United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600 1-79-004b
January 1979
Research and Development
Carcinogenic
Potential of
Rotenone
Phase II:
Oral and
Intraperitoneal
Administration to
Rats
1 V"
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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/l-79-004b
January 1979
CARCINOGENIC POTENTIAL OF ROTENONE
PHASE II: ORAL AND INTRAPERITONEAL ADMINISTRATION TO RATS
by
A. P. Leber and D. C. Thake
Battelles1, Columbus Laboratories
Columbus, Ohio 43201
Contract No. 68-02-1715
Project Officer
Ronald L. Baron
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
-------
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.
-------
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 estab-
lishment 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, epidemio-
logy, and clinical studies using human volunteer subjects. These studies
address problems in air pollution, non-ionizing radiation, environmental
carcinogenesis and the toxicology of pesticides as well as other chemical
pollutants. The Laboratory participates in the development and revision
of air quality criteria documents on pollutants for which national ambient
air quality standards exist or are proposed, provides the data for registra-
tion of new pesticides or proposed suspension of those already in use,
conducts research on hazardous and toxic materials, and is primarily respon-
sible for providing 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 endanger-
ment of their health.
Rotenone, the extract of derris root, has long been used as an insecti-
cide and fish poison. A recent report by the Spanish investigators Gosalvez
and Merchan has shown rotenone to produce mammary tumors in rats by intra-
peritoneal or dietary dosage. The present study was carried out to determine
the possible carcinogenic effect of rotenone in two different rat strains.
F. G. Hueter, Ph.D
Director
Health Effects Research Laboratory
m
-------
Abstract
A published report has described mammary tumors in rats dosed
with rotenone either intraperitoneally or in the diet. The present
study was carried out to determine the possible carcinogenic effect
of the compound in two different rat strains.
In the intraperitoneal study, test groups of male and female
Sprague-Dawley rats were given daily doses of 0, 1.7 or 3.0 mg/kg
of rotenone for 42 days. The animals were observed for an additional
18 months until death or sacrifice. The high rotenone dosage groups
showed some decrease in weight gain but there was no effect on mortality.
The only non-neoplastic lesions which occurred with substantially greater
frequency in rotenone-treated animals as compared to controls included
myocardial fibrosis and/or lymphoreticular myocarditis in both high and
low dosage groups of both sexes and cystic ductular dilatations in
mammary glands of the female high dosage group. There were numerous
mammary gland neoplasms, mostly fibroadenomas, detected but they occurred
with similar frequency among control and treatment groups. Except for
two lymphosarcomas which occurred in high dose females, all other neoplasms
were rare and/or not dosage related.
In the oral study, groups of male and female Wistar rats were given
daily doses of 0, 1.7 or 3.0 mg/kg of rotenone by gavage for 42 days.
The rats were observed for an additional 12 months until death or sacrifice.
There were no appreciable effects of rotenone dosage on body weight, mortality,
or non-neoplastic disease. Neoplasms were observed in mammary glands of
three female rats. Multiple adenomas were present in two animals, one
from the control group and one from the low dosage group. A small, early
carcinoma and an adenocarcinoma were present in one animal in the low
dosage group. Ductal ectasias and cysts were slightly more prevalent
in mammary glands of dosed females as compared to controls. Adrenal
cortical adenomas occurred in slightly greater frequency in dosed than
in control animals. Fibrosarcomas occurred in subcutaneous sites in
three males from the high dosage group and one fibroma was observed in
a male rat from the low dosage group.
There was no evidence from either the intraperitoneal or oral project
that rotenone induced mammary neoplasia in the rat strains studied. The
significance of the slight increases in fibrosarcomas and fibromas in both
the intraperitoneal and oral studies and in adrenal cortical adenomas in
the oral study was inconclusive.
iv
-------
CONTENTS
Foreword iii
Abstract iv
Tables vi
Figures vi
1. Introduction 1
2. Objectives, Methods, and Observations 2
Rotenone Chemistry and Handling 2
Animal Care and Handling 3
Pathology 3
Data and Test Materials Storage 5
Palatability Study 5
Methods 5
Observations 6
Eighteen-Month Carcinogenesis Study 6
Methods 6
Observations 8
Fourteen-Month Carcinogenesis Study 18
Methods 18
Observations 18
3. Discussion 37
4. Conclusions 39
-------
LIST OF TABLES
Page
Table 1. Rats Surviving Following Rotenone Intraperi-
toneal Injections ....... .... 10
Table 2. Neoplastic and Hyperplastic Lesions by Dose
Group, Sex, and Animal Number (Intraperi-
toneal Study) ............ H
Table 3. Incidence of Neoplastic/Hyperplastic Lesions
by Sex and Dosage Group (Intraperitoneal
Administration) ........... 16
Table 4. Incidence of Non-Neoplastic Lesions by Sex
and Dosage Group (Intraperitoneal
Administration) ............ 20
Table 5. Rats Surviving Following Rotenone Oral Gavage . . 23
Table 6. Neoplastic and Hyperplastic Changes by Dose
Group, Sex, and Animal Number (Oral Study) ... 24
Table 7. Incidence of Neoplastic and Other Growth
Changes Including Mammary Gland Cysts and
Ectasias by Sex and Dosage Group (Oral
Administration) ........... 27
Table 8. Incidence of Non-Neoplastic Lesions by Sex
and Dosage Group (Oral Administration ) . 29
Table 9. Comparative Incidence of Mammary Gland Neoplasia
in Rats Given Rotenone by Oral and Intraperi-
toneal Routes ..... . ..... 38
LIST OF FIGURES
Figure 1. Rodent Numbering System 4
Figure 2. Mean Body Weights and Feed Consumption Data-
Rotenone Palatability Feeding Study .... 7
Figure 3. Rat 18-Month Mean Body Weights-Rotenone
(Intraperitoneal) Carcinogenicity Study ... 9
Figure 4. Rat 14-Month Mean Body Weights-Rotenone
(Oral Feeding) Carcinogenicity Study .... 22
vi
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INTRODUCTION
In a brief communication published in 1973 (Cancer Research 3^3; 3047),
Gosalvez and Merchan described experiments in which mammary tumors were found
in rats which were fed rotenone. In these studies, 4 series of female albino
rats of a strain which reportedly has a natural mammary tumor incidence of
0.5 tumors per 1000 rats per year, were given intraperitoneal injections of
rotenone (K & K Laboratories, Inc., Plainview, N.Y., 1.7 mg/kg body weight) dis-
solved in 0.1 ml sunflower oil on 42 consecutive days. Control animals received
0.1 ml of vehicle. At study initiation the rats were 35 days old (approximately
100 grams each), and were observed for up to 19 months after treatment. In the
first series, 8 out of 8 rats exhibited mammary tumors between months 6 and 11
after treatment while 10 controls had no tumors after 19 months. Several of these
tumors were histologically diagnosed as "adenomas with accentuated interstitial
fibrosis and showed localized areas with adenocarcinomatous transformation:
1 was diagnosed as a differentiated adenocarcinoma. All tumors were encapsulated
and did not show metastasis." Four to five successful tumor transplants out of
30 trials were made to normal rats. Primary and transplanted tumors developed
slowly, requiring 7 to 12 months after initial detection before full development
was achieved.
-------
In order to further evaluate the carcinogenic potential of rotenone
in rats, and in attempt to confirm the findings of Gosalvez and Merchan, long
term studies were performed in Sprague-Dawley and Wistar rats treated by
intraperitoneal injection or oral gavage.
Studies included in this report and inclusive performance dates
are shown below:
Title and Description
(1) Fourteen-Day Feed Palatability Study
(2) Eighteen-Month Carcinogenicity Study
Rotenone Administered Intraperi-
toneally for 42 Days, Followed by
17-Month Observation Period
(3) Fourteen-Month Carcinogenicity Study
Rotenone Administered by Oral
Gavage for 42 Days, Followed by
12-Month Observation Period
Receipt of
Test Animals
1/7/76
1/27/76
6/8/76
and
6/15/76
Dose
Initi-
ation
1/14/76
2/9/76
7/6/76
Final
Sacrifice
of Animals
2/4/76
8/23/77
8/25/77
OBJECTIVES, METHODS, AND OBSERVATIONS
Rotenone Chemistry and Handling
The rotenone for all phases of these studies was obtained from
S. S. Penick and Company. The material was reported by the supplier to be
98+ percent pure with traces of other rotenoids. High pressure liquid chroma-
tographic (HPLC) analyses performed at Battelle according to a published
procedure (J. Chromatography 134:207, 1977), demonstrated the chemical to
be 95+ percent pure. The chemical was stored in desiccators under nitrogen at
-20°C until used.
Rotenone was prepared for oral gavage by adding appropriate amounts
of the chemical to corn oil, followed by stirring the mixture to suspend the
chemical. Corn oil (Mazola) was purchased from a local grocery store. Fresh
gavage suspensions were prepared daily. In the rotenone palatability. study,
a suspension of rotenone in corn oil was poured onto the surface of pre-weighed
amounts of Purina Rat Chow Meal in a twin shell blender, and mixed for 30
minutes. Previous feed analyses by HPLC indicated that rotenone degraded very
rapidly following its addition to feed, with a half-life of less than 2 days.
-------
However, when rotenone was suspended in corn oil, and then added to feed in a
final corn oil concentration of 1 percent, 92 percent of the rotenone could be
recovered after one week. Subsequently, all rotenone diets were made weekly
and included 1 percent corn oil for stability.
Animal Care and Handling
Rats were housed in polycarbonate cages which measured 19.0 x 10.5 x 6.2
inches. Water and Purina Rat Chow were provided ad_ libitum throughout all
studies. Environmental conditions included relative humidity - 45 to 55 percent,
temperature - 71 to 73°F, and 12 hours light - 12 hours dark lighting cycles.
Cage bedding was changed once per week when rats were singly housed, twice
weekly for multiple-housed rats. Rats were identified by a toe clipping-ear
punching scheme as illustrated in Figure 1.
Pathology
Necropsies were performed on animals which died spontaneously and
those which were terminated at the end of the exposure period. Necropsies
were performed by technicians skilled in rodent necropsy techniques with a
supervising pathologist present for interpretation and recording of lesions.
Tissues were placed in 10-percent neutral buffered formalin for fixation prior
to processing and slide preparation. Processing was conducted in the usual
manner and slides were stained with hematoxylin and eosin as described in the
BCL Manual on Histopathology Procedures.
The following tissues were preserved for microscopic examination:
Abdominal skin Eye Seminal Vesicle Pancreas
Mammary Gland Bone Marrow Salivary Gland Liver
Trachea Tonsil Esophagus Thyroid
Lung Kidney Stomach Parathyroid
Heart Urinary Bladder Duodenum Adrenal
Bronchial L.N. Ovary Jejunum Rib & Femur
Mandibular L.N. Uterus Ileum Brain
Mesenteric L.N. Testicle Cecum Spinal Cord
Spleen Epididymis Colon Sciatic Nerve
Thymus Prostrate Muscle
-------
Left ear 20
7000
4000
Left Front Foot
2 Right ear
200
too
Right Front Foot
FIGURE 1. RODENT NUMBERING SYSTEM
-------
Weights for the following organs were obtained at necropsy: heart,
pituitary, adrenal and thyroid glands, brain, testicles, ovaries, spleen,
liver, and kidneys.
Microscopic examination of tissues from these animals were performed
by board certified or board eligible pathologists. Tissues from the groups of
rats treated via intraperitoneal injection were evaluated by Drs. Steve Weisbrode
and Edward Hoover, Veterinary Pathology Consultants, 1925 Coffey Road, Columbus,
Ohio 43210.
Data and Test Materials Storage
All records, tissues, paraffin blocks, microscopic slides, and reports
for this study will be stored in Battelle's archives.
Palatability Study
The following study was performed to test the palatability of 500 and
1000 ppm rotenone diets in rats,and was conducted as a preliminary task leading
to the verification of work performed at other laboratories.
Methods
Twenty Sprague-Dawley derived rats were obtained from Laboratory
Supply Company. Body weights ranged from 125 to 145 grams (females) and 150
to 175 grams (males) on Day 0 of the dosed feeding period. Rats were individually
housed, and quarantined for 7 days prior to exposure to rotenone.
Five rats of each sex were assigned to 1 of 2 dosage groups. Dosed
feeds were prepared which contained either 500 or 1000 ppm rotenone and 1 percent
corn oil. These feeds were presented to the rats for 2 weeks, followed by 1
week exposure of the rats to control feed (no corn oil) . Individual rats and
feed remaining in feed pans were weighed weekly to obtain body weight and feed
consumption values.
-------
Observations
The mean body weights and feed consumption data are presented in
Figure 2. Mean body weight gains were suppressed in females fed either 500 or
1000 ppm rotenone in the diet. Females appeared to be more sensitive to the
chemical than male rats in this respect. Male rats experienced smaller weight
gains when fed 1000 ppm rotenone as opposed to 500 ppm diets.
Feed consumption was slightly higher for rats fed 500 ppm rotenone
as compared to those fed 1000 ppm (Days 7-14). When rats were returned to
normal diets (Days 14-21), feed consumption increased by a factor of 2. It
is concluded that feed containing rotenone at a concentration as low as 500 ppm
is less palatable to rats than normal feed, and that its ingestion at this
concentration leads to depressed body weight gains in immature rats.
Eighteen-Month Carcinogenesis Study
The intent of this phase of the project was to duplicate, in a
different strain of rats, the earlier study carried out by Gosalves and Merchan
(see INTRODUCTION) in which mammary tumors were found in rats following
intraperitoneal administration of rotenone.
Methods
One hundred and thirty Sprague-Dawley rats were obtained from the
Charles River Company, 65 of each sex. Body weights on the first day of dosing"
were 67 to 156 grams for females, and 110 to 169 grams for males. Rats were
housed in groups of 2 rats per cage. Rats were held in-house for quarantine for
13 days prior to dose initiation.
Following quarantine, the rats were assigned to one of 3 treatment
groups. Rotenone was prepared for injections by pre-weighing amounts of the
chemical which were determined to be appropriate for the following week. These
amounts were calculated ftom the mean weights of the rats in a specific group
using the previous week's body weights, and the estimated weight increase for
the following week, using supplier's growth-rate data. All samples were suspended
in corn oil on the day of dosing and administered by intraperitoneal injection
in a volume of 0.1 ml. Test groups, 25 males and 25 females each, received doses
of 1.7 or 3.0 mg of rotenone per kilogram body weight. Control rats, 15
-------
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FIGURE 2. MEAN BODY WEIGHTS AND FEED CONSUMPTION DATA —
ROTENONE PALATABILITY FEEDING STUDY
-------
male and 15 female, received 0.1 ml corn oil injections. Injections were made
on 42 consecutive days. Following the dosing period, the rats were observed for
a period of 17 months at which time the surviving animals were necropsied.
Observations
The mean body weight data are presented in Figure 3. Male rats which
received 3.0 mg/kg rotenone exhibited up to 25 percent lower body weights than
controls during the 18-month study, while the 1.7 mg/kg group maintained
body weights intermediate to those of controls and the high dose animals.
The high dose-treated female rats also experienced reduced body weights which
were no less than 87 percent of controls.
Table 1 presents the rat survival data for the study. No significant
increases in deaths resulted from rotenone exposure. Control, low dose and high
dose groups experienced 23, 16 and 30 percent mortalities respectively, within
the 18-month study period. No substantial difference in mortalities were
experienced between the 2 sexes.
There were numerous mammary gland neoplasms observed both macro-
scopically and microscopically in animals from the control group and both
treatment groups. Individual (Table 2) and group (Table 3) data are presented
on the following pages. These were for the most part fibroadenomas and they
occurred with similar frequency among control and treatment groups. In addition
to the fibroadenomas, 2 adenomas occurred in females given 3.0 mg/kg and 3
in females given 1.7 mg/kg, the latter occurred in conjunction with fibroadenomas.
Mammary carcinoma was present in 1 female given 3.0 mg/kg. Several fibroadenomas
were also present in males from the control and 1.7 mg/kg groups. Fibroadenomas
occurred in control rats with a frequency equal to or greater than that of treated
groups, and the incidence of all female animals bearing mammary neoplasms was
53 percent, 52 percent, and 43 percent for the control, low and high dosage groups
respectively (Table 3). The highest incidence of mammary neoplasms in male
rats occurred in the control group. Other neoplasms which occurred in rats from
this study are listed in Table 3. Two lymphosarcomas occurred in females given
3.0 mg/kg; all other neoplasms occurred in only 1 animal from any given dosage
group with the exception of chromophobe adenomas of the pituitary gland and
adrenal cortical adenomas which occurred with similar frequency among control
and treatment groups.
8
-------
900
M 3 Bg/kg A&
F 3 »g/kg a~ A
M 1.7 s«/kg*—*
1.7 «g/kg Jdr—ai
M Control » i -
F Control •---•
48 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80
FIGURE 3. RAT 18-MONTH MEAN BODY WEIGHTS--ROTENONE (INTRAPERITONEAL) CARCINOGENICITY STUDY
-------
TABLE 1. RATS SURVIVING FOLLOWING ROTENONE INTRAPERITONEAL
INJECTIONS
Initiated
(2/76)
January 1,1977
February 1
March 1
April 1
May 1
June 1
July 1
August (Until
3.0
Male
25
21
21
21
20
18
18
17
17
mg/kg
Female
25
21
21
21
21
20
19
19
18
1.7
Male
25
24
24
24
24
24
23
23
22
mg/kg
Female
25
25
24
24
23
23
23
23
20
Male
15
15
15
14
12
12
12
11
11
0
Female
15
15
15
14
14
13
13
12
12
Necropsy)
10
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TABLE 2. NEOPLASTIC AND HYPERPLASTIC LESIONS BY DOSE GROUP. SEX , AND
ANIMAL NUMBER (INTRAPERITONEAL STUDY)
Mammary Gland, Fibroadenoma
Control - 1129, 1117, 1116. 1112, 1111, 1110, 1109, 1108, 1107, 1104,
1102
1.7 - 1086. 1075, 1069, 1068, 1067, 1066, 1065, 1064, 1062, 1061,
1059, 1057, 1054, 1052
3.0 - 1021, 1003, 1019, 1017, 1016, 1014, 1002
Mammary Gland, Adenoma
Control - 1108
1.7 - 1075, 1065, 1054
3.0 - 1020
Mammary Gland, Hyperplasia
Control - None
1.7 - 1070, 1068
3.0 - None
Mammary Gland, Carcinoma
Control - None
1.7 - None
3.0 - 1016
Pituitary, Chromophobe Adenoma
Control - 1105, 1111
1.7 - 1054, 1055, 1087
3.0 - 1005, 1014, 1039
Pituitary, Chromophobe Hyperplasia
Control - 1101, 1102, 1113
1.7 - 1059, 1067, 1076, 1084, 1093, 1099
3.0 - 1033, 1036, 1004
(a) Males - Underlined.
11
-------
TABLE 2. (Continued)
Adrenal, Cortical Adenoma
Control - HOI, 1102, 1103, 1105
1.7 - 1052, 1058, 1062, 1068, 1074
3.0 - 1003, 1021
Skin Keratoacanthoma
Control - 1122
1.7 - None
3.0 - None
Skin. Souamous Cell Carcinoma
Control - None
1.7 -1091
3.0 - None
Skin. Pseudoepitheliomatous Hyperplasia
Control - None
1.7 - 1066
3.0 - None
Skin. Fibroma
Control - None
1.7 - 1082
3.0 - None
Skin, Fibrosarcoma
Control - None
1.7 - 1091
3.0 - None
12
-------
TABLE 2. (Continued)
Skin. Sebaceous Adenoma
Control - None
1.7 - 108A
3.0 - None
Bone, Osteosarcoma
Control - 1119
1.7 - None
3.0 - None
Multiple Tissues, Lymphosarcoma
Control - None
1.7 - None
3.0 - 1024, 1002
Kidney, Pelvis, Hyperplasia
Control - None
1.7 - None
3.0 - 1009
Bladder, Transitional Cell Papilloma
Control - None
1.7 - 1099
3.0 - None
Testicle, Interstitial Cell Tumor
Control - None
1.7 - 1078
3.0 - None
13
-------
TABLE 2. (Continued)
Uterus, Adenocarcinema
Control - None
1.7 _ None
3.0 - 1002
Liver, Hepatocellular Carcinoma
Control - None
1.7 - 1089
3.0 - 1040
Liver, Focus of Cellular Alteration
Control - None
1.7 - 1065
3.0 - None
Liver, Biliary Hyperplasia
Control - 1101, 1115
1.7 - 1071, 1074 , 1077
3.0 - None
Pancreas, Islet Hyperplasia
Control - None
1.7 - 1095
3.0 - 1012
Pancreas, Ductular Hyperplasia
Control - 1103
1.7 - 1065, 1076
3.0 - None
14
-------
TABLE 2. (Continued)
Pancreas, Islet Adenoma
Control - None
1.7 - 1077, 1090
3.0 - None
Stomach, Pseudoepithelial Hyperplasia
Control - 1109
1.7 - 1067
3.0 - None
Salivary Gland, Hyperplasia
Control - 1107
1.7 - 1090
3.0 - None
Lymph Node, Lymphoplasmatic Hyperplasia
Control - 1118, 1123> 1126
1.7 - None
3.0 - 1001, 1026. 1032
Thyr o id, Ad enoma
Control - 1120
1.7 - 1088
3.0 - None
15
-------
TABLE 3. INCIDENCE OF NEOPLASTIC/HYPERPLASTIC LESIONS BY SEX AND DOSAGE
GROUP (INTRAPERITONEAL ADMINISTRATION)
3mg/kg IP
Organ and Lesion
Kidney
Hyperplasia, pelvic epithelium
Bladder
Transitional cell papillozna
Thyroid
Adenoma
Pituitary
Chromophobe hyperplasia
Chromophobe adenoma
Adrenal
Cortical adenoma
Spleen
Extreme hematopoietic hyperplasia
Lymph node
Lymphoplasmatic hyperplasia
Salivary gland
Ductular hyperplasia
Stomach
Pseudoepitheliomatous hyperplasia
of squamous epithelium
Pancreas
Islet hyperplasia
Islet adenoma
Ductular hyperplasia
Liver
Hepatocellular carcinoma
Focus of cellular alteration
Biliary hyperplasia
Male Female
(n-23) (n-21)
0
0
0
2
1
0
0
2
0
0
0
0
0
1
0
0
1
0
0
1
2
2
1
1
0
0
1
0
0
0
0
0
Group
1.7 mg/kg IP
Male
-------
TABLE 3. (Continued)
Organ and Lesion
3mg/kg IP
Male Female
(n-23) (n-21)
Group
1.7 mg/kg IP
Male Female
(n-24) (n-25)
Control
Male Female
(n-14) (n-15)
Testicle
Interstitial cell tumor
Skin
Keratoacanr.homa
Polyp
Squamous cell carcinoma
Pseudoepitheliomatous hyperplasia
Fibroma
Fibrosarcoma
Sebaceous adenoma
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
Mammary gland
Fibroadenoma
Glandular hyperplasia
Adenoma
Carcinoma
Bone (rib)
Osteosarcoma
Lymph node, spleen, liver, ovary
Lymphosarcoma
7
0
1
1
13
2
3
0
17
-------
The only non-neoplastic lesions which occurred with substantially
greater frequency in rotenone-treated animals as compared to controls included
myocardial fibrosis and/or lymphoreticular myocarditis in both the 1.7 and
3.0 mg/kg groups and cystic ductular dilatations in mammary glands of females
given 3.0 mg/kg (Table 4).
Fourteen-Month Oral Carcinogenesis Study
A study was performed to examine the potential carcinogenicity of
rotenone in a second strain of rats. This study employed oral gavage as the
route of administration, again in an attempt to duplicate the studies of
Gosalvez and Merchan.
Methods
One hundred and fifty Wistar rats were obtained from Charles River
Company, 75 of each sex. The rat weights on the first day of dosing ranged from
75 to 145 grams for females, and from 81 to 156 grams for'males. Animals were
caged in pairs.
Rotenone was administered by oral gavage using a stainless steel
feeding needle. The rotenone suspension was prepared as described in the 18-
month study methods section. The rotenone in corn oil was given in 0.25 ml
volumes daily for 42 consecutive days. Doses of 0, 1.7 or 3.0 mg rotenone per
kilogram body weight were administered to groups of 25 males and 25 females.
Following the 42-day administration period, the rats were observed for 12 months
at which time the rats were necropsied.
Observations
The data in Figure 4 depict the body weights of the rats during the
14-month study. No appreciable differences are seen in body weights between
the different groups throughout the study. Although this study was not as
lengthy, nor did it use the same route of administration as the previous one
described in this report, the Wistar strain appears to display lower sensitivity
to rotenone administration as measured by body weights.
Survival data are presented in Table 5. Again, no significant number
of deaths can be attributed to rotenone treatment in this study. Because of this
18
-------
shorter observation period for this study, it is difficult to make comparisons on
the lethality of rotenone in Wistar and Sprague-Dawley rats.
Neoplasms were observed in mammary glands of 3 female rats from this
study, as indicated in Tables 6 and 7. Multiple adenomas (2) were present
in 2 animals, one from the control group and one from the 1.7 mg/kg dosage
group. A small early carcinoma and an adenocarcinoma were present in 2 differ-
ent mammary glands from one animal in the 1.7 mg/kg dosage group. There were
other small masses observed grossly in which mammary cysts, ductal or glandular
ectasias or mild hyperplasias were observed microscopically. Ductal or glandular
ectasias and cysts were slightly more prevalent in females from the 1.7 or 3.0
mg/kg dosage groups as compared to the control group.
Adrenal cortical adenomas occurred in slightly greater numbers in
both the 1.7 and 3.0 mg/kg dosage groups as compared to the controls. These
occurred in greater numbers in the females and occurred with similar frequency
among females from both the high and low dosage levels.
Fibrosarcomas occurred in subcutaneous sites in 3 males from the 3.0
mg/kg dosage group and one fibroma was observed in a male rat from the 1.7 mg/kg
dosage groups. Neither fibromas nor fibrosarcomas were observed in control
groups in this study.
Bile duct hyperplasias were observed in 3 females and one male from
the high dose group and one male from the control group. These were extremely
mild changes and were not considered significant.
All other neoplastic or hyperplastic lesions occurred with similar
or greater frequency in the control groups as compared to treated groups.
Other non-neoplastic changes (Table 8) generally occurred with similar
frequency among control and treated groups. Chronic renal disease consisting
of glomerular scleroses, thickening of tubular basement membranes with associated
renal tubular epithelial regenerative changes, and in some instances chronic
interstitial inflammatory changes (some or all of these changes) occurred in
high percentages of male rats from all dosage groups and to a lesser extent in
females. Likewise, respiratory disease was common in many animals: peribronchial
and peribronchiolar lymphoid nodules were present in essentially all animals
and were not listed in the diagnoses unless associated with other changes.
Subacute enteritis, hepatic congestion, and lymphadenitis of mesenteric and
mandibular lymph nodes occurred with slightly greater frequency in the 3 mg/kg
dosage group than in controls; however, these variations were considered to be
relatively insignificant.
19
-------
TABLE 4. INCIDENCE OF NON-NEOPLASTIC LESIONS ff? SEX
AND DOSAGE GROUP (INTRAPERITONEAL ADMINISTRATION)
Group
3mg/kg IP
Organ and Lesion
Lung
Pneumonia
Bronchiectasis
Thrombosis
Heart
Myocardial fibrosis, lymphoreticular
myocarditis
Vessels
Pyogranulomatous vasculitis
Kidney
Nephritis (all developmental stages)
Bladder
Cystitis
Stomach
Granulomatous gastritis
Liver
Hepatic necrosis
Vacuolar degeneration
Ovary
Folllcular cyst
Uterus
Endometritis
Cystic glandular dilatation/hydrometra
Prostate
Prostatitis
Muscle
Myositis
Atrophy
Skin
Dermatitis (all types)
Epidermoid cyst
Dilated hair follicles
Male Female
(n-23) (n-21)
2
0
0
11
0
16
0
0
0
0
—
_
-
0
0
0
3
2
1
0
0
0
4
0
2
2
0
0
0
2
2
1
-
0
0
0
0
0
1.7 mg/kg IP
Male
(n-24)
2
1
0
22
1
20
0
0
c
0
-
_
-
1
1
1
2
1
0
Female
(n-25)
0
0
0
8
0
it
0
1
1
2
0
0
1
-
1
0
1
0
0
Control
Male
(n-14)
0
0
1
4
C
12
0
0
0
0
—
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-
C
0
0
4
0
0
Female
(n-15)
0
0
0
0
0
6
0
0
2
1
1
0
1
-
0
0
0
0
0
20
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800
M 3 Kg/kg
F 3 «g/kg
M 1.7 ng/kg
F 1.7 mg/kg
M Control
F Control «---»
I -
End of 42-
S Dosing Schedul
4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
WEEK OF STUDY
FIGURE 4. RAT 14-MONTH MEAN BODY WEIGHTS—ROTENONE (ORAL FEEDING) CARCINOGENICITY STUDY
22
-------
TABLE 5. RATS SURVIVING FOLLOWING ROTENONE
ORAL GAVAGE
Initiated
(6/76)
January 1,
February 1
March 1
April 1
May I
June 1
July i
3.0
Male
25
1977 25
25
25
24
24
23
23
mg/kg
Female
25
24
24
24
24
24
24
24
1.7
Male
25
25
25
25
25
25
25
24
mg/kg
Female
25
24
24
24
24
24
24
24
Male
25
25
25
25
25
25
25
25
0
Female
25
25
25
25
25
25
25
25
August (Until
Necropsy) 23
24
24
24
25
25
23
-------
TABLE 6. NEOPLASTIC AND HYPERPLASTIC CHANGES BY DOSE GROUP,
, AND ANIMAL NUMBER (ORAL STUDY)
Adrenal Cortical Adenoma
Control - 2260, 2261, 2267, 2296
1.7 - 2203, 2204, 2205, 2211, 2212, 2223, 2224, 2238
3.0 - 2153, 2154, 2157, 2162, 2167, 2171, 2176. 2184. 2194
Adrenal Cortical Hyperplasia
Control - 2253, 2254, 2261, 2262, 2291. 2279. 2282, 2283, 2285,
2287, 2292, 2293, 2295, 2296, 2299, 2300
1.7 - 2205, 2206, 2208, 2214, 2220, 2232. 2237. 2238. 2243.
2247
3.0 - 2154, 2157, 2166, 2169, 2185. 2188. 2193. 2197
Adrenal Cortical Carcinoma
1.7 - 2212
Pituitary, Chromophobe Adenoma
Control - 2262, 2265, 2270, 2271
1.7 - 2202, 2205, 2215, 2225
3.0 - 2158, 2159, 2163, 2170. 2184. 2195. 2197
Pituitary Chromophobe Hyperplasia
Control - 2267, 2286. 2290, 2255, 2256, 2281
1.7 - 2214, 2230, 2216, 2223, 2224
3.0 - 2153, 2155, 2165, 2166, 2168, 2173. 2192
Pituitary Focus of Cellular Alteration
Control - 2264 , 2256
1.7 - 2213, 2219
3.0 - 2161
Pituitary Adenoma, Pars Intermedia
1.7 - 2234
(a) Males - Underlined.
24
-------
TABLE 6. (Continued)
Mammary Gland Ductal or Glandular Ectasia
Control - 2256
1.7 - 2202, 2204, 2211, 2213
3.0 - 2153, 2161, 2163, 2167, 2169, 2171
Mammary Gland Cyst
Control - 2258, 2262, 2268, 2271
1.7 - 2210, 2214, 2217
3.0 - 2151, 2155, 2157, 2169, 2173, 2174
Mammary Gland Adenoma
Control - 2255
1.7 - 2223
Mammary Gland Adenocarcinoma and Carcinoma
1.7 - 2215
Mammary Gland Hyperplasia
1.7 - 2223
3.0 - 2156, 2175
Skin - Fibroma
1.7 - 2245
Skin Fibrosarcoma
3.0 - 2177, 2183, 2188
Lympho s a r c oma
Control - 2270, 2279
Liver, Neoplastic Nodule
Control - 2291
3.0 - 2166
Liver, Focus of Cellular Alteration
Control - 2292
3.0 - 2198
Thyroid Gland Follicular Adenocarcinoma
3.0 - 2166
25
-------
TABLE 6. (Continued)
Thyroid Gland Follicular Hyperplasia
Control - 2282, 2284
3.0 - 2197
Thyroid Gland Medullary Carcinoma
Control - 2286
Liver, Bile Duct Hyperplasia
Control - 2300
3.0 - 2190, 2153, 2155, 2171
Diaphragm, Carcinoma of Undetermined Origin
Control - 2300
Skin Trichoepithelioma
3.0 - 2186
26
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DISCUSSION
There was a striking difference between the incidence of mammary
neoplasia among rats in the groups treated orally as compared to those which
were exposed by intraperitoneal administration (Table 9). The incidence of
mammary fibroadenomas in the intraperitoneal treatment group was as high in the
control animals as in those given 1.7 mg/kg, and higher than the group given
3.0 mg/kg. The incidence of animals from the intraperitoneal study with mammary
neoplasms was similar for rats in the 1.7 mg/kg and control groups since the
three adenomas occurred in animals which also had fibroadenomas. The lowest
incidence was recorded in those rats given 3.0 mg/kg. We interpret these data
as showing no evidence that rotenone enhanced or induced mammary neoplasia in
animals from the intraperitoneal study. Likewise, there was no evidence of
enhanced induction of mammary neoplasia in animals from the oral treatment study.
The significantly higher incidence of mammary neoplasia in animals
from intraperitoneal as compared to the oral treatment group reflects the
difference in incidence of spontaneous mammary neoplasms in Wistar and
Sprague-Dawley rats. The large numbers of spontaneous fibroadenomas commonly
present in Sprague-Dawley rats was evident in this study and was in sharp
contrast to the absence of this tumor type in the Wistar rats.
The absence of any evidence of increased tumor incidence in rotenone-
treated animals from either the oral or intraperitoneal study is significantly
different from results reported by Gosalvez and Merchan who reported an incidence
of mammary tumors ranging from 60-100 percent which developed 6-11 months
following intraperitoneal injections of 1.7 mg/kg of rotenone in 0.1 ml of sun-
flower oil for 42 days. The reason for the variation in test results is not known.
The significance of three fibrosarcomas and one fibroma which
occurred in male rats from the 3.0 and 1.7 mg/kg dosage groups in the oral
study is not clear. One fibroma and one fibrosarcoma also occurred in male
rats given 1.7 mg/kg in the intraperitoneal study. Similar neoplasms were not
present in control groups from either study. This is interpreted as suspicious
but inconclusive evidence that the fibromas and fibrosarcomas were induced by
the test compound.
37
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TABLE 9. COMPARATIVE INCIDENCE OF MAMMARY GLAND NEOPLASIA
IN RATS GIVEN ROTENONE BY ORAL AND INTRAPERITONEAL
ROUTES
Route
Oral
Oral
Oral(a>
Oral(a)
I. P.
I. P.
I. P.
I. P.
I. P.
I.P.(b)
I. P.
I. P.
I. P.
Type of Neoplasm
Adenoma
Adenoma
Carcinoma
Adenocar-
cinoma
Fibroadenoma
it
M
"
"
Adenoma
"
H
Carcinoma
No. Treatment Level (No. in Group) Sex
1
1
1
1
8
3
13
1
7
3
1
1
1
Control
1.7 mg/kg
1.7 mg/kg
1.7 mg/kg
Control
Control
1.7 mg/kg
1.7 mg/kg
3.0 mg/kg
1.7 mg /kg
3.0 mg/kg
Control
3.0 mg/kg
(25)
(24)
(24)
(24)
(15)
(U)
(25)
(24)
(21)
(25)
(21)
(15)
(21)
F
F
F
F
F
M
F
M
F
F
F
F
F
Incidence (%)
4.0%
4.2%
4.2%
4.2%
53.3%
21.4%
52.0%
4.2%
33.3%
12.0%
4.7%
6.6%
4.8%
(a) Occurred in same animal.
(b) All three adenomas in this group occurred in animals which also had fibroadetiomas.
38
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A greater Incidence of adrenal cortical adenomas occurred in the
treated groups when compared to control groups from the oral study. The
significance of these differences is difficult to interpret in view of the
small number of animals included in these groups and the relatively high
incidence of these changes which were present in control groups.
CONCLUSIONS
1. There was no evidence that rotenone induced mammary
neoplasia in animals from these studies.
2. The correlation between rotenone treatment and the
fibrosarcomas and fibromas induced in both oral and
intraperitoneal studies is inconclusive.
3. The significance of the increased numbers of adrenal
cortical adenomas in animals from treated groups in the oral
study is unclear.
39
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TECHNICAL REPORT DATA
/Please read Instructions on the reverse before completing)
REPORT NO.
£PA-600/l-79-004b
3. RECIPIENT'S ACCESSION NO.
'4. TITLE AND SUBTITLE
CARCINOGENIC POTENTIAL OF ROTENONE
PHASE II: ORAL AND INTRAPERITONEAL ADMINISTRATION TO
RATS
5. REPORT DATE
January 1979
6. PERFORMING ORGANIZATION CODE
7. AUTHCR(S)
A. P. Leber, D. C. Thake
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Battelle
Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
68-02-1715
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
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
EPA 600/11
15. SUPPLEMENTARY NOTES
Principal Investigator
A. P. Leber
16. ABSTRACT
In the intraperitoneal study, test groups of male and female Sprague-Dawley
rats were given daily doses of 0,1.7 or 3.0 mg/kg of rotenone for 42 days. The
high rotenone dosage groups showed decrease in weight gain but there was no effect
on mortality. There were numerous mammary gland neoplasms, mostly fibroadenomas,
detected but they occurred with similar frequency among control and treatment groups.
Except for two lymphosarcomas which occurred in high dose females, all other neoplasms
were rare and/or not dosage related.
In the oral study, groups of male and female Wistar rats were given daily doses
of 0, 1.7 or 3.0 mg/kg of rotenone by gavage for 42 days. There were no appreciable
effects of rotenone dosage on body weigh, mortality, or non-neoplastic disease.
Ductal ectasias and cysts were slightly more prevalent in mammary glands of dosed
females as compared to controls.
There- was no evidence from either the intraperitoneal or oral project that
rotenone induced mammary neoplasia in the rat strains studied. The significance of
the slight increases in fibrosarcomas and fibromas in both the intraperitoneal and
oral studies and in adrenal cortical adeomas in the oral study was inconclusive.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Carcinogens
Toxicity
Rotenone
06, T, F
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport)
UnclassifipH
21. NO. OF PAGES
49
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
40
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