18
20
15
15
15
18
14
14
11
TA1538
16
16
15
168
22
28
24
25
22
17
22
14
16
20
13
16
30
22
n
O
•e
o
CO
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Dinoseb
u>
yg Compound
Added/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
97
80
69
59
57
67
82
91
Killing
79
82
94
83
87
104
TA1535
15
15
12
12
17
17
12
7
Killing
13
14
12
14
13
12
TA1537
12
16
21
16
18
17
16
17
Killing
19
18
17
15
17
10
TA1538
21
17
• 15
168
15
14
20
17
15
19
Killing
15
14
15
17
14
7
Killing Killing Killing Killing
§
w
ID
o-
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
DSMA
Hg Compound
Added/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
56
72
50
56
51
66
71
53
43
54
85
50
55
50
60
53
TA1535
15
14
12
10
20
15
16
13
12
22
7
16
17
13
15
14
TA1537
12
9
7
10
10
11
7
12
7
8
8
—
11
9
5
2
TA1538
7
15
- 10
250
4
5
3
8
8
7
9
8
15
3
5
7
10
8
o
en
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toiuidine
Azinphos-methyl
NJ
Ul
yg Compound
Added/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
101
102
66
74
74
73
75
107
104
76
69
68
81
65
84
119
TA1535
26
26
39
22
23
30
49
30
31
23
23
24
22
24
30
24
TA1537
6
3
4
3
2
3
4
2
3
1
2
3
3
2
0
0
TA1538
13
24
13
78
10
11
9
11
10
10
13
20
25
28
21
19
24 '
23
g"
01
I
n>
ct
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Fenthion
fO
ov.
yg Compound
Added/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
94
80
64
97
105
112
100
107
90
114
97
81
90
98
83
89
TA1535
36
20
31
34
32
36
38
42
32
15
17
9
16
14
22
20
TA1537
10
9
8
11
15
15
16
10
6
10
12
9
12
7
8
10
TA1538
12
12
15
168
13
17
12
14
14
14
12
12
10
17
21
13
10
15
o
3
-------
Table 91 (continued)
Compound
Metabolic
Activation
yg Compound
Added/Plate
Average Number of
Histidine-Positive Revertants/Plate
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Folpet
ISJ
25
25
1
5
10
25
50
100
500
1000
1
5
10
25
50
100
500
1000
TA100
72
93
127
150
244
300
550
286
Killing
Killing
112
173
241
420
720
532
Killing
Killing
TA1535
19
15
20
35
39
48
111
110
Killing
Killing
20
51
79
70
218
216
Killing
Killing
TA1537
3
7
7
0
1
0
2
0
0
0
2
1
5
6
10
3
0
0
TA1538
8
20
6
183
5
8
11
14
7
2
Killing
Killing
30
26
35
36
45
48
Killing
Killing
o
•0
-------
Table 91 (continued)
Compound
Metabolic
Activation
yg Compound
Added/Plate
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Malathion
N3
00
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
89
92
54
48
85
99
81
82
61
65
61
99
92
75
90
66
TA1535
10
10
8
7
7
8
7
12
10
5
6
9
8
9
8
7
TA1537
8
10
11
12
10
6
7
5
7
6
8
7
7
6
12
10
TA1538
7
7
6
183
3
3
5
7
5
7
4
9
5
4
6
5
4
4
ff
M
01
rt
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Methomyl .
yg Compound
Added/Plate
Average Number of
Histidine-Positive Revertants/Plate
NJ
VO
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
TA100
128
149
123
112
98
109
110
119
105
145
115
126
129
132
133
122
TA1535
18
14
19
20
16
18
21
17
13
12
10
12
10
13
10
14
TA1537
33
20
28
35
28
27
34
23
24
18
18
21
19
20
20
24
TA1538
17
22
17
206
14
10
18
14
24
26
21
15
15
13
20
19
18
14
8s
rt
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Monuron
yg Compound
Added/Plate
Average Number of
Histidine-Positive Revertants/Plate
u>
o
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
TA100
128
149
126
- 98
108
122
114
122
125
125
142
119
116
108
104
123
TA1535
17
12
15
24
17
19
19
20
22
10
13
17
15
15
15
11
TA1537
17
15
15
12
12
11
15
18
14
15
15
13
19
16
15
15
TA1538
17
22
6
177
19
18
22
21
29
29
19
21
15
18
21
19
12
17
g
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
MSMA
yg Compound
Added/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
56
72
79
69
62
52
41
53
48
79
64
65
67
53
66
68
TA1535
15
14
15
15
14
17
15
17
13
11
15
7
7
12
14
10
TA1537
12
9
7
13
11
11
12
8
12
11
8
10
14
8
7.
10
TA1538
7
15
10
250
6
4
6
6
7
5
5
10
10
9
7
8
8
10
-------
Table 91 (continued)
Compound
Metabolic
Activation
yg Compound
Added/Plate
Negative control
Positive control, 4-o-tolylazo-o-toluidine
ParathioE
Co
N>
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
95
114
94
138
85
98
87
110
107
56
75
69
76
88
105
103
TA1535
19
21
12
12
13
13
15
13-
14
11
16
14
14
17
15
12
TA1537
6
13
7
7
4
4
4
4
3
12
7
5
6
7
8
6
TA1538
6
7
6
177
8
7
7
8
6
8
13
15
15
19
8
5
9
12
o
3
-------
Table 91 (continued)
Compound
Metabolic
Activation
Vig Compound
Added/Plate
u>
OJ
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Parathion-methyl
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
84
77
64
75
86
75
72
78
46
61
53
60
69
66
67
56
TA1535
10
10
12
9
8
10
9
7
3
4
11
8
10
10
6
4
TA1537
10
6
6
7
8
8
9
7
5
8
9
9
12
4
6
7
TA1538
7
7
6
177
8
8
9
13
10
20
18
14
10
8
11
13
14
16
p>
ft
H>
O
1
rt
-------
Table 91 (continued)
Compound
Metabolic
Activation
Ug Compound
Added/Plate
Average Number of
Histidine-Positive Revertants/Plate
OJ
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Quintozene (PCNB)
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
TA100
96
101
99
85
90
87
94
100
107
90
95
91
100
87
119
106
TA1535
33
24
32
29
34
30
37
27
35
20
25
18
18
23
22
30
TA1537
9
6
6
7
6
6
5
5
6
9
7
6
3
4
1
1
TA1538
(3
25,
6
177
15
12
13
7
11
14
11
25
27
29
25
20
26
27
.0
Lntozeni
o
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Phorate
LO
Ln
yg Compound
Added/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
96
118
70
65
85
65
72
70
58
101
103
79
89
79
59
70
TA1535
15
17
17
15
17
17
11
14
14
14
11
.13
15
15
16
19
TA1537
8
11
8
8
7
5
7
6
7
11
8
10
9
6
6
8
TA1538
8
11
6
177
16
11
11
8
9
6
9
15
10
12
11
7
11
5
s1
n
pi
rt
(D
-------
Table 91 (continued)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Simazine
u>
yg Compound
Added/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
Average Number of
Histidine-Positive Revertants/Plate
TA100
98
106
83
72
73
87
85
71
69
84
90
82
83
87
89
120
TA1535
10
7
7
5
7
7
8
3
4
9
4
8
9
8
4
2
TA1537
8
8
15
10
20
10
12
7
11
11
11
16
9
11
16
10
TA1538
25
26
22
266
22
20
20
22
17
25
22
22
22
15
14
15
15
18
N
5-
-------
Table 91 (concluded)
Compound
Metabolic
Activation
Negative control
Positive control, 4-o-tolylazo-o-toluidine
Trifluralin
yg Compound
Added/Plate
Average Number of
Histidine-Positive Revertants/Plate
25
25
1
5
10
50
100
500
1000
1
5
10
50
100
500
1000
TA100
96
80
73
81
74
93
86
76
95
72
80
78
90
81
79
81
TA1535
15
15
11
12
16
19
18
18
13
10
13
14
15
8
12
12
TA1537
17
20
18
24
29
23
25
22
18
13
16
18
14
16
13
15
TA1538
14
8
15
168
15
14
14
16
15
11
15
9
9
14
13
15
11
10
H
i-l
H-
Ml
H>
C
i-J
0>
-------
Table 92
RESULTS OF ASSAYS WITH ESCHERICHIA COLI WP2
Compound
Negative control
Positive control,
AF-2
Moncro tophos
u>
,00
Bromacil
Metabolic
Activation
_^
+
-
+
-
-
-
-
-
- •
+
+
+
+
+
+
_
^.
^
_
_
-
+
+
+
+
+
+
yg of Compound
Added per Plate
0.05
0.05
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
Average Number of Tryptophan
Positive Revertants per Plate
68
73
204
220
89
83
76
77
61
70
90
88
76
73
75
95
65
74
71
70
70
67
71
73
66
70
70
71
-------
Table 92 (continued)
Metabolic jjg of Compound Average Number of Tryptophan-
Compound Activation Added per Plate Positive Revertants per Plate
Cacodylic Acid - 1 lu
10 102
50 89
100 82
500 91
1000 85
+ 1 95
+ 10 76
+ 50 79
w + 100 89
*° + 500 81
+ 1000 85
Captan - l 124
5 381
10 733
15 1358
25 1755
50 2600
+ 1 89
+ 5 182
+ 10 423
+ 15 699
+ 25 955
+ 50 1712
-------
Table 92 (continued)
Metabolic yg of Compound Average ^Timber of Tryptophan-
Compound Activation Added per Plate Positive Revertants per Plate
Chloropyrifos
Dinoseb
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
57
57
49
71
52
42
60
73
53
49
61
49
64
73
58
55
44
Toxic
73
69
63
65
49
Toxic
-------
Table 92 (continued)
Compound
Metabolic
Activation
yg of Compound
Added per Plate
Average Number of Tryptophan-
Positive Revertants per Plate
DSMA
Fenthion
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
86
81
67
68
68
71
69
62
67
73
81
79
59
63
62
56
70
71
64
50
67
64
64
81
-------
Table 92 (continued)
Compound
Metabolic
Activation
yg of Compound
Added per Plate
Average Number of Tryptophan-
Positive Revertants per Plate
Folpet
Azinphos-methyl
1
5
10
25
50
100
1
5
10
25
50
100
1
10
50
100
500
1000
1
10
50
100
500
1000
65
162
170
424
720
1260
74
167
202
900
1680
1880
92
87
83
89
68
88
83
74
87
86
73
79
-------
Table 92 (continued)
Compound
Metabolic
Activation
Malathion
u>
Methomyl
yg of Compound
Added per Plate
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
Average Number of Tryptophan-
Positive Revertants per Plate
62
54
60
60
54
48
58
50
55
59
75
64
61
76
83
57
68
71
70
81
78
83
63
74
-------
Table 92 (continued)
Metabolic yg of Compound Average Number of Tryptophan-
Compound Activation Added per Plate Positive Revertants per Plate
Monuron - 1 72
10 68
50 57
100 63
500 65
1000 63
+ 1 60
+ 10 59
+ 50 47
+ . 100 71
+ 500 50
+ 1000 61
MSMA - 1 55
10 64
50 57
100 76
500 60
1000 63
+ 1 55
+ 10 71
+ 50 73
+ 100 71
+ 500 61
+ 1000 72
-------
Table 92 (continued)
Compound
Metabolic
Activation
Parathion
yg of Compound
Added per Plate
Average Nimber of Tryptophan-
Positive Revertants per Plate
Parathion-methyl
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
71
64
66
70
64
64
69
53
76
57
72
66
53
56
60
68
63
52
64
83
60
65
53
71
-------
Table 92 (continued)
Compound
Metabolic
Activation
Quintozene (PCNB)
Phorate
yg of Compound
Added per Plate
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
Average Number of Tryptophan-
Posltlve Revertants per Plate
54
58
70
60
57
62
78
67
54
57
59
62
63
64
65
49
71
60
78
86
83
73
90
70
-------
Table 92 (concluded)
Metabolic pg of Compound Average Number of Tryptophan-
Compound Activation Added per Plate Positive Revertants per Plate
Simazine
Trifluralin
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
1
10
50
100
500
1000
55
51
73
54
54
53
64
66
72
56
71
83
75
73
81
86
69
60
58
63
63
65
70
70
-------
Table 93
MICROBIAL INHIBITION IN ESCHERICHIA COLI.AND BACILLUS SUBTILIS
00
Diameter of Zone
Compound
Positive control,
l-phenyl-3,-
dimethyltriazene
Negative control,
chloramphenacol
Monocrotophos
Bromacil
Cacodylic acid
Cap tan
Chloropyrifos
Dinoseb
DMSA
Fenthion
Folpet
Azinphos-methyl
mg of Compound
Added to Disc
1.0
0.03
1
1.0
1
0.1
2.5
1
1
1
0.1
1
E.
W3110
37
34. 5
6 .
6.5
6
6.5
6
10
6
6
6.5
6
coli
p3478
52
34
6
6.5
6
11
10
17
6
6
10
6
of Inhibition (mm)
B.
HI 7
40
32
6
6.5
6
9
6
8.5
6
6
6.5
6
subtilis
m45
61
31
6
6.5
f>
19
11
11
6
6
7.5
6
-------
Table 93 (concluded)
Diameter of Zone of Inhibition (mm)
vo
Compound
Malathion
Methomyl
Monuron
MSMA
Parathion
Parathion-methyl
Quintozene (PCNB)
Phorate
Simazine
Trif luralin
mg of Compound
Added to Disc
1
1
1
1
1
1
1
1
1
1
E.
W3110
6
6
6
6
6
6
6
6
6
6
coli
P3478
6
6
6
6
6
6
6
6
6
6
B.
H17
6
6
6
6
6
6
6
6
6
6
subtilis
m45
6
6
6
6
6
6
6
6
6
6
-------
Ln
O
Table 94
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MONOCROTOPHOS
Percent Survivors
Compound
Negative control
Positive control
1, 2, 3, 4-Diepoxybutane
Monocrotophos
Negative control
Monocrotophos
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7 )
EXPERIMENT 1
5.7
+ 5.8
0.1 5.8
+ 0.1 4.6
5 5.7
+ 5 4.7
EXPERIMENT 2
9.1
+ 8.6
5 6.3
+ 5 4.8
Percent of
Control
100
100
102
79
100
81
100
100
69
56
Mitotic Recombinants
per ml
(x 10-3)
4.5
4.5
1,650
1,435
44
30
8
9.5
26
40
per 10 5
Survivors
7.9
7.8
2,845
3,120
77.2
63.8
8.8
11.0
41.2
83.3
-------
Table 95
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - BROMACIL
Compound
Metabolic
Activation
EXPERIMENT 1
Negative control
Positive control
1,2,3,4-Diepoxybutane
Bromacil
EXPERIMENT 2
Negative control
Positive control
1,Z,3,4-Diepoxybutane
Bromacl1
Percent
Concentration
(w/v or v/v)
0.04
0.04
0.005
0.01
0.05
0.10
0.50
0.005
0.01
0.05
0.10
0.50
0.04
0.04
0.05
0.10
0.25
0.50
0.05
0.10
0.25
0.50
Survivors
Cells/ml
(x 10 ')
4.8
4.7
3.4
3.5
5.0
4.5
5.0
4.4
2.0
4.4
3.9
4.3
3.8
2.4
4.5
4.2
4.5
4.2
5.7
5.4
5.5
4.9
4 .9
4.7
4.8
4.9
Percent of
Control
100
100
71
74
104
94
104
92
42
94
83
91
81
51
100
100
100
100
126
120
122
108
117
112
114
117
Mitotic Recombinants
per ml
(x 10 3)
3
3
745
683
3
2
3
1
1
5
3
3
1
3
5
3
870
653
7
5
5
1
4
5
6
1
per 10 5
Survivors
6.3
6.4
2191
1951
6.0
4.4
6.0
2.3
10.0
11.4
7.7
7.0
2.6
12.5
11.1
7.1
1933
1555
12.3
9.3
9.1
2.0
8. 2
1O.6
12.5
2.0
-------
Table 96
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - CACODYLIC ACID
Percent Survivors
Compound
Negative control
Cacodylic acid
Negative control
Cacodylic acid
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7 )
EXPERIMENT 1
7.4
+ 7.7
5 5.6
+ 5 6.3
EXPERIMENT 2
7.1
+ 6.5
5 15.5
+ 5 17.5
Percent of
Control
100
100
76
82
100
100
217
270
Mitotic
per ml
(x 10~3
7.5
5
20
11
3.5
3
1,187
1,159
Recombinants
per 10s
) Survivors
10.1
6.5
35.7
17.5
4.9
4.6
766
662
-------
Ln
OJ
Table 97
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - CAPTAN
Compound
Negative control
Cap tan
Negative control
Cap tan
Percent
Metabolic Concentration
Activation (w/v or v/v)
EXPERIMENT 1
-
+
0.003
+ 0 .003
EXPERIMENT 2
_
+
0.003
+ 0.003
Survivors
Cells/ml
(x ID'7)
7.1
6.5
6.0
9.1
7.5
6.0
.77
5.1
Percent of
Control
100
100
84
140
100
100
10
85
Mitotic Recombinants
per ml
(x 10-3)
3.5
3.0
205
145
1.5
4
37
58
per 10 5
Survivors
4.9
4.6
342
159
2.0
6.7
481
114
-------
tn
Table 98
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - CHLOROPYRIFOS
Percent Survivors
Compound
Negative control
Chloropyrifos
Negative control
Positive control,
1,2,3, 4-diepoxybutane
Chloropyrifos
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7 )
EXPERIMENT 1
7.5
+ 6.0
5 7.7
+ 5 8.0
EXPERIMENT 2
6.3
+ 7.4
0.1 3.8
+ 0.1 5.2
5 7.9
+ 5 7.4
Percent of
Control
100
100
103
133
100
100
60
70
125
100
Mitotic Recombinants
per ml
(x 10-3)
1.5
4
7
10
1.5
3.5
1,045
903
3
10
per 10 5
Survivors
2.0
6.7
9.1
12.5
2.4
4.7
2,750
1,737
3.8
13.5
-------
Table 99
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - DINOSEB
01
Compound
Negative control
Positive control,
1,2,3, 4-diepoxybutane
Dinoseb
Negative control
Dinoseb
Percent
Metabolic Concentration
Activation (w/v or v/v)
EXPERIMENT 1
-
+
0.1
+ 0.1
0.2
+ 0.2
0.3
+ 0.3
EXPERIMENT 2
_
+
0.1
+ 0.1
0.2
+ 0.2
Survivors
Cells/ml
(x 10-7)
6.3
7.4
3.8
5.2
6.2
4.0
.3
2.4
5.5
5.2
4.4
4.0
4.1
4.3
Percent of
Control
100
100
60
70
98
54
5
32
100
100
80
77
75
83
Mitotic Recombinants
per ml
(x 10-3)
1.5
3.5
1,045
903
9
1
5
5
2.5
2.0
3
4
8
8
per 10 5
Survivors
2.4
4.7
2,750
1,737
14.5
2.5
167
20.8
4.5
3.8
6.8
10.0
19.5
18.6
-------
t_n
Table 100
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - DSMA
Percent Survivors
Compound
Negative control
DSMA
Negative control
DSMA
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7)
EXPERIMENT 1
7.4
+ 7.7
4.5 1.1
+ 4.5 5.2
EXPERIMENT 2
7.1
+ 6.5
5 4.7
+ 5 3.4
Percent of
Control
100
100
15
68
100
100
66
52
Mitotic Recorabinants
per ml
(x 10-3)
7.5
5
0
0
3.5
3
3
7
per 10s
Survivors
10.1
6.5
4.9
4.6
6.4
20.6
-------
Ui
Table 101
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - FENTHION
Percent Survivors
Compound
Negative control
Positive control,
1,2,3, 4-diepoxybutane
Fenthion
Negative control
Fenthion
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7)
EXPERIMENT 1
6.3
+ 7.4
0.1 3.8
+ 0.1 5.2
5 6.6
+ 5 7.5
EXPERIMENT 2
7.5
+ 6.0
5 7.8
+ 5 7.1
Percent of
Control
100
100
60
70
105
101
100
100
104
118
Mitotic Recombinants
per ml
(x 10-3)
1.5
3.5
1,045
903
9
5
1.5
4
4
6
per 10 5
Survivors
2.4
4.7
2,750
1,737
13.6
6.7
2.0
6.7
5.1
8.5
-------
Ui
oo
Table 102
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - FOLPET
Compound
Negative control
Folpet
Negative control
Folpet
Percent
Metabolic Concentration
Activation (w/v or v/v)
EXPERIMENT 1
-
+
0 .003
+ 0 .003
EXPERIMENT 2
_
+
0 .003
+ 0 .003
Survivors
Cells/ml
(x 10-7)
7.5
6.0
4.0
3.8
6.3
7.4
9.5
9.1
Percent of
Control
100
100
53
63
100
100
151
123
Mitotic
per ml
(x 10-3
1.5
4
119
«5
1.5
3.5
89
82
Recombinants
per 10s
) Survivors
2.0
6.7
298
171
2.3
4.7
94
90
-------
Ui
VO
Table 103
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 -AZINPHOS-METHYL
Percent Survivors
Compound
Negative control
Positive control
1,2, 3, 4-Diepoxybutane
Azinphos-methyl
Negative control
Azinphos-methyl
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7 )
EXPERIMENT 1
5.7
+ 5.8
0.1 5.8
+ 0.1 4.6
4.5 5.3
+ 4.5 5.8
EXPERIMENT 2
9.1
+ 8.6
5 5.7
+ 5 6.2
Percent of
Control
100
100
102
79
93
100
100
100
63
72
Mitotic Recombinants
per ml
(x 10-3)
4.5
4.5
1,650
1,435
15
15
8
9.5
68
80
per 10 5
Survivors
7.9
7.8
2,845
3,120
28.3
25.9
8.8
11.0
119.3
129
-------
Table 104
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MALATHION
Percent Survivors
Compound
Negative control
Positive control,
1,2,3, 4-diepoxybutane
Malathion
Negative control
Malathion
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7)
EXPERIMENT 1
5.7
+ 5.8
0.1 5.8
+ 0.1 4.6
5 7.8
+ 5 6.3
EXPERIMENT 2
9.1
+ 8.6
5 8.1
+ 5 7.6
Percent of
Control
100
100
102
79
137
109
100
100
89
88
Mitotic Recombinants
per ml
(x 10-3)
4.5
4.5
1,650
1,435
11
7
8
9.5
13
8
per 10 5
Survivors
7.9
7.8
2,845
3,120
14.1
11.1
8.8
11.0
16.0
10.5
-------
Table 105
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - METHOMYL
Compound
Negative control
Positive control,
1,2,3, 4-diepoxybutane
Methomyl
Negative control
Methomyl
Percent
Metabolic Concentration
Activation (w/v or v/v)
EXPERIMENT 1
-
+
0.1
+ 0.1
2.0
+ 2.0
3.0
+ 3.0
EXPERIMENT 2
_
+
3
+ 3
Survivors
Cells/ml
(x 10-7)
6.6
5.8
1.8
1.5
5.0
2.7
3.7
4.1
5.5
5.2
4.7
4.4
Percent of
Control
100
100
27
29
76
50
56
76
100
100
85
85
Mitotic Recorabinants
per ml
(x 10-3)
4.5
2.5
266
184
4
0
8
6
2.5
2.0
15
10
per 10 5
Survivors
6.8
4.6
1,478
1,227
8.0
21.6
14.6
4.5
3.8
31.9
22.7
-------
N>
Table 106
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MONURON
Percent Survivors
Compound
Negative control
Positive control,
1,2,3, 4-diepoxybutane
Monuron
Negative control
Monuron
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7)
EXPERIMENT 1
6.6
+ 5.4
0.1 1.8
+ 0.1 1.5
5 3.5
+ 5 3.8
EXPERIMENT 2
5.5
+ 5.2
5 6.9
+ 5 6.2
Percent of
Control
100
100
27
29
53
70
100
1OO
125
119
Mitotic Recombinants
per ml
(x 10-3)
4.5
2.5
266
184
3
1
2.5
2.0
2
9
per 105
Survivors
6.8
4.6
1,478
1,227
8.6
2.6
4.5
3.8
2.9
14.5
-------
Table 107
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MSMA
Percent Survivors
Compound
Negative control
MSMA
Negative control
MSMA
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7)
EXPERIMENT 1
7.4
+ 7.7
5 4.3
+ 5 5.4
EXPERIMENT 2
7.1
+ 6.5
5 4.9
+ 5 5.8
Percent of
Control
100
100
58
70
100
100
69
89
Mitotic Recombinants
per ml
(x 10-3)
7.5
5
1
3
3.5
3
10.2
10.4
per 10 5
Survivors
10.1
6.5
2.3
5.6
4.9
4.6
20.8
17.9
-------
Table 108
"IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - PARATHION
Percent Survivors
Compound
Negative control
Positive control,
1,2,3, 4-d iepoxybutane
Parathion
Negative control
Parathion
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7)
EXPERIMENT 1
5.7
+ 5.8
0.1 5.8
+ 0.1 4.6
5 6.5
+ 5 5.8
EXPERIMENT 2
9.1
+ 8.6
5 8.8
+ 5 8.2
Percent of
Control
100
100
102
79
114
100
100
100
96
95
Mitotic Recombinants
per ml
(x 10-3)
4.5
4.5
1,650
1,435
3
5
8
9.5
4
5
per 105
Survivors
7.9
7.8
2,845
3,120
4.6
8.6
8.8
11.0
4.5
6.1
-------
Ui
Table 109
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - PARATHION-METHYL
Percent Survivors
Compound
Negative control
Positive control,
1,2,3, 4-diepoxybutane
Parathion-methyl
Negative control
Parathion-methyl
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7 )
EXPERIMENT 1
5.7
+ 5.8
0.1 5.8
+ 0.1 4.6
5 7.7
+ 5 5.4
EXPERIMENT 2
9.1
+ 8.6
5 7.4
+ 5 7.2
Percent of
Control
100
100
102
79
135
93
100
100
81
84
Mitotic Recombinants
per ml
(x 10-3)
4.5
4.5
1,650
1,435
16
15
8
9.5
19
25
per 10 5
Survivors
7.9
7.8
2,845
3,120
20.8
27.8
8.8
11.0
25.7
34.7
-------
Table 110
_IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - QUINTOZENE (PCNB)
Percent Survivors
Metabolic Concentration Cells/ml
Compound Activation (w/v or v/v) (x 10~7 )
EXPERIMENT 1
Negative control - 5.7
+ 5. a
Positive control, - 0.1 5.8
1,2,3,4-diepoxybutane + Q ^ 4 6
c* Quintozene (PCNB) _ 2 3.7
+ 2 4.2
EXPERIMENT 2
Negative control - 9.1
+ 8.6
Quintozene (PCNB) - 1 5.8
+ 1 7.0
2 6.8
+ 2 7.5
Percent of
Control
100
100
102
79
65
72
100
100
64
81
75
87
Mitotic Recombinants
per ml
(x ID'3)
4.5
4.5
1,650
1,435
3
4
8
9.5
4
7
3
10
per 10 5
Survivors
7.9
7.8
2,845
3,120
8.1
9.5
8.8
11.0
6.9
10.0
4.4
13.3
-------
Table 111
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - PRORATE
Percent Survivors
Compound
Negative control
Phorate
Negative control
Phorate
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7)
EXPERIMENT 1
9.1
+ 8.6
5 8.7
+ 5 7.5
EXPERIMENT 2
5.7
+ 5.8
5 7.5
+ 5 7.2
Percent of
Control
100
100
96
87
100
100
132
124
Mitotic
per ml
(x 10-3
8
9.5
9
3
4.5
4.5
4
7
Recombinants
per 10 5
) Survivors
8.8
11.0
10.3
4.0
7.9
7.8
5.3
9.7
-------
01
oo
Table 112
VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - SIMAZINE
Percent Survivors
Compound
Negative control
Positive control,
1,2,3, 4-diepoxybutane
Simazine
Negative control
Simazine
Metabolic Concentration Cells/ml
Activation (w/v or v/v) (x 10~7 )
EXPERIMENT 1
6.6
+ 5.4
0.1 1.8
+ 0.1 1.5
5 3.8
+ 5 2.0
EXPERIMENT 2
5.5
+ 5.2
5 7.0
+ 5 7.0
Percent of
Control
100
100
27
29
58
37
100
100
127
135
Mitotic Recombinants
per ml
(x 10-3)
4.5
2.5
266
184
3
1
2.5
2
7
4
per 10 5
Survivors
6.8
4.6
1,478
1,227
7.9
5.0
4.5
3.8
10.0
5.7
-------
£
Table 113
IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - TRIFLURALIN
Compound
Percent
Survivors
Mitotic Recombinants
Metabolic Concentration Cells/ml Percent of per ml per 10
Activation (w/v or v/v) (x 10~7 ) Control (x 10~3) Survivors
EXPERIMENT 1
Negative control
7.5
6.0
100
100
1.5
4
2.0
6.7
Trifluralin
+
5
5
8.4
6.0
112
100
5
2
5.9
3.3
VO
EXPERIMENT 2
>
CD
m
CD
Negative control
Positive control,
1,2,3,4-diepoxybutane
Trifluralin
+
o.i
o.i
5
5
6.3
7.4
3.8
5.2
8.7
8.4
100
100
60
70
138
114
1.5
3.5
1,045
903
7
3
2.4
4.7
2,750
1,737
8.0
3.6
-------
Table 114
IN VITRO MUTAGENESIS WITH SALMONELJA TYPHIMURIUM
SUMMARY DATA FOR EPA PESTICIDES
Positive Response, +; Negative Response, -
TA100
TA1535
TA1537
TA1538
Pesticide
- Metabolic + Metabolic
Activation Activation
- Metabolic + Metabolic - Metabolic + Metabolic - Metabolic + Metabolic
Activation Activation Activation Activation Activation Activation
Monocrotophos
Bromacil
Cacodylic Acid
Captan
Chlorpyrifos
Dinoseb
DSMA
Fenthion
Folpet
Azinphos-methyl
Malathion
Me thorny 1
Monuron
MSMA
Parathlon
Parathion-methyl
Quintozene (PCNB)
Phorate
Simazine
Trifluralin
-------
APPENDIX A
MUTAGENESIS STUDIES OF PESTICIDE COMPOUNDS
MOUSE HERITABLE TRANSLOCATION TEST
CAPTAN
171
-------
SUMMARY
SRI conducted a heritable translocation study of Captan in mice to
investigate whether heritable mutagenic events pccur when the compound
is ingested repeatedly over an extended period.
For 8 weeks, adult male mice were administered Captan in their diet;
60 mice received 2500 ppm, and 61 received 5000 ppm. A control group
of 60 adult male mice received an untreated diet during this time.
A positive control group containing 66 adult male mice was treated as
a control group for 4 weeks and then received the known mutagen tri-
ethylenemelamine (TEM) in the drinking water for A weeks. After
treatment, all males were bred with two virgin females each to produce
an FI generation, the males of which were raised to maturity. Selected
(200 per group) F males were bred to three virgin females each, and
presumptive translocates were rebred to three additional females each.
A third breeding was conducted with selected nonbreeder and/or presump-
tive males.
Evaluation of the data on fertility, breeding, and litter size
distribution for Fn and F generations does not suggest the presence
of translocation heterozygotes in control or Captan-treated male mice.
Data on dead implants and -rebreeding did, however, suggest the presence
of translocation heterozygotes in the group treated with 5000 ppm Captan.
Meiotic cell preparations of the testes of the presumptive males
were evaluated cytogenetically. Normal meiotic chromosomes were found
in the following numbers of F males derived from the group specified:
8 of 8 controls, 8 of 8 from the 2500 ppm Captan group, 8 from the
5000 ppm Captan group, and 2 of 2 from the 5000 ppm Captan-treated group
derived from traumatized F females. Five of 5 TEM-treated F males
and 1 of 8 from the 5000 ppm Captan-treated males showed reciprocal
translocations.
172
-------
The results of this study show that under the experimental procedures
employed, Captan at 5000 ppm in the diet of male mice for 8 consecutive
weeks can produce a heritable mutagenic event in F_ generation male mice.
173
-------
INTRODUCTION
The EPA is reviewing and evaluating the health hazard of pesticides
and of substitute candidate pesticides according to available data.
Additionally, the Agency is obtaining supplemental laboratory data.
The objective is to enable the EPA to select those chemicals that are
minimally hazardous when used according to labeling restrictions. SRI
is participating in this Substitute Chemical Program by investigating
the mutagenic potential of selected materials by in vitro and in vivo
procedures.
Captan has been shown to respond in a positive manner in Salmonella
typhimurium, Escherichia coli WP2, Saccharomyces cerevisiae, JJ. coli
(relative toxicity), Bacillus subtilis, WI-38 unscheduled DNA synthesis
(UDS) with metabolic activation, and Drosophila melanogaster experiments.
It was not positive in a mouse dominant-lethal test. Based on positive
responses in both Tier I (in vitro test) and Tier II (Drosophila)
mutagenic studies, it was recommended that a heritable translocation
test (Tier III) in the mouse be conducted to further assess the mutagenic
potential of Captan.
In this study, young adult male ICR/SIM mice from a closed, random-
bred colony were administered Captan in the diet for 8 weeks. After
treatment, each male was mated to two virgin females to produce an F
generation, the males of which were raised to maturity and bred to three
virgin females each. Pregnant females were evaluated against pre-
determined selection criteria for identification of suspect FI males,
which were rebred and evaluated again. Presumptive F males were
examined cytogenetically.
Through this procedure, a heritable mutagenic response can be
detected. Potential mutagenic effects were identified by examination
of fetuses during the middle to later stages of gestation. Cytogenetic
174
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examinations were made of meiotic cell preparations of the testes from
suspect males for confirmation of findings obtained from the breeding
studies.
Reported here are the results of the heritable translocation
study of Captan.
175
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MOUSE HERITABLE TRANSLOCATION TEST
Background
Human populations frequently are exposed to man-made chemicals,
often at barely detectable levels, for extended periods. To evaluate
the genetic hazards of such chemicals, a prudent approach is to study
them in mammalian systems so as to maximize detection of a mutagenic
response. The study reported here was such an investigation of Captan
for its potential to produce heritable genetic defects.
Chemical induction of chromosomal aberrations in the mouse is a
valuable and important experimental aid in understanding the many
genetic defects due to chromosomal anomalies in humans. To date,
mammalian evaluations of chemically induced chromosomal aberrations
have been attempted with the dominant-lethal test and cytogenetic studies
of somatic and germinal cells. Although these procedures can provide
useful information, they do not measure heritable genetic effects, the
most important mutagenic occurrences that are permanent and transmissible.
A need exists for a method to reliably identify compounds that cause
heritable chromosomal aberrations in mammalian systems. The mouse
translocation procedure appears to be such a system.
A well-defined translocation test will demonstrate the fertility
of an F male population derived from F males treated with a test agent.
Confirmation of a nonbreeder, sterile, or partially sterile response
can be obtained by cytological examination of the germ cells from suspected
males. Sterility and partial sterility are closely correlated with the
induction of translocation heterozygotes.
The procedure used in conducting this translocation test was based
on experimental techniques described by Leonard and DeKnudt,1 Cattanach
et al.,2 Falconer et al.,3 and Generoso.4 We modified this approach,
in consultation with government and industry scientists actively
engaged in mutagenesis research.
176
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Materials and Methods
Animals
Male and female ICR/SIM mice were purchased from Simonsen Laboratories,
Gilroy, California. The F males were 8 to 10 weeks old. The females
used in the breeding phases were 10- to ^.2-week-old virgin stock.
Chemical Supply
A supply of Captan sufficient for all aspects of the experimental
program was received from Battelle Columbus Laboratory and EPA-RTP.
Lot number SX-640, Chevron Chemical Company, was used for all treatment
periods. The excess material has been placed in storage in case it is
needed for future reference.
Dosage Selection and Compound Administration
SRI and EPA staff selected the two dosage levels of Captan to
be used in this experimental program. For 8 weeks, Captan was fed in
the diet at 2500 and 5000 ppm.
An appropriate amount of Captan was dissolved and/or suspended in
corn oil. Then the compound-oil concentrate was added at a level of
3% to a finely ground commercial diet (Purina) of known composition.
The use of corn oil assured even distribution of Captan and prevented
its stratification in an otherwise dry diet. Diets prepared at 2-week
intervals were refrigerated at 4°C until fed to the animals. The diet
was replaced in the feed containers twice weekly to minimize the
possibility of compound loss. Body weights and food consumption were
recorded weekly during the 8-week exposure period.
Reference Control
Males in the reference control group were fed the Purina diet with
only corn oil added at a level of 3%. These mice were treated in the
same manner as those in the compound test groups. Body weights were
recorded weekly, as was food consumption.
177
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Positive Control
For the positive control group, the known mutagen triethylenemelamine
(TEM) was administered in the drinking water at 0.32 mg/liter for 2 weeks
and then at 0.124 mg/liter for 2 weeks. TEM treatment was initiated after
the males had been on the control diet for 4 weeks. Body weights and
food consumption were recorded weekly. TEM is one of the chemical
mutagens that have the demonstrated effect of inducing translocations in
the F progeny of F treated males.
Genetic Tests
After 8 weeks of treatment, the males in each treatment group were
mated to two adult virgin females each. After 1 week, each female was
housed individually and allowed to deliver its litter. The FQ males
were discarded. All litters were raised to weaning age, at which time
the females were discarded. The F males were raised to maturity. At
maturity (10 to 12 weeks of age), 200 FI males from each experimental
group were selected randomly and housed individually.
Three adult virgin females were housed with each F.. male for the
first breeding. They were examined daily for the presence of vaginal
plugs. These females were sacrificed 14 days after mating, and a
uterine analysis was performed for determination of the number of total,
live, and dead implants. Males bred to females that produced litters
fitting our criteria for presumptive classification as sterile, partially
sterile, or nonbreeder were rebred to three new virgin females each.
The same evaluation was made for the second breeding.
Our criteria for presumptive classification of a male as "partially
sterile," "sterile," or "nonbreeder" are:
• "Partially Sterile" Male
- If all 3 females are pregnant, each must have 9 or fewer
live implants, with at least 1 having 6 or fewer live implants.
- If only 2 of 3 females are pregnant, both must have 9 or
fewer live implants, with 1 having 6 or fewer live implants.
178
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- If only 1 of 3 females is pregnant, this female must have
6 or fewer live implants.
• "Sterile" Male
- None of 3 females pregnant—previously identified by presence
of vaginal plug.
• "Nonbreeder" Male
- None of 3 females pregnant—pot previously identified by
presence of vaginal plug.
Any F male that did not fit one of these descriptions was
considered "normal" and was discarded. For each F male in the control
and compound-treated groups suspected of being a translocate or nonbreeder
after 2 or 3 breedings, a cytogenetic evaluation was made of meiotic cell
preparations of its testes. Five males from the positive control group
were also subjected to cytogenetic evaluation.
Evaluation of Breeding Data
F males were identified as sterile, partially sterile, or non-
breeders by the methods outlined above. Individual data were totaled
to give the number of observed (presumptive) translocations per treatment
group, using a data base of 600 to 800 females per group. Also, for an
accurate review of such findings, the F breeding and litter data were
thoroughly evaluated. The various measured evaluated included percentage
of pregnancies, average litter size, average number of males and females,
average number of males with females having zero to five or more dead
implants, average number of females having zero to five or more dead
implants, percentage of females with plugs, and percentage of pregnancies
with and without plugs.
Meiotic Cell Cytogenetic Studies
Cytogenetic examinations were made of the testes of 31 F.. mice, with
the two testes from each mouse being examined separately. The procedures
179
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used for the cytogenetic preparations are as follows. C0_ was used
to sacrifice the mice. The testes were removed, weighed, and placed in
an isotonic solution of 2.2% sodium citrate. The tunica of each testis
was punctured to release the tubules, which were then rolled on a glass
plate to release the cell contents into the isotonic solution. The
resulting cell suspension was centrifuged at 800 rpm for 5 minutes; the
supernatant was removed, and each pellet of cells was resuspended in
5 ml of 1% sodium citrate hypotonic and held at room temperature for
15 minutes. The cells were centrifuged again at 800 rpm for 5 minutes
and the supernatant was discarded. The cells were then treated with
Carnoy's fixative (3 parts methyl alcohol and 1 part glacial acetic
acid) to give a total volume of 5 ml, and immediately centrifuged again
at 800 rpm for 5 minutes. This procedure was performed twice. Then
the cells, suspended in an appropriate amount of fixative, were dropped
onto clean, wet microscope slides and allowed to air-dry. The slides
were stained with 2% buffered Giemsa for 5 minutes. Coverslips were
attached with Permount. The slides were coded to preclude bias on the
part of the scorers.
180
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RESULTS AND DISCUSSION
General
Table 1 presents the average body weights for mice in the various
groups. The body weights of the control, TEM, and 2500 ppm Captan
group were within normal limits and comparable throughout the experiment.
The 5000 ppm Captan-treated group showed a depressed body weight for
5 weeks before demonstrating a recovery trend during Weeks 6 to 8. |
This body weight depression appeared to be due to the inability of the
male mice to acclimate to such a high level of compound in their diet.
Table 2 summarizes the average food consumption by treatment group.
Both Captan-treated groups (2500 and 5000 ppm) showed a lower average
weekly food intake than did the control and TEM-treated groups.
During the week immediately following the 1-week F generation
mating, one animal rack holding some females that had been mated with
the mice given 5000 ppm Captan was accidentally tipped and some cages
were spilled onto the fllor, resulting in our inability to identify
which males had been mated with these females. There were, however,
sufficient numbers of F generation males from those females that were
not traumatized to allow us to randomly select 200 F males for use in
subsequent F generation breedings. In addition, we held all females
that had been traumatized by the tipping of the rack and maintained
them throughout the remainder of the study as a separate group. From
this' separate group we selected 50 F males (at least one per female
retained) for use in the F.. generation breedings and evaluations.
F Generation
Information on the breeding performance, litter size, sex distribu-
tion, and clinical effects of the F generation should be included in
the evaluation of translocation data, because it may provide valuable
reference data.
181
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Table 3 summarizes the breeding and litter performance of the F
generation. No adverse effects were observed in the control and 2500
ppm Captan groups. The two 5000 ppm Captan groups showed a reduced
pregnancy rate; the rate for the traumatized females was 29% below that
of the controls. Litter sizes for the 5000 ppm groups were slightly
below control values. As expected, the TEM group had a reduced pregnancy
rate and a litter size 47% below the control level,
Table 4 presents litter-size distributions of live young from, the
F generation mating. Although the distribution patterns for the control
and Captan-treated animals were within normal ranges for our strain of
mouse, there was evident a definite pattern of decreasing litter size
and increasing variance and standard deviations between the different
experimental groups. As expected, the TEM-treated animals showed the
classic shift toward smaller litters. Figure 1 graphically presents
the data on the Fn generation litter-size distribution.
F Generation
Table 5 summarizes breeding data from the first mating of the F
generation male mice. In the females mated with TEM males and with
males from the 5000 ppm Captan group of traumatized F mothers, there
were 10% fewer females with mating plugs and an increased percentage
of nonpregnant females in comparison with control values; also, these
two male groups had an increased percentage of males with no pregnant
females. Results from males in the 2500 and 5000 ppm Captan groups
were within normal limits for this strain of mouse and comparable with
values from control males.
Litter-size distributions of live implants derived from the first
mating of F.. generation males are presented in Table 6. Responses of
control and Captan groups were within normal limits and readily
comparable. The TEM group showed approximately a 13% reduction in
litter size. Mean litter sizes were 11.72 for the control group,
11.73 for the 2500 ppm Captan group, 11.56 for the 5000 ppm Captan
group, 11.88 for the 5000 ppm (traumatized F female) group, and
182
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10.24 for the TEM-treated group. The data on the F generation litter-
size distribution are presented graphically in Figure 2.
Tables 7 and 8 summarize the data on dead implants per F.. male
and dead implants per female, respectively. The 2500 and 5000 ppm
Captan groups showed a slight increase in total dead implants for both
males and females at the 4, 5, and >5 levels when compared with controls.
TEM animals showed significant increases in dead implants for both males
and females.
Table 9 summarizes the breeding results by treatment of those F..
males classified as presumptive sterile, partially sterile, or non-
breeders after three breedings. Table 10 identifies these F males
individually by number and treatment.
Details of the breeding and rebreeding data for presumptive F
males are presented in Table 11. In the reference control group, 24
of the 200 males were considered as presumptive translocates. When
rebred, 12 males remained in this classification. A third mating of
selected questionable and/or nonbreeder males reduced the number of
presumptive males to eight: 1 nonbreeder, 1 presumptive sterile, and
6 partially sterile (3 of which were questionable partially sterile).
For the TEM group, 83 of 200 F males were identified as presumptive
mutants after the first breeding. When rebred, 53 still met the original
criteria. A third breeding reduced this number to 49; 4 continued to
be nonbreeders, 14 were presumptive sterile, and 31 were partially sterile
(6 of which were questionable partially sterile).
In the 2500 ppm Captan group, 30 of 200 F males were identified
as presumptive mutants after the first breeding. When rebred, 11
still met the criteria: 5 were nonbreeders, 1 was a presumptive
sterile, and 2 were partially sterile (1 of which was questionable
partially sterile).
The 5000 ppm Captan group also had 30 of 200 F males identified
as presumptive mutants after the first breeding. The second mating
reduced this number to 9. After a third breeding, 8 males still met the
183
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original criteria: 2 were nonbreeders, 1 was a presumptive sterile,
and 5 were partially sterile.
For the group of F.. males derived from traumatized F females
and males treated with 5000 ppm Cap tan, 12 of 50 F.. males were identified
as presumptive mutants after the first mating. A second breeding
reduced this number to 4 and a third breeding further reduced to 2 the
number of F males that still met the original criteria; both were
partially sterile, with one of them being questionable partially sterile.
The data on the F_ and F generations' fertility, breeding, and
litter-size distribution as well as the data on the F^ generation's
dead implants and rebreeding show that Captan tends to induce dose-
related effects on the reproductive performance of male mice. The data
also suggest the presence of translocation heterozygotes in the 5000 ppm
Captan group.
Review of the data on dead implants, breeding, and rebreeding for
the F.. generation of the TEM-treated group showed, as expected, the
potential for the presence of translocation heterozygotes in 24.5%
of the F- males.
Cytogenetic Studies
Table 12 presents the findings from the cytogenetic evaluation of
meiotic cell preparations from F.. males in the Captan groups characterized
as nonbreeder, presumptive sterile, or partially sterile. Also, eight
control males and 5 of 49 TEM males were evaluated.
Whenever possible, 25 spermatocytes per testis were scored. The
slides were decoded only after all scoring was completed. The results
are summarized as follows:
• All eight males examined in the control group were cyto-
genetically normal.
• The five TEM males all showed positive reciprocal translocations.
• All eight males in the 2500 ppm Captan group were cyto-
genetically normal.
• Seven males in the 5000 ppm Captan group were cytogenetically
normal; however, the eighth male (No. 657) showed as a
positive reciprocal translocation.
184
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• The two males examined in the 5000 ppm Captan group derived
from traumatized F females were cytogenetically normal.
Discussion
Increased use of the translocation procedure has revealed that a
meaningful relationship exists between the incidence of dead implants
in F matings and the occurrence of a heritable translocation event.
Previous experiments at SRI and at Oak Ridge National Laboratories have
demonstrated this correlation. The following paragraphs discuss
occurrences of dead implants in this study.
When females had a total implant count of less than six or when
all their implants were identified as dead, we generally considered
this to be a result of first breeding or of some factor other than
compound treatment (such as background incidence) and excluded those
females from this evaluation. Tables 13 through 17 present the total,
dead, and live implantation data for suspect translocates of the control,
the TEM group, and the three Captan groups.
The control group (Table 13) showed a normal implant distribution,
with the exception of one F male (No. 122) for whom the number of
implants was high. In the TEM group (Table 14), the expected increase
in dead implants and the resultant decrease in live implants occurred,
although the numbers of total implants were generally normal. This
pattern occurred during all breeding periods.
The 2500 ppm Captan group (Table 15) contained seven males in
the first breeding with females having high dead implant counts but
normal live litter size, according to the criteria. Also, 3 males
showed high dead implant counts during the first breeding, with live
implant counts fitting the criteria for partially sterile males. This
increase in dead implant occurrence was not repeated in subsequent
breeding, and all FI males were classified as normal after the breeding
phases.
185
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Five F males in the 5000 ppm Captan group (Table 16) showed an
increase in dead implants during the first breeding. In the rebreeding
of these males, only male No. 657 continued to show the increase in
dead implants and the resultant decrease in live implants. All other
F- males in this group had a normal distribution of dead and live
implants for all breedings.
Table 17 presents the implant data for FI males derived from
traumatized females in the 5000 ppm Captan group. With the exception
of an occasional female showing an increase in dead implants, the
distribution of total, dead, and live implants was normal.
The numbers of total implantations were generally within normal
limits for all experimental groups in all breedings.
186
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REFERENCES
1. A. Leonard and G. H. DeKnudt. Mutation Res. £, 127 (1970).
2. B. M. Cattanach, C. E. Pollard, and J. H. Isaacson. Mutation
Res. j>, 297 (1968).
3. D. S. Falconer, D. M. Slizynski, and C. Auerbach. J. Genet. 51,
81 (1952).
4. W. M. Generoso. Proc. Second Annual Environmental Mutagen Soc.,
p. 9 (March 1971).
187
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Table 1
AVERAGE BODY WEIGHTS IN GRAMS FOR MICE RECEIVING
VARIOUS LEVELS OF CAPTAN IN THEIR DIETS
Dietary Levels
of Captan
Week of
Test
Initial
1
2
3
4
5
6
7
8
Control
33.8
32.5
33.7
34.8
34.6
34.8
37.3
37.7
39.2
TEM
33.4
32.9
34.6
35.4
35.8
35.9
38.1
38.9
39.8
(ppm
2500
33.8
33.3
33.7
34.4
34.4
36.0
37.5
39.3
38.9
diet)
5000
33.6
31.8
31.4
32.1
31.4
33.0
35.3
35.8
36.9
188
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Table 2
AVERAGE FOOD CONSUMPTION FOR MICE RECEIVING
VARIOUS LEVELS OF CAPTAN IN THEIR DIETS
(Grams of Food Consumed/Mouse/Day)
Dietary Levels
of Captan
Week of
Test
1
2
3
4
5
6
7
8
Control
4.01
4.82
4.91
5.28
5.08
5.36
5.81
5.55
TEM
4.05
5.00
5.08
5.33
5.55
5.56
6.14
5.74
(ppm
2500
3.74
4.72
4.90
4.93
5.44
4.95
5.63
5.13
diet)
5000
3.25
4.38
4.40
4.45
4.95
4.75
5.27
4.83
189
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Table 3
TRANSLOCATION STUDY OF CAFTAN
F0 GENERATION MICE
SUMMARY OF BREEDING AND LITTER DATA
VO
o
Parameter
Number of F^ males
Number of F females
Number pregnant
Percent pregnant
Number of nonbreeder males
.Percent nonbreeders
Average live litter size
Average number of males weaned/litter
Control
60
120
93
77.5
8
13.3
12.30
5.84
TEM
66
132
77
58.3
19
28.8
6.55
3.26
2500 ppm
60
119
91
76.5
8
13.3
12.16
5.68
5000 ppm
31
61
38
62.3
5
16.1
11.68
6.29
5000 ppma
30
59
33
55.9
--
--
11.33
5.25
Group of females accidentally tipped off rack following 1 week of mating with 5000 ppm-treated
mates. These traumatized females, and their offspring, most of which could not be identified
back to a particular FQ male, were considered as a separate group for the remainder of the
s tudy.
-------
Table 4
TRANSLOCATION STUDY OF CAPTAN
MOUSE LITTER-SIZE DISTRIBUTION OF LIVE YOUNG
DERIVED FROM FQ GENERATION ADULTS
Litter
Size
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Mean (p,)
Variance
(o?)
Standard
Deviation
(a)
Captan
(ppm diet)
Control
0
0
0
0
0
0
1
2
9
6
8
18
22
18
6
3
0
0
12.30
3.89
1.97
.TEM
1
2
3
8
10
15
12
8
8
2
3
1
0
1
0
0
0
0
6.55
5.84
2.42
2500
0
0
0
0
1
2
0
3
3
8
9
24
20
10
6
3
2
0
12.16
4.97
2.23
5000
0
1
0
0
0
1
0
0
1
6
5
8
9
6
1
0
0
0
11.68
5.73
2.39
5000a
0
0
0
1
0
1
1
0
3
3
7
5
8
1
3
0
0
0
11.33
6.09
2.47
Traumatized F. females.
191
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24
— — 2500 ppm Captan
> • — 5000 ppm Captan
5000 ppm Captan
(Traumatized Females)
8 10
LITTER SIZE
FIGURE 1 LITTER SIZE DISTRIBUTION F0 GENERATION
192
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VO
OJ
Table 5
TRANSLOCATION STUDY OF CAPTAN
F! GENERATION MICE
SUMMARY OF BREEDING DATA—FIRST BREEDING
Captan (ppm)
Parameter Control TEM 2500 5000 5000a
Number of Fl males 200 200 200 200 50
Number of females 600 600 600 600 150
Number of mating plugs 450 376 442 439 95 -
Percent mating plugs 75 63 74 73 63
Number pregnant 492 383 472 477 112
Percent pregnant 82 64 79 80 75
Number pregnant with plug 434 326 419 413 94
Percent pregnant with plug 88 85 89 87 84
Number pregnant without plug 58 57 53 64 18
Percent pregnant without plug 12 15 11 13 16
Number not pregnant 108 217 128 123 38
Percent not pregnant 18 36 21 20 25
Number not pregnant with plug 16 50 23 26 1
Percent not pregnant with plug 15 23 18 21 3
Males with no pregnant females 13 39 14 9 7
Percent males with no pregnant females 6.5 19.5 7.0 4.5 14.0
Traumatized FQ females.
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Table 6
TRANSLOCATION STUDY OF CAPTAN
MOUSE LITTER SIZE DISTRIBUTION OF LIVE YOUNG
DERIVED FROM FI GENERATION ADULTS--FIRST BREEDING
Litter
Size
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Mean (|i)
Variance
(a2)
Standard
Deviation
(a)
Cap tan (ppm)
Control
5
4
2
4
5
8
7
14
35
37
73
80
89
72
32
16
7
1
0
0
11.72
8.13
2.85
TEM
4
9
12
17
21
11
8
17
25
31
53
61
52
27
21
8
1
1
1
0
10.24
13.59
3.69
2500
4
0
5
6
4
5
6
13
35
40
77
75
87
54
36
14
7
2
0
1
11.73
8.68
2.95
5000
3
3
1
5
6
8
11
19
17
53
77
85
93
55
19
11
7
3
0
0
11.56
7.40
2.72
5000a
0
0
0
1
1
4
3
4
2
16
14
15
17
14
13
5
1
0
0
0
11.88
7.20
2.68
Traumatized Fo females.
194
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120
110 —
100 —
— — — — — 2500 ppiTi Captan
——••—•»— 5000 ppm Capian
5000 ppm Captan
(Traumati?od Females)
10 12 14 16 18 20
FIGURE 2 LITTER SIZE DISTRIBUTION F, GENERATION — FIRST MATING
195
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Table 7
TRANSLOCATION STUDY OF CAPTAN
SUMMARY OF DEAD IMPLANTS PER Fl MALE
Number of Males Cap tan (ppm)
with Females Having
0 Dead implants
1 Dead implant
2 Dead implants
3 Dead implants
4 Dead implants
5 Dead implants
> 5 Dead implants
Control
68
54
30
18
8
2
7
TEM
37
36
25
16
9
6
32
2500
69
44
31
13
18
4
7
5000
49
68
36
18
9
3
8
5000a
20
11
7
2
0
2
1
a
Traumatized FQ females.
196
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Table 8
TRANSLOCATION STUDY OF CAPTAN
SUMMARY OF DEAD IMPLANTS PER FEMALE
Number of Dead Captan (ppm)
Implants/Female
0
1
2
3
4
5
> 5
Total Pregnant
Females 492 383 472 477 112
Control
320
109
39
18
3
0
3
TEM
182
83
35
16
11
11
45
2500
295
113
37
16
7
2
2
5000
284
130
41
9
3
2
8
5000a
79
23
9
0
0
0
1
Traumatized F0 female.
197
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Table 9
TRANSLOCATION STUDY OF CAPTAN
SUMMARY OF PRESUMPTIVE TRANSLOCATION Fx MALES AFTER THREE BREEDINGS
Captan (ppm)
Control TEM 2500 5000 5000a
Total number of FI males 200 200 200 200 50
Number of nonbreeder males 1 4 520
Number of presumptive sterile males 1 14 110
Number of partially sterile males 6(3?) 31(6?) 2(1?) 5 2(1?)
M
OO
Traumatized Fo females
-------
Table 10
TRANSLOCATION STUDY OF CAPTAN
INDIVIDUAL IDENTIFICATION OF PRESUMPTIVE FX MALES
AFTER THREE BREEDINGS
Partially Presumptive
Treatment Sterile Sterile Nonbreeder
Control 16? 72 220
65
122?
164?
189
190
TEM 215 202 220
216 231 241
232 251 391
235? 256 396
238 268
245 273
262 288
264 317
269 321
280 326
281 339
290 343
292 369
299? 389
300
314
315
327?
344
345
350
359
360?
361
371
375
376
388
390
399?
400?
(Continued)
199
-------
Table 10
(Concluded)
Treatment
Captan
2500 ppm
Captan
5000 ppm
Captan
5000 ppma
Partially
Sterile
480?
526
635
657
760
779
780
805
837?
Presumptive
Sterile
474
736
Nonbreeder
449
479
496
523
583
634
733
Traumatized Fo females.
200
-------
Table 11
TRANSLOCATION STUDY OF CAFTAN
BREEDING AND REBREEDING SUMMARY OF PRESUMPTIVE FI MALES
(Live Implants Only)
Fj_
Male
Treatment No.
Control 11
16
24
39
43
50
59
65
67
72
86
102
122
129
139
144
152
164
179
189
190
192
200
Totals
TEM 202
215
216
220
226
227
228
229
230
First Breeding
(3
_**
-
_
9
-
4
0
4
-
-
-
-
6
-
-
-
3
-
-
0
1
7
-
0
-
-
-
-
0
-
7
-
Females)
-
_
0
-
7
-
0
-
-
-
-
2
-
-
-
9
-
-
1
4
(14)
-
24
0
-
-
-
4
(9)
0
-
.
(13)
_
-
-
6
-
5
-
-
-
(ID
13
-
-
-
9
-
-
0
6
-
-
0
-
(9)
-
-
2
-
9
-
Second Breeding
(3
13
-
0*
0
-
12
10
3
14
0
(12)
9
7
-
14
9
10
-
10
0
1
12
13
0
7
-
-
12
12
14
14
11
Females)
8 i
-
11
-
-
11
9
6
11
-
-
5
7
-
-
10
8
-
-
0
0
11
-
12
0
4
-
-
2
4
12
14
10
(15)1"
-
_
-
-
12
-
2
-
-
-
16
14
-
-
-
13
(9)
-
0
0
-
-
0
-
-
-
-
8
11
13
-
Third
Breeding
(3 Females)
_
12 11 11
10
- _ .
12 12 4
14
_
010
Oil
8
4 - -
_
(Continued)
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
*"( )" indicates all implants were in early stages of development and impossible
to determine if they were live or dead upon gross observation.
201
-------
Male
Treatment No.
TEM 231
232
233
235
237
238
239
241
244
245
251
253
254
256
258
262
264
267
268
269
270
273
280
281
283
286
288
290
292
294
299
300
302
314
315
317
318
321
322
Table 11
(Continued)
First Breeding
(3 Females)
0*
3
-
8
5
3
-
-
-
®
0
3
4
0
0
2
3
-
-
-
0
-
9
®
9
-
0
5
6
8
8
6
9
-
0
0
4
0
9
_**
4
-
7
9
0
-
-
-
0
0
10
-
-
-
8
5
-
-
-
-
-
7
6
9
-
0
5
3
(18) t
-
(15)
-
-
3
0
4
0
-
.
1
-
6
8
1
-
-
-
1
0
-
-
-
-
5
-
-
-
-
-
-
2
-
8
-
0
4
0
9
-
-
-
-
0
0
3
-
-
(Continued)
i
Second Breeding
(3 Females)
0
8
11
7
10
1
13
-
10
1
0
14
13
-
13
5
®
13
-
6
8
0
8
4
14
-
0
7
3
10
9
8
13
2
0
0
9
0
11
0
2
15
7
10
2
-
-
13
0
0
11
14
-
0
4
5
©
-
7
-
-
8
1
0
-
0
5
3
12
4
5
12
1
0
0
4
0
11
0
5
6
13
14
©1"!"
-
-
11
3
0
-
11
-
-
5
9
12
-
-
-
-
5
2
_
_
0
0
8
11
9
2
15
4
0
-
11
_
-
Third
Breeding
(3 Females)
10
*
**
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
"( )" indicates all implants were in early stages of development and impossible
to determine if they were live or dead upon gross observation.
"®" indicates female was pregnant but had no live implants.
202
-------
FI
Male
Treatment No.
TEM 326
327
333
334
339
343
344
345
346
349
350
355
356
359
360
361
363
367
369
371
372
375
376
381
382
385
387
388
389
390
391
396
397
399
400
Totals
Table 11
(Continued)
First Breeding
(3 Females)
_**
0
0
-
0
0
3
6
-
-
4
-
-
9
(15)
4
5
-
0
4
(16)
3
5
_
(13)
9
-
3
-
4
-
-
-
5
2
-
6
_
-
0
5
4
-
-
3
-
-
4
-
4
-
-
-
6
-
-
0
-
(ID
5
-
4
-
3
-
-
-
4
2
83
-
-
_
-
0
-
5
-
(12)
2
-
8
-
9
-
-
-
-
-
-
6
-
-
(6)
-
0
-
5
-
-
(14)
(11)
(12)
(Continued)
Second Breeding
(3 Females)
0*
0
10
_
0
0
5
4
12
10
®tt
13
11
7
2
3
12
4
-
5
10
5
1
11
-
10
15
3
-
3
-
-
11
5
4
(10)t
0
_
0
0
4
4
11
8
0
0
-
3
®
7
14
5
-
5
9
-
-
0
-
12
-
3
-
5
-
-
0
2
5
53
-
_
_
0
0
5
3
_
10
1
-
(15)
7
-
®
-
11
-
5
-
7
1
-
-
7
-
4
-
3
-
-
0
4
2
Third
Breeding
(3 Females)
1
12
(9)
12
11 10
49
~K
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
"( )" indicates all implants were in early stages of development and impossible
to determine if they were live or dead upon gross observation.
4.4-
" (?) " indicates female was pregnant but had no live implants.
203
-------
Table 11
(Continued)
Treatment
Captan
2500 ppm
1
Captan
5000 ppm
Male
No.
430
432
436
449
450
451
452
455
461
469
472
474
479
480
484
489
496
518
523
526
528
546
561
568
569
582
583
585
588
590
Totals
601
604
620
622
626
627
634
635
"0" indicates a
**
"-" indicates a
' II t VII •
First Breeding
(3
**
4
9
_
0*
7
9
-
.
-
8
-
-
(3)
4
11
-
-
-
7
0
-
-
8
-
-
-
9
-
8
-
7
8
-
-
8
-
-
plug was
plug was
Females)
_
(17)
5
_
1
0
10
-
.
-
10
-
-
0
14
12
-
-
-
3
-
-
-
(9)
-
-
-
10
-
0
30
(11)
9
-
-
-
8
-
-
_
f 9
8
_
14
_
10
-
-
-
9
-
-
-
11
6
-
-
-
(1)
-
-
-
(13)
-
-
-
12
(12)
9
(8)
9
-
(9)
-
8
-
-
(Continued)
Second Breeding
(3
12
11
11
_
10
0
13
10
13
-
0
0
-
(13)
11
9
-
0
-
0
14
-
-
9
11
8
-
12
9
0
11
9
9
0
(12)
10
-
-
observed for a female
not
detected and
Females)
8
10
12
_
13
0
11
12
11
-
11
-
-
-
0
11
-
11
-
5
-
-
.
11
-
13
-
13
-
11
11
0
10
-
12
12
6
-
®
that was
the female was
12
12
13
_
13
10
13
11
9
11
10
-
-
8
12
14
-
13
-
0
-
-
-
8
10
7
-
13
-
12
-
14
-
-
•4*4
/B\ 1 1
-
-
not p
not
Third
Breeding
(3 Females)
9
15
13
11
11
"( )" indicates all implants were in early stages of development and impossible
to determine if they were live or dead upon gross observation.
"(P)" indicates female was pregnant but had no live-implants.
204
-------
Treatment
Captan
5000 ppm
Captan
5000 ppm
Fl
Male
No.
638
646
653
657
660
668
671
672
679
722
732
733
734
736
743
760
761
765
767
769
779
780
Totals
805
806
815
816
817
818
822
823
837
841
846
847
Table 11
(Concluded)
First Breeding
10
10
0*
2
0
-
-
-
6
-
4
-
-
-
0
7
9
9
10
8
2
4
-
-
6
-
-
10
-
-
-
.
5
(14)
(3 Females)
6
**
(13) *
5
10
-
-
-
0
-
-
-
-
-
-
5
-
10
-
0
0
1
30
-
-
9
(10)
-
6
-
-
-
-
-
-
6
_
-
1
8
6
-
-
8
_
-
-
-
-
(15)
8
-
-
-
(12)
1
5
-
-
7
(ID
-
-
-
-
-
-
-
-
Second Breeding
(3
10
11
14
2
14
9
14
-
7
_
12
_
11
_
14
1
5
12
12
12
0
0
-
12
13
9
7
11
6
13
7
13
10
10
Females)
12
13
13
1
9
12
11
-
12
_
-
_
12
-
10
0
13
11
13
13
4
2
9
.
14
11
-
0
8
-
-
-
10
12
10
11
9
10
4
12
12
_
14
3
_
-
_
-
_
10
0
11
9
12
10
-
4
2
10
10
-
-
12
-
-
-
-
12
14
Third
Breeding
(3 Females)
12
11
10
2 13
12 12
Totals
12
Traumatized Fo females.
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development and impossible
to determine if they were live or dead upon gross observation.
205
-------
Table 12
TRANSLOCATION STUDY OF CAFTAN
CYTOGENETIC EVALUATION OF Fl MALE MICE
Fl
Male
Treatment No.
Control
NJ
o
16
17
65
72
122
164
Body
Weight (g)
64.6
58.5
38.8
68.0
48.7
55.8
TEM
Captan
2500 ppm
189
190
232
262
290
345
361
449
474
479
480
496
523
526
583
49.6
47.5
54.6
41.4
62.6
54.1
50.4
60.2
56.5
65.1
58.7
60.9
50.7
54.1
50.7
Testes
Weight (mg)
265
307
237
232
289
314
245
222
276
243
222
294
278
256
175
287
307
242
348
295
297
Classification-Based
Upon Breeding Data
Partially sterile
(questionable)
Nonbreeder
Partially sterile
Presumptive sterile
Partially sterile
(questionable)
Partially sterile
(questionable)
Partially sterile
Partially sterile
Partially sterile
Partially sterile
Partially sterile
Partially sterile
Partially sterile
Nonbreeder
Presumptive sterile
Nonbreeder
Partially sterile
(questionable)
Nonbreeder
Nonbreeder
Partially sterile
Nonbreeder
Cytogenetic Classification
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Positive reciprocal
Positive reciprocal
Positive reciprocal
Positive reciprocal
Positive reciprocal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
translocation
translocation
translocation
translocation
translocation
-------
Treatment
Captan
5000 ppm
Captan
5000 ppma
Fl
Male
No.
634
635
657
733
736
760
779
780
805
837
Body
Weight (g)
57.1
56.0
61.0
62.2
57.9
55.9
54.8
53.0
70.0
56.9
Testes
Weight (mg)
299
312
231
256
256
298
224
295
268
303
Table 12
(Concluded)
Classification-Based
Upon Breeding Data
Nonbreeder
Partially sterile
Partially sterile
Nonbreeder
Presumptive sterile
Partially sterile
Partially sterile
Partially sterile
Partially sterile
Partially sterile
(questionable)
Cytogenetic Classification
Normal
Normal
Positive reciprocal translocation
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Traumatized Fo females.
-------
Table 13
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES
Control Group
F, Male
Number
First 11
breeding
16
17
24
39
43
50
59
65
67
72
86
102
122
Vc
"n" l nA t oat-A0 Q
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
-**
-
-
(13)t
.
.
9*
0
.
-
4
8
7
0
-
-
5
0
5
-
.
.
-
(H)
16
4
13
Dead
Implantations
-
-
-
-
1
.
-
.
0
0
-
-
0
1
1
0
-
1
0
0
.
-
-
-
-
9
10
2
0
Live
Implantations
-
.
-
(13)
-
-
-
-
9
0
-
-
4
7
6
0
-
-
4
0
5
-
-
-
.
-
-
(11)
6
2
13
Initial Classification
Nonbreeder
Partially sterile
(questionable)
Nonbreeder
Nonbreeder
Normal
Nonbreeder
Partially sterile
Presumptive sterile
Partially sterile
Nonbreeder
Nonbreeder
Nonbreeder
Partially sterile
(questionable)
Normal
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
208
-------
Table 13 (Continued)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES
Control Group
First
breeding
(concl.)
Second
breeding
FI Male
Number
129
139,
144
152
164
179
189
190
192
200
11
16
17
Female
Number
1
2
3
I
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
**
-
-
-
7
12
9
-
-
0*
1
0
1
4
6
7
(14) +
"
13
9
(15)
-
Dead Live
Implantations Implantations
-
-
-
-
-
..
4 3
3 9
0 9
-
.
-
0 0
0 1
0 0
0 1
0 4
0 6
0 7
? (14)
-
0 13
1 8
? (15)
-
Initial Classification
Nonbreeder
Nonbreeder
Nonbreeder
Partially sterile
Nonbreeder
Nonbreeder
Partially sterile
Partially sterile
Partially sterile
(questionable)
Nonbreeder
Final Classification
Normal
'' Rebred
b
Rebred
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
*"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live 01 dead upon gross observation.
See third breeding for final classification.
209
-------
Table 13 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES
Control Group
Second
breeding
(cont.)
F, Male
Number
24
39
43
50
59
65
67
72
86
102
122
129
139
144
1-inai-ac a
Female
Number
1
2
3
\
2
3
I
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
0*
12
**
0
-
-
-
12
11
12
10
12
3
6
2
15
11
-
0
-
(12) +
10
6
16
14
14
2
_
-
15
-
-
9
10
Dead
Implantations
0
1
-
0
-
-
0
0
0
0
3
-
0
0
0
1
0
0
-
-
?
-
1
1
0
7
7
8
1
-
0
0
Live
Implantations
0
11
-
0
-
.
-
-
12
11
12
10
9
3
6
2
14
11
-
0
-
-
(12)
-
-
9
5
16
7
7
4
-
-
14
-
-
9
10
Final Classification
Normal
b
Rebred
h
Rebred
Normal
Normal
Partially sterile
Normal
b
Rebred
b
Rebred
Normal
Normal
Rebredb
Normal
Normal
t,,
'-" indicates a plug was not detected and the female was not pregnant.
()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
See third breeding for final classification.
210
-------
Table 13 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES
Control Groun
Second
breeding
(concl.)
Third
breeding
?i Male
Number
152
164
179
189
190
192
200
16
17
39
43
72
86
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1 1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
11
8
14
_**
(9)t
11
-
-
0*
0
0
1
0
0
13
12
13
Dead
Implantations
1
0
1
?
1
0
0
0
0
0
0
1
1
-
0
Live
Implantations
10
8
13
„
(9)
10
-
0
0
0
1
0
0
12
! 11
-
13
-
Final Classification
Normal
Rebred
Normal
Rebred
Rebred
Normal
Normal
Partially sterile
(questionable)
-
-
12
12
11
10
-
-
14
14
5
_
0
1
0
0
-
-
2
2
1
-
.
12
11
11
10
-
-
12
12
4
Nonbreeder
Normal
Normal
Presumptive
Normal
sterile
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
See third breeding for final classification.
211
-------
Table 13 (Concluded)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES
Control Group
F, Male
Number
Third 129
breeding
(concl.)
164
189
190
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
14
**
-
-
-
0*
1
-
0
1
1
Dead
Implantations
0
-
-
-
0
0
-
0
0
0
Live
Implantations
14
-
-
.
-
<•
0
1
-
0
1
1
Final Classification
Normal
Partially sterile
(questionable)
Partially sterile
Partially sterile
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
212
-------
Table 14
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE FI MALES
TEM Group
F, Male
Number
First 202
breeding
215
216
220
226
227
228
229
230
231
232
233
235
237
Female
Number
]•
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
0*
0
0
**
.
-
(9) +
-
-
-
_
-
0
11
11
-
(9)
8
0
10
-
0
12
9
10
15
11
11
5
12
12
Dead
Implantations
0
0
0
_
-
7
.
-
-
0
7
9
7
1
0
1
-
0
9
5
9
7
4
5
0
3
4
Live
Implantations
0
Q
Q
_
-
-
(9)
-
-
0
4
2
(9)
7
0
9
.
-
0
-
3
4
1
-
-
-
8
7
6
5
9
8
Initial Classification
Presumptive sterile
Nonbreeder
Partially sterile
(questionable)
Nonbreeder
Nonbreeder
Partially sterile
Partially sterile
(questionable)
Normal
Nonbreeder
Presumptive sterile
Partially sterile
Nonbreeder
Partially sterile
Partially sterile
'"0" indicates n plug was observed for a female that was not pregnant.
"""-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
213
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES
TEM Group
F Male
Number
First 238
breeding
(cont.)
239
241
244
245
251
253
254
256
258
262
264
267
268
269
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
14
0*
9
_**
-
.
-
.
-
-
6
0
4
0
0
0
12
12
-
6
-
-
0
-
0
-
-
12
12
15
8
8
.
-
_
Dead
Implantations
11
0
8
-
-
-
-
_
-
6
0
3
0
0
0
9
2
-
2
0
-
-
0
-
10
4
10
5
3
-
-
-
_
-
-
Live
Implantations
3
P
\
,
.
-
-
-
-
.
-
0
0
1
0
0
0
3
10
-
4
-
-
0
0
-
-
2
8
5
3
5
-
_
-
_
-
-
_
-
Initial Classification
Partially sterile
Nonbreeder
Nonbreeder
Nonbreeder
Partially sterile
Presumptive sterile
Normal
Partially sterile
Presumptive sterile
Presumptive sterile
Partially sterile
Partially sterile
Nonbreeder
Nonbreeder
Nonbreeder
"0" indicates a plug was observed for a female that was not pregnant.
k
"-" indicates a plug was not detected and the female was not pregnant.
214
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F,
MALES
TEM Group
?i Male
Number
First 270
breeding
(cont.)
273
280
281
283
286
288
290
292
294
299
300
302
314
Female
Number
1
2
3
I
2
3
1
2
3
1
£
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
0*
-**
-
.
-
13
12
3
4
12
-
12
11
9
-
0
0
0
9
14
11
14
10
0
8
(18)t
11
11
-
9
(15)
-
11
-
-
-
-
Dead
Implantations
0,
-
_
.
-
4
5
1
4
6
3
2
1
.
-
0
0
0
4
9
7
8
7
0
0
7
2
3
-
-
3
7
-
2
-
-
-
-
Live
Implantations
0
-
-
w
.
-
9
7
2
0
6
-
9
9
8
.
-
0
0
0
5
5
4
6
3
0
8
(18)
9
8
-
-
6
(15)
-
9
-
-
-
-
-
Initial Classification
Presumptive sterile
Nonbreeder
Partially sterile
Partially sterile
Normal
Nonbreeder
Presumptive sterile
Partially sterile
Partially sterile
Partially sterile
(questionable)
Normal
Partially sterile
(questionable)
Normal
Nonbreeder
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
'()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
215
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES
F, Male
Number
First 315
breeding
(cont.)
317
318
321
322
326
327
333
334
339
343
344
345
346
349
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
0*
3
0
0
_**
-
9
13
13
0
0
-
10
-
-
-
0
-
0
11
-
-
-
-
0
-
0
0
0
10
12
14
10
10
.
-
(12) +
Dead
Implantations
0
0
0
0
5
9
10
0
0
-
1
.
-
_
-
-
0
-
0
5
-
_
-
0
-
0
0
0
7
7
-
8
6
5
-
-
-
7
Live
Implantations
0
3
0
0
-
4
4
3
0
0
-
9
-
-
.
.
0
-
0
6
-
.
-
-
0
0
0
0
' 3
5
-
6
4
5
-
-
-
(12)
Initial Classification
Partially sterile
Presumptive sterile
Partially sterile
Presumptive sterile
Normal
Nonbreeder
Presumptive sterile
Partially sterile
Nonbreeder
Presumptive sterile
Presumptive sterile
Partially sterile
Partially sterile
Nonbreeder
Partially sterile
(questionable)
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
216
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES
TEM Group
First
breeding
(cont.)
F Male
Number
350
355
356
359
360
361
363
367
369
371
372
375
376
381
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3 '
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
12
11
8
-**
-
-
15
12
16
(15) +
-
12
14
11
9
-
-
_
-
0*
-
10
13
(16)
-
-
14
-
11
0
10
_
Dead
Implantations
8
8
6
6
8
8
•7
-
8
10
2
4
-
-
.
-
0
-
6
7
7
11
-
6
0
4
Live
Implantations
4
3
2
.
-
9
4
8
(15)
-
-
4
4
9
5
-
-
0
-
4
6
-
(16).
3
5
0
6
Initial Classification
Partially sterile
Nonbreeder
Nonbreeder
Partially sterile
Partially sterile
(questionable)
Partially sterile
Partially sterile
Nonbreeder
Presumptive sterile
Partially sterile
Partially sterile
(questionable)
Partially sterile
Partially sterile
Nonbreeder
"0" indicates a plug was observed for a female that was not pregnant.
f>.
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
217
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OE PRESUMPTIVE F
MALES
TEM Group
F. Male
Number
First 382
breeding
(concl.)
385
387
388
389
390
391
396
397
399
400
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
(13) +
(11)
.**
9
6
(6)
.
-
-
14
9
0*
_
-
-
11
12
14
-
-
-
.
.
-
.
-
(14)
11
9
(11)
12
9
(12)
Dead
Implantations
7
?
0
1
?
.
-
-
11
5
0
-
-
-
7
9
8
-
-
-
-
-
7
6
5
7
10
7
7
Live
Implantations
(13)
(11)
9
5
(6)
.
-
-
3
4
0
_
-
4
3
5
.
-
-
.
-
-
-
(14)
5
4
(11)
2
2
(12)
Initial Classification
Partially sterile
(questionable)
Partially sterile
Nonbreeder
Partially sterile
Nonbreeder
Partially sterile
Nonbreeder
Nonbreeder
Partially sterile
(questionable)
Partially sterile
(questionable)
Partially sterile
(questionable)
Second
breeding
202
215
0
0
0
11
9
Final Classification
Presumptive sterile
Partially sterile
"0" indicates a plug was observed for a female that was not pregnant.
indicates a plug
and the female was
pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
218
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F,
MALES
Second
breeding
(cont.)
FI Male
Number
216
220
226
227
228
229
230
231
232
233
235
237
238
239
241
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
.**
_
-
14
5
-
12
11
8
14
12
12
15
14
13
11
10
-
0*
0
0
17
12
15
11
15
7
11
13
14
11
14
14
9
12
13
13
-
-
.
-
Dead
Implantations
.
-
2
3
-
0
7
0
0
0
1
1
0
0
0
0
0
0
0
9
10
10
0
1
1
4
6
1
1
4
1
8
10
13
0
-
-
Live
Implantations
.
.
-
12
2
-
12
4
8
14
12
11
14
14
13
11
10
0
0
0
8
2
5
11
15
6
7
7
13
10
10
14
1
2
0
13
-
-
-
Final Classification
Rebredb
Rebredb
Normal
Normal
Normal
Normal
Normal
Presumptive sterile
Partially
Normal
Normal
Normal
Partially
Normal
Rebred
sterile
sterile
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
See third breeding for final classification.
219
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE FI MALES
TEM Group
Second
breeding
(cont.)
j
*»n" 4nj
F. Male
Number
244
245
251
253
254
256
258
262
264
267
268
269
270
273
280
.
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
12
13
11
1
0*
7
0
0
0
16
11
.**
13
14
11
_
-
-
13
0
-
8
12
10
9
11
12
14
1
14
.
-
6
7
8
.
-
0
-
-
11
11
10
_1 1 C _
Dead
Implantations
2
0
0
0
0
4
0
0
0
2
1
-
0
0
0
-
-
0
0
-
3
8
5
9
6
3
1
1
2
_
-
-
0
0
-
0
.
-
0
-
-
3
3
5
Live
Implantations
10
13
11
1
0
3
0
0
0
14
11
-
13
14
11
_
-
-
13
0
-
5
4
5
0
5
9
13
0
12
.
-
6
7
-
8
-
0
-
8
8
5
Final Classification
Normal
Partially
sterile
Presumptive sterile
Normal
Normal
Rebred
Normal
Partially
Partially
Normal
Rebredb
Partially
_ . ,b
Rebred
Rebredb
Partially
sterile
sterile
sterile
sterile
"-" indicates a plug was not detected and the female was not pregnant.
See third breeding for final classification.
220
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F,
MALES
TEM Group
F, Male
Number
Second 281
breeding
(cont .)
283
286
288
290
292
294
299
300
302
314
315
317
318
321
Female
Number
1
2
3
I
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
11
12
10
14
0*
.**
.
0
0
0
13
8
0
9
14
11
11
12
12
14
12
12
13
9
12
13
13
15
7
12
12
0
0
0
0
0
-
14
9
13
0
0
Dead
Implantations
7
11
8
0
0
-
.
-
0
0
0
6
3
0
6
11
3
1
0
1
5
8
3
5
4
10
0
1
0
5
11
8
0
0
0
0
0
5
5
2
0
0
Live
Implantations
4
1
2
14
0
-
_.
-
0
0
0
7
5
0
3
3
8
10
12
11
9
4
9
8
5
2
13
12
15
2
1
4
0
0
0
0
0
-
9
4
11
0
0
"
Final Classification
Partially sterile
Normal
Rebred
Presumptive sterile
Partially sterile
Partially sterile
Normal
Normal
Partially sterile
(questionable)
Normal
Partially sterile
Partially sterile
Presumptive sterile
Normal
Presumptive sterile
"0" indicates a plug was observed for a female that was not pregnant.
if
"-" indicates a plug was not detected and the female was not pregnant.
See third breeding for final classification.
221
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F,
MALES
TEM Group
F. Male
Number
Second 322
breeding
(cont.)
326
327
333
334
339
343
344
345
346
349
350
355
356
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
' 1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
11
11
**
0*
13
(10)t
-
12
0
-
-
-
-
0
0
0
0
0
0
10
10
11
6
12
4
12
12
-
10
11
12
8
0
10
13
0
-
11
-
(15)
Dead
Implantations
0
0
-
0
-
-
13
7
2
0
-
-
-
0
0
0
,0
0
0
5
6
6
2
8
1
0
1
-
0
3
2
8
0
9
0
0
-
0
.
7
Live
Implantations
11
11
-
0
-
-
0
(10)
10
0
-
-
0
0
0
0
0
0
5
4
5
4
4
3
12
11
-
10
8
10
0
0
1
13
0
-
11
-
(15)
Final Classification
Normal
Presumptive
b
Rebred
Normal
b
Rebred
Presumptive
Presumptive
sterile
sterile
sterile
Partially sterile
Partially sterile
Normal
Normal
Partially sterile
Normal
Normal
t
"0" indicates a plug was observed for a female that was not pregnant.
*
indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
b
See third breeding for final classification.
222
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE FI MALES
TEM Group
Second
breeding
(cont.)
F Male
Number
359
360
361
363
367
369
371
372
375
376
381
382
385
387
388
""0" indicates a
it*
"-" ind
1 1 r R t p s a
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
plug was
nine was
Total
Implantations
10
9
16
10
13
-**
13
13
8
14
14
10
11
11
10
11
14
11
9
5
11
1
4
11
0*
10
12
7
15
12
6
12
observed for
not detected
Dead
Implantations
3
6
9
8
13
10
6
8
2
0
-
6
6
0
-
5
6
9
1
0
0
4
0
3
0
0
-
-
0
0
0
0
9
3
8
Live
Implantations
7
3
7
2
0
-
3
7
0
12
14
4
5
11
-
5
5
5
10
9
5
7
1
1
11
0
-
-
10
12
7
15
-
3
3
4
Final Classification
Partially sterile
Partially sterile
(questionable)
Partially sterile j
Normal
Normal
b
Rebred
Partially sterile
Normal
Partially sterile
Partially sterile
Normal
b
Rebred
Normal
Normal
Partially sterile
a female that was not pregnant.
and the female v
jas not pregnant
See third breeding for final classification.
223
-------
Table 14 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES
TEM Group
Second
breeding
(concl.)
Third
breeding
*"fl" ,'r,^
F. Male
Number
389
390
391
396
397
399
400
216
220
241
256
268
270
273
.
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
_**
-
-
14
12
12
.
-
-
-
-
-
11
0*
0
14
7
4
9
13
3
9
-
.
-
-
.
-
_
-
0
-
9
-
-
_
-
Dead
Implantations
-
11
7
9
.
-
0
0
0
9
5
0
5
8
1
5
-
-
-
_
-
«
-
0
-
0
-
-
_
-
«T . 1 _ ,_1__._
Live
Implantations
_
-
-
2
5
3
_
-
_
-
-
11
0
0
5
2
4
4
5
2
4
-
-
_
.
-
_
.
-
_
-
0
-
-
9
-
-
_
.
Final Classification
b
Rebred
Partially sterile
Rebred
Rebred
Normal
Partially sterile
( ques tionab le)
Partially sterile
(questionable)
Partially sterile
(questionable)
Nonbreeder
Nonbreeder
Presumptive sterile
Presumptive sterile
Normal
Presumptive sterile
"-" indicates a plug was not detected and the female was not pregnant.
See third breeding for final classification.
224
-------
Table 14 (Concluded)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES
TEM Group
Third
breeding
(concl.)
F. Male
Number
286
327
334
369
382
389
391
396
*"0" indicates a
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
plug was
Total
Implantations
11
_**
11
(9)t
14
12
-
-
••
11
10
-
0*
~
-
_
observed for a
Dead
Implantations
1
10
2
0
-
_
0
0
-
0
~
-
-
-
Live
Implantations
10
1
(9)
12
12
-
"
11
10
-
0
.
.
-
-
-
Final Classification
Normal
Partially sterile
(questionable)
Normal
Presumptive sterile
Normal
Presumptive sterile
Nonbreeder
Nonbreeder
female that was not pregnant.
t,,
"-" indicates a plug was not detected and the female was not pregnant.
()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
225
-------
Table 15
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F,
MALES
2500 ppm Group
F, Male
Number
First 430
breeding
432
436
449
450
451
452
455
461
469
472
474
479
480
484
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
_**
-
-
4
(17)t
9
14
7
11
-
-
0*
5
14
11
0
10
12
13
-
-
-
.
-
-
9
11
12
_
-
-
-
-
(3)
0
-
11
16
11
Dead
Implantations
.
-
4
9
9
5
2
3
.
-
0
4
0
4
0
-
1
2
3
-
-
-
-
-
1
1
3
_
-
-
_
-
?
0
7
2
0
Live
Implantations
-
-
4
(17)
9
9
5
8
.
.
-
0
1
14
7
0
-
9
10
10
.
-
-
_
-
_
-
8
10
9
_
-
-
_
-
-
(3)
0
-
4
14
11
Initial Classification
Nonbreeder
Partially sterile
(questionable)
Partially sterile
Nonbreeder
Normal
Normal
Normal
Nonbreeder
Nonbreeder
Nonbreeder
Normal
Nonbreeder
Nonbreeder
Partially sterile
Normal
"0" indicates a plug was observed for a female that was not pregnant.
>r»
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
226
-------
Table 15 (Continued)
TRANSIJOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES
2500 ppm Group
F, Male
Number
First 489
breeding
(concl.)
496
518
523
526
528
546
561
568
569
582
583
585
588
590
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
14
13
8
_**
-
-
-
-
7
4
(l)t
0*
-
.
-
-
-
12
(9)
(13)
.
-
10
11
15
-
(12)
11
0
9
Dead
Implantations
3
1
2
-
-
-
.
-
0
1
7
0
-
-
-
.
-
4
•?
7
-
-
-
-
1
1
3
7
3
0
0
Live
Implantations
11
12
6
-
-
_
-
_
-
7
3
(1)
0
-
.
-
-
8
(9)
(13)
.
-
-
.
-
-
-
9
10
12
-
(12)
8
0
9
Initial Classification
Normal
Nonbreeder
Nonbreeder
Nonbreeder
Partially sterile
Presumptive sterile
Nonbreeder
Nonbreeder
Partially sterile
(questionable)
Nonbreeder
Nonbreeder
Nonbreeder
Normal
Partially sterile
(questionable)
Normal
"0" indicates a plug was observed for a female that was not pregnant.
•>Wf
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
227
-------
Table 15 (Continued)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES
2500 ppm Group
F. Male Female
Number Number
Second 430
breeding
432
436
449
450
451
452
455
461
469
472
474
479
480
*"0" indicates a p
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
lug was
Total Dead Live
Implantations Implantations Implantations Final Classification
12
8
14
12
11
13
12
12
13
_**
-
-
11
13
1 14
0*
0
11
14
11
13
10
12
12
13
11
11
.
-
12
0
12
11
0
-
_
.
-
(13)t
-
8
observed for
0
0
2
1
1
1
1
0
0
-
-
-
1
0
1
0
0
1
1
0
0
0
0
1
0
0
2
.
-
1
0
1
1
0
_
-
-
•7
0
a female that was
12
8
12
11
10
12
11
12
13
-
-
-
10
13
13
0
0
10
13
11
13
10
12
11
13
11
9
.
-
11
0
11
10
0
-
_
_
-
(13)
-
8
not pregns
Normal
Normal
Normal
b
Rebred
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Rebredb
Rebredb
Rebred
mt.
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
b
See third breeding for final classification.
228
-------
Table 15 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE 7 MALES
2500 ppm Group
FA Male
Number
Second 484
breeding
(cont.)
489
496
518
523
526
528
546
561
568
569
582
583
585
588
Female
Number
1
2
3
I
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
11
0*
14
9
13
14
**
0
11
13
-
-
0
5
0
15
-
.
-
-
-
11
11
8
11
10
7
15
10
-
-
12
13
13
9
-
-
Dead
Implantations
0
0
2
0
2
0
.
.
0
0
0
-
-
0
0
0
1
-
-
-
-
2
0
0
0
0
0
2
2
-
0
0
0
0
Live
Implantations
11
0
12
9
11
14
,
_
-
0
11
13
-
0
5
0
14
-
-
-
-
-
-
9
11
8
11
-
10
7
13
8
12
13
13
9
Final Classification
Normal
Normal
Rebred
Normal
b
Rebred
b
Rebred
Normal
Rebred
b
Rebred
Normal
Normal
Normal
b
Rebred
Normal
b
Rebred
"0" indicates a plug was observed for a female that was not pregnant.
iff
"-" indicates a plug was not detected and the female was not pregnant.
b
See third breeding for final classification.
229
-------
Table 15 (Concluded)
i
TRANSLOGATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F,
MALES
2500 ppm Group
Second
breeding
(concl.)
Third
breeding
Fj^ Male
Number
590
449
474
479
480
496
523
526
546
561
583
588
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
0*
13
12
_**
_
-
.
.
-
10
-
-
.
-
-
-
-
2
1
0
9
13
-
15
11
-
-
-
12
-
Dead
Implantations
0
2
0
.
-
-
_
-
-
.
-
-
8
-
-
_
-
-
-
0
0
0
0
0
-
0
0
-
_
-
-
1
-
Live
Implantations
0
11
12
_
-
-
.
-
-
-
-
2
-
-
.
-
-
.
-
-
2
1
0
9
13
-
15
11
-
_
-
11
-
Final Classification
Normal
Nonbreeder
Presumptive sterile
Nonbreeder
Partially sterile
(questionable)
Nonbreeder
Nonbreeder
Partially sterile
Normal
Normal
Nonbreeder
Normal
"0" indicates a plug was observed for a female that was not pregnant.
!t
"-" indicates a plug was not detected and the female was not pregnant.
230
-------
Table 16
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F,
MALES
5000 ppm Group
F. Male
Number
First 601
breeding
604
620
622
626
627
634
635
638
646
653
657
660
668
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
_**
(ll)t
(8)
7
11
14
8
-
(9)
9
11
8
-
10
6
6
12
-
0*
(13)
-
9
13
8
0
11
8
7
Dead
Implantations
_
7
2
0
2
5
0
-
-
?
-
1
3
0
-
-
T
0
0
0
2
0
?
-
7
8
7
0
1
0
1
Live
Implantations
_
(11)
(8)
7
9
9
8
(9)
-
-
8
8
8
-
-
10
6
6
10
0
(13)
2
5
1
0
10
8
6
Initial Classification
Partially sterile
(questionable)
Normal
Normal
Partially sterile
(questionable)
Nonbreeder
Normal
Nonbreeder
Nonbreeder
Normal
Normal
Partially sterile
(questionable)
Partially sterile
Normal
Partially sterile
"0" indicates a plug was observed for a female that was not pregnant.
**
"-" indicates a plug was not detected and the female was not pregnant.
'"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
231
-------
Table 16 (Continued)
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE f MALES
5000 ppm Group
First
breeding
(cont.)
F, Male
Number
671
672
679
722
732
733
734
736
743
760
761
765
767
769
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3 '
Total
Implantations
_**
.
-
-
6
0*
12
_
-
4
-
-
.
-
.
-
0
-
(15)t
8
5
9
12
-
12
11
-
12
-
-
8
-
(12)
Dead
Implantations
.
-
-
.
-
0
0
4
_
-
0
-
.
-
_
-
-
0
-
7
1
0
1
3
-
3
1
2
-
8
-
9
Live
Implantations
-
-
.
-
-
6
0
8
_
.
-
4
-
.
-
-
-
-
-
-
0
-
(15)
7
5
8
9
.
-
9
10
-
10
-
-
8
-
(12)
Initial Classification
Nonbreeder
Nonbreeder
Partially sterile
Nonbreeder
Partially sterile
Nonbreeder
Nonbreeder
Nonbreeder
Partially sterile
(questionable)
Partially sterile
Normal
Normal
Normal
Partially sterile
(questionable)
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
232
-------
Table 16 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES
5000 ppm Group
F Male
Number
First
breeding
(concl.)
Second
breeding
779
780
601
604
620
622
626
627
634
635
638
646
653
Female
Number
1
2
3
I
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total Dead
Implantations Implantations
2
0*
1
4
1
5
12
0
_**
9
10
15
10
0
12
(12)t
12
13
6
1
-
-
1
10
12
11
11
13
10
14
13
10
0
0
0
0
0
0
1
0
-
0
0
1
1
0
0
-
?
0
3
0
1
-
1
0
0
0
0
0
1
0
0
0
Live
Implantations Initial Classification
2
0
1
4
1
5
11
0
9
10
14
9
-
-
0
12
-
(12)
12
10
6
0
-
-
-
0
-
10
12
11
11
13
9
14
13
10
Partially sterile
Partially sterile
Final Classification
Normal
Normal
Normal
Normal
Normal
Normal
b
Rebred
b
Rebred
Normal
Normal
Normal
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
See third breeding for final classification.
233
-------
Table 16 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES
5000 ppm Group
Second
breeding
(cont .)
*
"n" ir,,
F. Male
Number
657
660
668
671
672
679
722
732
733
734
736
743
760
761
765
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
7
9
5
14
10
12
9
13
13
14
11
.**
-
14
7
12
4
-
12
-
-
-
-
11
12
-
-
-
-
14
10
11
1
0*
0
5
14
12
15
11
9
Dead
Implantations
5
8
1
0
1
0
0
1
1
0
0
-
-
0
0
0
1
-
-
0
-
-
0
0
-
0
0
1
0
0
0
0
1
1
3
0
0
Live
Implantations
2
1
4
14
9
12
9
12
12
14
11
-
-
14
7
12
3
-
12
-
11
12
-
-
-
-
14
10
10
1
0
0
5
13
11
12
11
9
Final Classification
Partially sterile
Normal
Normal
Normal
Normal
Normal
b
Rebred
Normal
b
Rebred
Normal
b
Rebred
Normal
Partially sterile
Normal
Normal
"-" indicates a plug was not detected and the female was not pregnant.
See third breeding for final classification.
234
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Table 16 (Concluded)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES
5000 ppm Group
Second
breeding
(concl.)
Third
breeding
Fj^ Male
Number
767
769
779
780
634
635
722
733
736
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
14
13
12
12
13
10
0*
4
_**
0
2
5
.
-
-
13
-
0
Dead
Implantations
2
0
0
0
0
0
0
0
0
0
1
-
1
-
-
0
-
-
Live
Implantations
12
13
12
12
13
10
0
4
0
2
4
-
12
-
-
0
-
Final Classification
Normal
Normal
Partially sterile
Partially sterile
Nonbreeder
Partially sterile
Normal
Nonbreeder
Presumptive sterile
"0" indicates a plug was observed for a female that was not pregnant.
!f
"-" indicates a plug was not detected and the .female was not pregnant.
235
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Table 17
TRANSLOCATION STUDY OF CAPTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES
5000 ppm Group
First
breeding
Second
breeding
Fj^ Male
Number
805
806
815
816
817
818
822
823
837
841
846
847
805
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
**
-
-
6
9
7
(10)t
(11)
-
-
10
6
-
-
-
-
.
-
_
-
5
-
(14)
-
"
-
2
Dead
Implantations
-
-
-
0
0
0
?
1
-
0
0
-
-
-
-
_
-
0
-
7
-
0
Live
Implantations
.
-
-
-
6
9
7
-
(10)
(11)
-
10
6
-
-
-
.
-
-
-
-
-
5
-
(14)
-
~
2
Initial Classification
Nonbreeder
Nonbreeder
Partially sterile
Partially sterile
(questionable)
Nonbreeder
Normal
Nonbreeder
Nonbreeder
Nonbreeder
Nonbreeder
Partially sterile
Partially sterile
(questionable)
Final Classification
Rebred
Indicates traumatized FQ females.
•,-
"0" indicates a plug was observed for a female that was not pregnant.
"-" indicates a plug was not detected and the female was not pregnant.
"()" indicates all implants were in early stages of development, and thus difficult to
determine if they were live or dead upon gross observation.
b
See third breeding for final classification.
236
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Table 17 (Continued)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES
5000 pptn Group
Second
breeding
(concl.)
Third
breeding
V Male
Number
806
815
816
817
818
822
823
837
841
846
847
805
817
822
Female
Number
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Total
Implantations
13
14
10
14
11
13
11
_**
-
11
0*
11
8
13
6
13
-
8
-
15
10
10
12
12
11
10
14
-
13
9
13
10
12
12
Dead
Implantations
1
0
0
1
0
3
2
-
4
0
0
0
1
0
-
0
1
2
0
0
0
0
1
0
0
-
2
7
0
0
0
0
Live
Implantations
12
14
10
13
11
10
9
-
7
0
11
8
12
6
-
13
.
-
7
-
13
10
10
12
12
10
10
14
_
11
2
13
10
12
12
Final Classification
Normal
Normal
Normal
Rebred
Normal
b
Rebred
Normal
b
Rebred
Normal
Normal
Normal
Partially sterile
Normal
Normal
Indicates traumatized F- females .
*
"0" indicates a plug was observed for a female that was not pregnant.
)V
"-" indicates a plug was not detected and the female was not pregnant.
See third breeding for final classification.
237
-------
Table 17 (Concluded)
TRANSLOCATION STUDY OF CAFTAN
IMPLANTATION SUMMARY OF PRESUMPTIVE FI MALES
5000 ppm Group
FA Male
Number
Third 837
breeding
(concl.)
Female Total Dead
Number Implantations Implantations
2
3
Live
Implantations Final Classification
Normal
Indicates traumatized Fn females.
"-" indicates a plug was not detected and the female was not pregnant.
238
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/1-77-028
3. RECIPIENT'S ACCESSION NO.
. TITLE ANDSUBTITLE
EVALUATION OF SELECTED PESTICIDES AS CHEMICAL MUTAGENS
In Vitro and In Vivo Studies
5. REPORT DATE
May 1977
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
Vincent F. Simmon, Ann D. Mitchell and Ted A. Jorgenson
8. PERFORMING ORGANIZATION REPORT NO.
LSU-3493
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Stanford Research Institute
Menlo Park, California 94025
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
68-01-2458
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, N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
twenty pesticides being reviewed as part of the EPA Substitute Chemical Program
were studied for mutagenic activity by several in vitro and in vivo test procedures.
The pesticides reviewed were: monocrotophos, bromacil, cacodylic acid, captan,
chlorpyrifos, dinoseb, DSMA, fenthion, folpet, azinphos-methyl, malathion, methomyl,
monuron, MSMA, parathion, parathion-methyl, quintozene (PCNB), phorate, simazine,
and trifluralin.
Ten of the twenty compounds were evaluated in vivo by the mouse dominant lethal
test. All twenty compounds were tested in vitro. None of the ten compounds tested
in the mouse produced a dominant lethal response. Ten of the twenty compounds were
mutagenic in one or more in vitro assays. Two were mutagenic in all of the in vitro
assays: captan and folpet.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
pesticides
in vivo analysis
in vitro analysis
mutagens
06
06
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
249
20. SECURITY CLASS (This page)
22. PRICE
EPA Form 2220-1 (9-73)
239
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