EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600 1 -79 004a
January 1979
Research and Development
Carcinogenic
Potential of
Rotenone
Phase I:
Dietary
Administration to
Hamsters
P 600/1
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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-79-004a
January 1979
CARCINOGENIC POTENTIAL OF ROTENONE
PHASE I: DIETARY ADMINISTRATION TO HAMSTERS
by
A. P. Leber and R. L. Parsing
Battelles', 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
^
ijJL
LJ l.
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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.
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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 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 hamsters.
F. 6. Hueter, Ph.D.
Di rector
Health Effects Research Laboratory
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ABSTRACT
Studies were performed to evaluate the potential carcinogenicity rotenone
in the Syrian Golden hamster. Several ancillary range-finding studies were
carried out including 14-day feeding trials and a reproduction experiment.
The latter experiment indicated that rotenone at a level of 500 ppm in the
maternal diet was embryo-toxic (depressed birth weight and fetal deaths) and
resulted in cannabalism of the young by the maternal animals. A level of
1000 ppm led to sterility in one or both sexes. The 18-month carcinogenesis
study indicated that 1000 ppm rotenone in the diet was toxic to hamsters
(depressed body weights compared to controls) but no gross or histopatholog-
ical evidence was obtained which indicated that the test compound induced the
formation of any tumors. Three adrenal cortical carcinomas in 65 hamsters
observed in the 1000 ppm group could not be ascribed to rotenone treatment.
iv
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CONTENTS
Foreword i
Abstract iy
Figures vi
Tables vii
1. Introduction 1
2. Outline of Hamster Studies 2
3. Objectives, Methods, and Observations 4
Rotenone purity and dose preparation 4
Hamster care and handling 4
Data and test material storage 5
Prechronic study I 5
Methods 5
Observations 6
Prechronic study II 6
Methods 6
Observations 8
Oral dosing study 8
Methods 8
Observations 8
Reproduction study 8
The 1000 ppm diet study 10
The 0 ppm diet study (controls) 10
The 500 ppm diet group 11
Carcinogenesis study 11
Methods 12
Tissues removed at necropsy 13
Observations 14
4. References 32
V
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FIGURES
Number Page
1 Hamster group mean body weights during prechronic
study I 7
2 Group mean hamster body weights during prechronic
study II 9
3 Group mean body weights for hamsters in carcinogenesis
study 15
4 Group mean rotenone diet consumption in carcinogenesis
study 16
5 Group mean rotenone consumption during carcinogenesis
study 17
vi
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TABLES
Number Page
1 Hamsters Surviving While Maintained on Rotenone
Diets 18
2 Gross Lesions in Hamsters That Died Spontaneously
(Incidence and Percent Affected) 20
3 Gross Lesions in Hamsters Terminated at the End of the
Study (Incidence and Percent Affected) 21
4 Distribution of Hamsters Evaluated Microscopically . . 22
5 Microscopic Lesions by Dosage Group and Sex
(Excluding Neoplastic and Hyperplastic Lesions ... 24
6 Neoplastic and Hyperplastic Lesions 29
7 Microscopic Diagnoses of Neoplasia by Animal Number . . 30
vii
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INTRODUCTION
In a brief communication published in 1973 (Cancer Research 33:3047),
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 10 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.
This report presents results of a chronic feeding study which was
performed to investigate the carcinogenic potential of rotenone in Syrian Golden
hamsters. Included are observations made in ancillary studies on effects of
rotenone on hamster reproduction and acute toxicity trials.
The following page presents an outline o^ the different phases of the
study, and the dates associated with each phase.
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OUTLINE OF HAMSTER STUDIES
Title & Description
Prechronic Study I
To test feed palatability and
subacute toxicity of roterione:
rotenone administered 14-days
in feed at 63, 125, 250, 500
and 1000 ppm. After 14-day
observation period, gross
necropsies performed.
Dates
Receipt of
Test Animals
Initiation
Completion
8/25/75
9/3/75
10/9/75
Prechronic Study II
Repeat of prechronic Study I.
This study included addition
of corn oil to rotenone-feed
mixtures to increase cheriical
stability.
9/23/75
10/7/75
11/11/75
Oral Dosing Study
Rotenone was administered by oral
gavage in corn oil for 9 days in
a dose of 80 mg/kg. Animals
observed for gross toxic signs
9/16/75
9/30/75
10/8/75
Reproduction Study
Both males and females maintained
on rotenone diets: litter sizes,
offspring survival and pregnancy
rates were recorded.
Group Fed 1000 ppm
Group Fed 0 ppm
Group Fed 500 ppm
12/3/75
3/16/76
6/15/76
12/8/75
3/21/76
6/28/76
6/1/76
1/26/77
1/25/77
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OUTLINE OF HAMSTER STUDIES
(Continued)
Title & Description
Dates
Receipt of
Test Animals
Initiation
Completion
Carcinogenesis Study
Hamsters fed rotenone in
diets for 18 months;
histopathology performed on
0, 125, and 1000 ppm groups.
Group Fed 0 ppm
Group Fed 125 ppm
Group Fed 250 ppm
Group Fed 500 ppm
Group Fed 1000 ppm
1/13/76
1/27/76
1/21/76
1/6/76
12/29/75
1/26/76
2/9/76
2/2/76
1/19/76
1/12/76
7/28/77
8/17/77
7/29/77
7/22/77
7/15/77
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OBJECTIVES, METHODS, AND OBSERVATIONS
Rotenone Purity and Dose Preparation
Rotenone employed in this study was obtained from S. S. Penick and Co.,
Orange, New Jersey. Analysis of the chemical by high pressure liquid chromato-
graphy at Battelle according to published procedures [J. Chromatography 134:207
(1976)] determined the rotenone to be 95+ percent pure. The stock chemical
was stored at -20 C under nitrogen prior to dosage preparation.
Rotenone was prepared for oral gavage by adding appropriate amounts
of the chemical to corn oil, followed by high-speed stirring of the mixture.
Corn oil (Mazola) was obtained locally from retail grocery stores, and was
used without purity analyses.
Dosed feed preparations for Prechronic Study I were prepared by
adding rotenone to ^urina Laboratory Chow Meal, and mixing for 30 minutes in
a Patterson-Kelly twin shell blender. For subsequent studies, rotenone was first
suspended in corn oil and then blended with the feed. By instituting the
addition of a one percent corn oil concentration in the feed mixtures, the
stability and homogeneity of the test chemical were increased. Ninety-two
percent of added rotenone could be recovered after one week by employing this
procedure. Feed mixtures for the Prechronic II, Reproduction and Carcinogenesis
studies contained one percent corn oil, and were prepared weekly throughout the
studies.
Coded feed samples were sent to Penick Research and Applications
Development Center (Mr. Joseph Haus) for rotenone analyses. Values obtained
from these assays were generally within 20 percent of calculated values except
at the higher concentrations in which cases decreased extraction efficiency
was suspected as the cause of the low values. See Appendix I for these
results.
Hamster Care and Handling
Animals were housed individually throughout all studies except
during mating periods in the reproduction study. Hamsters were housed in
19 X 10.5 X 6.2 inch polycarbonate cages in rooms separate from those
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used for other studies. Cage bedding was changed once per week. Environ-
mental conditions were maintained under conditions suitable for the species
as outlined in Battelle's Manual on Standard Operating Procedures for
Animal Housing and Care, including temperature, 70 -74° and relative
humidity, 50-60%.
The Syrian Golden hamsters used in these studies were obtained
from Charles River Lakeview Hamster Colony. Each group of animals was
given a minimum of five days for quarantine before going on study. Water
was provided ad libitum throughout. Animal cages were equipped with
feeders that were suitable for periodic feed consumption determinations.
Hamsters were uniquely identified by a toe clipping-ear punching scheme.
Data and Test Material Storage
All records, tissues, paraffin blocks, microscopic slides, and
reports for this study will be stored in Battelle's archives.
Prechronic Study I
This four-week study was carried out prior to beginning chronic
studies to provide preliminary information on the palatability of rotenone
containing feeds, and any resulting gross toxicity which may occur in the
treated animals.
Methods
Rotenone was blended with Purina Hamster Chow Meal to yield
concentrations of 63. 125, 250, 500, and 1000 ppm. Hamster weights ranged
from 50 to 70 grams at the beginning of the study. Animals were given
access to control feed for 1 week prior to dosing. Hamsters in groups of
10 animals, (5 males and 5 females) were then fed rotenone-containing
feeds for 14 days, followed by 14 days of control feed. Animal body
weights and feed consumption were determined weekly. Gross necropsies
were performed 14 days following final exposure to rotenone.
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Observations
The mean body weight data indicate that there was a substantial
reduction in weight gain for males during the first week of feeding 1000 ppm
rotenone in the diet (Figure 1.) The effect was reversible with observed
weight gains greater than normal after return to control diet.
During the course of the preliminary study, no abnormal behavior
or symptomology was observed.
Gross observations at necropsy showed lungs of several 1000 ppm
hamsters to be congested and colored cherry red. Several small petechial
hemorrhagic areas were present on the lung surface. Also seen were con-
gestion of the duodenal mucosa, dilation of meningeal vessels and
hemorrhagic enteritis in the small intestines. Similar but less extensive
lesions were present in animals given 500 ppm. No lesions were found in
the 250 ppm or lower dosaee groups. None of the hamsters died during the
course of the study.
Prechronic Study II
A second range-finding feeding study was performed in an identical
manner to Study I except corn oil was used as a vehicle for dispersing ro-
tenone into the feed. This procedure was designed to provide for more
homogeneity and stability of the chemical in the feed.
Methods
Rotenone was suspended in corn oil and blended with Purina Lab
Chow. Final rotenone concentrations were 0, 63, 125, 250, 500, and 1000 ppm,
and corn oil was one percent. Ten hamsters were assigned to each feeding
group, 5 males and 5 females. Animals were fed control diet for 1 week,
rotenone diets 2 weeks, and again control diets for 2 weeks, Weekly body
weights and feed consumptions were recorded. Necropsies were performed
on fivp 1^00 ppm and two 500 ppm hamsters.
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Observations
Figure 2 shows that no substantial differences occurred for
mean body weight gains between treatment groups. No behavioral
changes or deaths were evident during the four week study. No detectable
lesions were found during gross necropsies carried out at the end of the
study.
This preliminary study confirmed that 1000 ppm rotenone in the
diet would be an appropriate high dose level to use in a chronic study.
Oral Dosing Study
This study was designed to determine the toxicity following
oral gavage of rotenone to hamsters.
Methods
Ten hamsters were given oral intubations of 80 mg/kg rotenone in
1.0 ml of corn oil daily. Observations and dosing continued for 9 days.
Observations
The animals soon became lethargic after the initial dose. Three
animals died during the first two days of the study, and exhibited an oily
substance in the peritoneal cavity, although no stomach punctures were
found. Other animals displayed signs of diarrhea, eye discharge, lameness,
shaggy coats, and mouth ulcerations. The lungs, liver, and G.I. tract
were congested with redness in the lungs and pleura. Intussussceptions
of the small intestine and rectal prolapses were commonly seen. Rats
treated with corn oil only exhibited mild diarrhea for four days which
subsided after that time.
Reproduction Study
In an effort to evaluate the toxic effects of rotenone on
reproduction, the following study was initiated. Male and female animals
were placed on rotenone diets, and the effects on number of offspring,
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X 20
-10
r
Termination of Kotenone Feeding
Necropsies
o Control
X 63 pp«
O 125pp.
+ 500pp«
• lOOOppa
— Female
— Male
1234
Weeks on Study
FIGURE 2. Group Mean Hamster Body Weights During Prechronic Study II
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number of pregnancies, and sex ratios for offspring were to be evaluated.
Fja offspring were to be maintained on rotenone diets to determine their
sensitivity to any potential carcinogenic effects of the chemical.
The 1000 ppm Diet Study
The first group of animals (mean body weights were 45 grams for
both sexes) to be mated were maintained on a 1000 ppm rotenone diet
containing 1% corn oil. Fifty females and 25 males were placed on test
diet, and were to be mated three months later.
The hamsters continued to increase in body weight the first 5
weeks of the study after which time feed consumption decreased. Sixteen
(3 males and 12 females) of the 75 animals on study died during the first
two months. Physical condition deteriorated and body weights decreased
in the survivors. At eight to nine weeks, the downward trend in body
weights reversed, and animals became more alert, and their coats developed
a sheen while feed consumption returned to normal.
Females were observed daily according to the Orsini technique
to monitor estrus cycles. During the fourth month of the study, mating
was initiated by housing one male with two females at the end of the first
day of the estrus cycle which is normally four days in duration.
Several vaginal plugs were observed. However, it was soon
determined that one or both sexes were nonfertile since no pregnancies
occurred. Males were observed grossly to have smaller than normal testicles.
Since attempts to produce offspring in the 1000 ppm treated
hamsters were unsuccessful, the study was terminated, and a lower dietary
level was scheduled for a subsequent study.
The 0 ppm Diet Study (Controls)
Hamsters (50 males and 50 females) were maintained on a control
diet (1% corn oil in Lab Chow Meal) throughout the entire study. One
female was caged with one male for mating. Pregnancies occurred, and
offspring were delivered.
10
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The F^a generation all appeared healthy through weaning.
Forty-three litters were delivered from 50 females. Average litter size
was 7 with a range from 2 to 10 pups. One to three pups were retained
from each litter, and placed in the test group to be observed for tumor
formation.
The second group of offspring (F^b) also appeared to be healthy
through weaning. There was an average of 12 pups per litter which ranged
in size from 9 to 17 pups.
The 500 ppm Diet Group
Fifty males and fifty females were maintained on a 500 ppm
rotenone diet containing 1% corn oil. The animals were fed test diet
for three months prior to and during the mating periods. Mating was carried
out as described previously.
The first generation of offspring (F^a) consisted of 45 litters
from 50 females. Only 7 survived through weaning as the dam often
cannabalized or totally neglected her young. The pups were all smaller
than normal. Average litter size was 9 pups, and ranged from 4 to 15
offspring.
The second litters from the 500 ppm group consisted of 21 litters.
ranging in size from 7 to 16 pups with an average of 12. The dams
continued to refuse to nurse their young and often cannabalized them.
Six months after the beginning of the 500 ppm rotenone feeding
study, and 10 months for the 0 ppm group,the studies were terminated
concurrently at the request of the EPA Project Officer. The high toxicity
of rotenone at the 500 ppm level as exhibited by large numbers of dead
births, maternal deaths, cannabalism of offspring, and offspring deaths
resulted in a small number of hamsters surviving weaning, and continuation
of the study at this dose level would not be productive.
Carcinogenesis Study
A long term (18-month) study to evaluate the potential
carcinogenicity of rotenone in hamsters was performed. Rotenone was
incorporated in the feed of the test animals for the duration of the study.
11
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Methods
Hamsters were divided into five groups of 50 males and 50 females
each. The groups were fed diets containing 1% corn oil and rotenone
concentrations of 0, 125, 250, 500, or 1000 ppm. Animals were weighed
weekly for the first 6 months, and bi- or tri-weekly thereafter. Feed
consumptions were measured weekly throughout the study. Daily observations
were made for mortality, behavior and appearance.
All hamsters in the feeding study (with the exception of animals
numbered 223 and 283 from the 0 ppm group, number 118 in 500 ppm group, and
numbers 63 and 82 from the 1000 ppm group) were subjected to complete
necropsy and examined for evidence of tumors at the time of their
spontaneous death, or following sacrifice with intraperitoneal injection
of pentobarbital sodium at the termination of the study. The above
five animals were inadvertently lost to necropsy evaluation early in the
study.
Tissues examined and removed at necropsy were placed in buffered
neutral 10% formalin. Tissues from animals in the 0, 125, and 1000 ppm
groups were processed, sectioned at 5 microns, and stained by the hematoxylin
and eosin method for histologic evaluation according to Battelle's Manual
for Standard Operating Procedures in Histology and Pathology. The groups
fed 250 and 500 ppm rotenone were excluded from initial histopathological
examination since these data would be superfluous if either one of two
situations existed: (1) chemical-related tumors were found in the 125 ppm
low dose group or (2) no chemical-related tumors were found in either the
125 ppm low dose or the 1000 ppm high dose groups. In the first situation,
the tumor-indueing threshold dose would be below 125 ppm, and data from
the 250 and 500 ppm groups would not add to the question of the carcinogenic
nature of rotenone. In the second circumstance, lack of tumors in the
high and low dose groups would suggest that the tumor-indueing threshold
for rotenone is above 1000 ppm, and that formation of tumors by the
intermediate dose would be extremely unlikely. If tumors were found in
the 1000 ppm rotenone group, but none in the 125 ppm group, the preserved
tissues could have been processed and examined for the establishment of a
dietary threshold.
12
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Tissues Removed at Necropsy
Skin (Thoracic)
Skin (Abdominal)
Mammary Gland
Trachea
Lung
Heart
Abdominal Aorta
Bronchial Lymph Node
Mandibular Lymph Node
Mesenteric Lymph Node
Spleen
Thymus
Kidney
Ureter
Bladder
Ovary
Uterus
Testicle
Epididymis
Prostate
Seminal Vesicle
Salivary Gland
Tongue
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Pancreas
Liver
Gall Bladder
Thyroid
Parathyroid
Adrenal
Rib
Femur
Muscle
Cerebrum
Cerebellum
Medulla
Pituitary
Spinal Cord
Sciatic Nerve
Eyes
Selected sections of kidney, spleen, liver, adrenal, and thyroid
glands were stained by the Bennhold Congo red method for amyloid detection.
Other selected samples of adrenal glands were stained by the Gomori chromaffin
staining technique for chromaffin granule detection.
Substantial spontaneous death losses were encountered in all groups
of hamsters from this study during the first 12 months of exposure. The
deaths were apparently not related to rotenone administration since the losses
were as high or higher in controls than in the treatment groups. Most deaths
during the first year of the study were associated with a syndrome of cecal
hemorrhage and dilatation which often involved the ileum and colon, and in a
few instances,the upper small intestine. The syndrome was characterized
clinically by sudden death in apparently healthy animals with no prodromal
clinical signs. Intestinal and cecal material from affected animals, when
passed through a 450 nm filter and injected into the peritoneal cavity of young
mice, caused death within 4 to 8 hours in dilutions up to 1:256. Similar
filtrates from non-affected hamsters did not produce death in mice. Clostridium
perfringens B, C, D, and E antitoxins would not protect against the lethality.
The lethal capacity of the filtrates was destroyed by heating to 56° F for
30 minutes. A pathogenic strain of E. coli was isolated from several animals
which died of this syndrome. This evidence indicates that a heat-labile
13
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enterotoxin of E_. coll may have induced this syndrome. However, unequivocal
evidence of this was not obtained since experimental production of the
syndrome with the suspected isolates or toxins produced by them was not
attempted in other hamsters. The identification of the enterotoxin
was not pursued at the request of the EPA Project Officer.
Spontaneous deaths from this syndrome decreased with age and
finally subsided after the animals reached approximately one year of age.
Spontaneous deaths after one year began to increase again and were primarily
a result of systemic amyloidosis which is common in Syrian hamsters.
Due to the relatively large number of early spontaneous deaths,
it was mutually agreed by the Project Officer from the Environmental
Protection Agency and Battelle's Principal Investigator that microscopic
evaluations were performed on animals that died or were terminated after
approximately one year on study. Gross pathological examinations were
performed on all animals for tumor formation. Tumors were diagnosed at
necropsy in 3 animals from either the 500 or 250 ppm dosage groups.
Data from microscopic evaluations of these animals are discussed later
in this report but are not included in the tabulated data.
Observations
The mean values for hamster body weights are presented in
Figure 3. Both 500 and 1000 ppm groups exhibited depressed body weights
relative to other test groups, particularly males fed the high dose. This
trend may have resulted in part from the decreased feed consumption for
these groups during the first six months of the study as seen in Figure 4.
Group mean rotenone consumption values are presented in Figure 5
expressed as milligrams of rotenone consumed per kilogram of body weight
per week. Data are plotted for alternate weeks.
Survival data for the hamsters on study are presented in Table 1.
Spontaneous deaths were not dose-related for rotenone, nor were there sex-
related differences for mortalities in the test groups. Female control animals
experienced a high death rate during the final 5 months of the study which
may have been related to the enteric infections which were prevalent in this
group early in the study.
14
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H 1000 pp« ,
F 1000 pp«tf a
M 500 ppm a o
F 500 pp« IT - -D
M 250 pp« * »
F 2SO pp«» »
H 125 pp«X K
F 125 ppojt ^
M Control * «
F Control •- —•
30 rt
0
12 16 20 24 28 32 36
40 44 48 "52 56 60 8 68 72 76 80 84
Weeks on Study
FIGURE 4. Group Mean Rotenone Diet Consumption in Carcinogenesis Study
92 96 100
16
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900
M lOOOppm A »
F lOOOppm S~~h
M 500ppm a—a
F SOOppm P--O
M 250ppm * *
F 250ppm *•-"•
M UBppm X K
F 125ppm »—•*
I
15 9 13 17 21 25 29 31 37 41 45 49 53 57 61 65 69 73 77 81 85 89 9:
Weeks on Study
FIGURE 5. Group Mean Rotenone Consumption During Carcinogenesis Study
17
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TABLE 1
HAMSTERS SURVIVING WHILE MAINTAINED ON ROTENONE DIETS
Date
1000 ppra
Male Female
500 ppm
Male Female
250 ppm
Male Female
12i ppm
Male Female
0 ppm
Male Female
Initial 50 50 50 50 50 50 50 50 50 50
January
1977
(12 months) 33 34 35 30 33 37 35 28 30 28
February 32 33 34 28 31 34 32 26 30 27
March 29 30 33 26 27 30 27 25 30 23
April 27 28 32 26 27 30 26 24 29 20
May 26 2~7 31 23 25 28 25 22 28 14
June 25 24 29 21 24 27 24 19 25 11
July 25 19 26 20 20 24 24 14 22 6
August
(until
necropsy) 24 19 25 18 19 20 21 9 22 2
18
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Gross visible lesions encountered at necropsy of hamsters
dying spontaneously and in euthanized animals at study termination are
presented in Tables 2 and 3 respectively. Notable lesions encountered
in animals dying spontaneously were nephrosis due to apparent amyloid
deposition, centrilobular congestion and necrosis of the liver, vegetative
thrombosis generally in the left atrium of the heart, pulmonary congestion,
hemorrhage and pneumonia, as well as atrophy or hypoplasia of the testicles.
Several tumors were also apparent at necropsy. Ovarian tumors
were noted in female animals number 94 of the 1000 ppm group, and number
285 of the 0 ppm group; masses involving leg muscles were found in animal
number 69 (female) of the 1000 ppm group, and animal number 291 (female)
of the 0 ppm group. A renal tumor was suspected in animal number 128 (male)
of the 500 ppm group, and a cystic adenoma was found in animal 472 (female)
of the 125 ppm group; possible thyroid tumors were found in female hamsters
451 of the 125 ppm group, and 284 and 286 of the 0 ppm group. An adrenal
tumor was suspected in animal number 61 (female) of the 1000 ppm group;
and a lymphoid neoplasm was present in several tissues from animal number
383 (female) of the 250 ppm group.
Gross lesions observed in hamsters euthanized at the termination
of the study are described in Table 3. Euthanized hamsters had fewer and
less severe inflammatory intestinal and pulmonary lesions than animals that
died spontaneously. The incidence of amyloid-induced nephrosis in hamsters
terminated at the end of the study was higher, and was more severe than that
present in hamsters with spontaneous deaths. The incidence of hepatic or
biliary cysts in the liver also increased with age. Grossly visible tumors
or focal hyperplasias of the adrenal glands were noted in male hamsters
4 and 21 of the 1000 ppm group, number 107 of the 500 ppm group, number 402
and 425 of the 125 ppm group, and number 220 of the 0 ppm group. A chondroma
of the costocondral junction of the rib was found in animal number 83 (female)
of the 1000 ppm group.
Results of Histopathological evaluations of animals from the 1000,
125 and 0 ppm dosage groups which died or were euthanized are summarized in Table 4.
19
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TABLE 2- CROSS LF.SIONS IN HAMSTERS THAT DIED SPONTANEOUSLY
( INCIDENCE AND PERCENT AFFECTED )
1OOO ppm
57 Z
Enter It la
Typhlitis
Colitis
Nephrosla
Liver CentrJ lobular Congestion and Necroals
Pulmonary Congestion and Hemaorhage
Pneumonia
Heart Arterial or Ventricular Thrombosis
Liver Hepatic or Biliary Cysts
Gastric Ulceratlon
Rectal Prolapae and Intussusception
Testlcular Atrophy or Hypoplasla
Ovarian Tumor or Cyst
Tumor ^fuscle of Leg)
Renal Tumor
Spleen Focal Hyperplasla or Tumor Mass
Thyroid Focal Hyperplaala or Tumor Mass
Adrenal Focal Hyperplasla or Tumor Masa
Salivary Gland Focal Hyperplasls or Tumor Mass
Tumor , Lymphonrcoma
26
27
8
10
9
8
10
4
2
2
6
1
1
1
1
45,
47.
14,
17,
15.
14
17,
7
3
3
10
1
1
1
1
.6
.3
,0
.5
.7
0
.5
.0
.5
.5
.5
.7
.7
.7
.7
Dosage
500 ppm
57 Z
23
33
9
8
12
20
4
2
1
1
1
40.3
57.8
15.7
14.0
21.0
35.0
7.0
3.5
1.7
1.7
1.7
Level and Number In Group
250
61
20
24
8
17
18
19
8
4
3
6
1
4
1 cyst
1
1
ppm
Z
32.
39,
13.
27,
29,
31',
13
6
4
9
1
6
1
1
1
,7
.3
.1
.8
.5
.1
.1
.5
.9
.8
.6
.5
.6
.6
.6
125
70
37
28
8
25
13
20
1
5
5
5
1
9
1 cyst
1
1
1
ppm
Z
52.
4O.
11.
35.
8
0
4
7
18.5
28.
1.
7.
7.
7.
1.
12.
1.
1.
1.
1.
5
4
1
1
1
4
8
4
4
4
4
0
76
22
33
6
28
17
29
4
7
10
1
2
1
1
1
2
2
ppm
Z
28.9
43.4
7.8
36.8
22.3
38.1
5.2
9.2
13.1
1.3
2.6
1.3
1.3
1.3
2.6
2.6
20
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-------
TABLE 4. DISTRIBUTION OF HAMSTERS
EVALUATED MICROSCOPICALLY
1000 ppm
Male Female
125 ppm
Male Female
0 ppm
Male Female
Spontaneous Deaths
Euthanized
Total
8 14
24 19
32 33
11 17
21 9
32 26
8 25
22 2
30 27
22
-------
The predominant non-neoplastic microscopic alterations encountered
in all groups reviewed were associated with changes due to age related
amyloid deposition in the kidneys, liver, spleen, and adrenals (Table 5).
The pattern of change found in the kidneys was retraction of the capsular
surface with associated tubular degeneration and regeneration with the
deposition of an amyloid material in the tubular interstitial tissues,
generally of the cortex. The mesangium of the glomerular tuft was often
thickened and contained a similar lightly-stained eosinophilic material;
Bowman's capsule was also thickened in some instances. The general picture
was that of atrophied misshapened nephron units with dilated collecting
tubules filled with brightly staining eosinophilic protein material. The
normal liver architecture was altered by rather thick deposits of light pink
amyloid-related material in and between the hepatocytes and vascular tissues
of the portal triads that often become confluent between adjacent triads.
The splenic red pulp was often completely filled with the pale eosinophilic
material with only small lymphoid nodules disturbing the monotonous pink
fields. The adrenal cortex was also a deposition site for the lightly
staining amyloid-like material, where it was found in or between cortical
cells. Female hamsters from all three dosage groups had a higher incidence
of amyloid-like material deposited between follicles in the thyroid than did
males. Thrombosis of the left atrium was present in all female groups but
was not found in any male reviewed microscopically. Pulmonary lesions
ranged from mild alveolar septal congestion to bronchiolar pneumonia of a
moderate severity. Varying degrees of enteritis, typhlitis, and colitis
were observed in all groups. Testicular atrophy was present to some extent
in all three groups of male animals. Numerous other lesions of lower
incidence, less significance, or random occurrence are listed in Table 5,
and will not be discussed further.
Tables 6 and 7 list the tumors and various types of hyperplasias
which occurred in the different dosage groups. Adrenal cortical carcinomas
were observed in the group given 1000 ppm (2 in females, 1 In male).
Adenomas and hyperplasias of the adrenal cortex were common in all dosage
groups, and were the most consistent lesions noted in the male and female
groups examined. Three animals from the medium dosage groups had tumors
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TABLE 7. MICROSCOPIC DIAGNOSES OF NEOPLASIA BY ANIMAL NUMBER*
Lesion 0 ppm 125 ppm 1000 ppm
Adrenal Cortex Adenoma 207 241 285 402 431 476 1 29 99
211 245 286 408 433 477 11 41 100
217 250 411 457 496 15_ 61"
226 257 413 461 2± 66
228 277 414 470 j!8 90
229 427 472 .34 94
Adrenal Cortex Carcinoma 4 73
60
Adrenal Medulla Pheochromocytoma 12
Trachea Mucosa Carcinoma _20_
Ovary Granulosa Cell 285 497 94
96
Spleen Hemangioma
Kidney Papillary Cyst Adenoma 472
Rib Chondroma
Thyroid Cystic Follicular Adenoma 451
Thyroid Follicular Carcinoma 415
Thyroid Parafollicular Cell Adenoma 284 242 245
Parathyroid Adenoma 286 415
Spleen Reticulum Cell Sarcoma *>'i7
Pituitary Pars Distal is Adenoma 279 286
Flbrosarcoma Leg 291
Undifferentiated Sarcoma NOS
Ji4
83
30
68
69
* Males are underlined.
30
-------
which were evident at necropsy. Microscopically these were diagnosed
as renal nephroblastoma for male hamster number 128 (500 ppm), lymph-
osarcoma in number 383 (female, 250 ppm), and splenic hemangioma in
number 386 (female, 250 ppm).
Acinar cell hyperplasia occurred in the mammary tissue of one
female from the 1000 ppm group. There were no neoplastic or other lesions
present in mammary tissue from hamsters in this study. Adrenal cortical
adenomas and hyperplasias occurred with similar frequency in all groups.
However, 3 adrenal cortical carcinomas occurred in the group that received
1000 ppm rotenone in their diet; one male (number 4) and one female
(number 73) went to study completion, while another female (number 60) died
June 1, 1977, two months prior to study termination. Analysis of these data
by a Chi-square 2X3 contingency scheme indicates that the appearance of
this tumor in the high dose group (compared to the non-occurence in the
0 and 125 ppm groups) is significant between the 90 and 95 percent
confidence levels. However, because the control group represented a younger
population of hamsters due to a larger number of early deaths, this comparison
is not valid since these tumors are known to develop spontaneously in aging
hamsters (3). The significance of these carcinomas in the highest dosage
group and their absence from other groups is not clear. Although a
correlation between these adrenal cortical carcinomas and rotenone treatment
cannot be ruled out, evidence for a cause-effect relationship is inconclusive.
The lesions encountered in this 18-month study are similar in nature
to those reported by Pour et al (1-4), Chesterman (5), and Gleiser et. al.
(6) in large hamster colonies retained for life time studies. Aging hamsters
are predisposed to development of renal, splenic, hepatic, and adrenal
lesions related to amyloid deposition. In this study, no substantial
histopathological differences were seen between treatment groups. Males
in this study had less amyloid depositions in the liver, spleen, adrenals,
and thyroid than females. No profound differences in the incidence of non-
neoplastic pathologic lesions were observed between rotenone-treated animals at
various dose levels and the control group. No pathologic alterations of note
were seen in any dose group relative to mammary gland changes.
31
-------
REFERENCES
1. Pour, P., N. Kmoch, E. Greiser, U. Mohr, J. Althoff, and A. Cardesa.
1976. "Spontaneous Tumors and Common Diseases in Two Colonies of
Syrian Hamsters. I. Incidence and Sites". J. National Cancer
Institute _56:931-935.
2. Pour, P., U. Mohr, A. Cardesa, J. Althoff, and N. Kmoch. 1976.
"Spontaneous Tumors and Common Diseases in Two Colonies of Syrian
Hamsters. II. Respiratory Tract and Digestive System". J. National
Cancer Institute 56:937-948.
3. Pour, P., U. Mohr, J. Althoff, A. Cardesa, and N. Kmoch. 1976.
"Spontaneous Tumors and Common Diseases in Two Colonies of Syrian
Hamsters. III. Urogenital System and Endocrine Glands". J. National
Cancer Institute 56:949-961.
4. Pour, U. Mohr, J. Althoff, A. Cardesa, and N. Kmoch. 1976. "Spon-
taneous Tumors and Common Diseases in Two Colonies of Syrian Hamsters.
IV. Vascular and Lymphatic System and Lesions of Other Sites". J.
National Cancer Institute 56:963-974.
5. Chesterman, F. C. 1972. "Background Pathology in a Colony of Golden
Hamsters". Progress in Experimental Tumor Research 16:50-68.
6. Gleiser, C. A., G. L. Van Hoosier, W. G. Sheldon, and W. K. Read.
1971. "Amyloidosis and Renal Paramyloid in a Closed Hamster Colony".
Laboratory Animal Science 21:197-202.
32
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO. 2.
EPA-600/l-79-004a
TITLE AND SUBTITLE
CARCINOGENIC POTENTIAL OF ROTENONE
PHASE I: DIETARY ADMINISTRATION TO HAMSTERS
AUTHOR(S)
A. P. Leber, R. L. Persing
PERFORMING ORGANIZATION NAME AND ADDRESS
Battel]e
Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
2. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
January 1979
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
68-02-1715
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/11
5. SUPPLEMENTARY NOTES
Principal Investigator A. P. Leber
6. ABSTRACT
Studies were performed to evaluate the potential carcinogenicity rotenone
in the Syrian Golden hamster. Several ancillary range-finding studies
were carried out including 14-day feeding trials and a reproduction
experiment. The latter experiment indicated that rotenone at a level of
500 ppm in the maternal diet was embryo-toxic (depressed birth weight and
fetal deaths) and resulted in cannabalism of the young by the maternal
animals. A level of 1000 ppm led to sterility in one or both sexes.
The 18-month carcinogenesis study indicated that 1000 ppm rotenone in
the diet was toxic to hamsters (depressed body weights compared to controls)
but no gross or histopathological evidence was obtained which indicated
that the test compound induced the formation of any tumors. Three
adrenal cortical carcinomas in 65 hamsters observed in the 1000 ppm
group could not be ascribed to rotenone treatment.
7. KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Carcinogens
Toxicity
8. DISTRIBUTION STATEMENT
Release to Public
b. IDENTIFIERS/OPEN ENDED TERMS
Rotenone
19. SECURITY CLASS (This Report!
Unclassified
20. SECURITY CLASS (This page)
Unclassified
c. COS AT I Field/Group
06, T, F
21. NO. OF PAGES
40
22. PRICE
PA Form 2220-1 (R*v. 4-77) PREVIOUS EDITION is OBSOLETE
33
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