oEPA
United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
P.O. Box 15027
Las Vegas NV 89114
EPA-600 3-80-031
February 1980
Research and Development
Selected Toxicological
Studies of Dimilin in
Weanling Male Rats
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EPA-600/3-80-031
February 1980
SELECTED TOXICOLOGICAL STUDIES OF DIMILIN IN WEANLING MALE RATS
Yogendra M. Patel and John A. Santolucito
Exposure Assessment Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89114
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
LAS VEGAS, NEVADA 89114
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring Systems
Laboratory, U.S. Environmental Protection Agency, and approved for publi-
cation. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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FOREWORD
Protection of the environment requires effective regulatory actions based
on sound technical and scientific data. The data must include the
quantitative description and linking of pollutant sources, transport
mechanisms, interactions, and resulting effects on man and his environment.
Because of the complexities involved, assessment of exposure to specific
pollutants in the environment requires a total systems approach that
transcends the media of air, water, and land. The Environmental Monitoring
Systems Laboratory at Las Vegas contributes to the formation and enhancement
of a sound monitoring-data base for exposure assessment through programs
designed to:
• develop and optimize systems and strategies for moni-
toring pollutants and their impact on the environment
• demonstrate new monitoring systems and technologies
by applying them to fulfill special monitoring needs
of the Agency's operating programs
This report provides significant new data on the toxicological response
of weanling male rats to the pesticide, Dimilin. This study specifically
deals with the effects of Dimilin on the circulating testosterone and the
development of the reproductive organs. For further information, the reader
should contact the Exposure Assessment Division.
George B. Morga
D*i rector
Environmental Monitoring Systems Laboratory
Las Vegas
iii
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ABSTRACT
Effects of subacute doses of Dimilin [l(4-chlorophenyl)-3-(2,6-
difluorobenzyl)urea] on the reproductive system of weanling male rats were
examined over a period of 96 days. The parameters evaluated were:
testosterone level in plasma, growth of reproductive organs (testes,
prostate, seminal vesicles) and adrenal glands, and histological examination
of tissues for pathological changes associated with the administration of
Dimilin. By intragastric intubation, groups of male rats, 25 days old, were
given 0, 15, 150, or 300 milligrams of Dimilin suspended in vegetable oil per
kilogram per day for a period of 0, 14, 28, 42, and 96 days.
The data indicate that Dimilin had no adverse effects on body weight or
organ weights of weanling rats, but decreased the levels of testosterone in
the plasma of animals of prepubertal age. However, this effect of Dimilin
began to disappear with the onset of puberty. Histological examination of
the test animals having lower levels of testosterone in plasma failed to
reveal any Dimilin-induced changes in interstitial or germinal cells. On the
basis of these observations, it is concluded that Dimilin, at dose levels of
15, 150, and 300 milligrams per kilogram per day, transiently depresses the
testosterone in plasma during the prepubertal period, yet has no delaying
effects on the development of the reproductive organs of male rats.
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CONTENTS
Disclaimer ii
Foreword iii
Abstract iv
Tables vi
1. Introduction 1
2. Conclusion 1
3. Experimental Procedures 2
4. Results 3
5. Discussion 6
References 7
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TABLES
Number Page
1. Experimental Design 2
2. Effect of Dimilin on Body Weight of Male Rats
Following Daily Administration of Dimilin 4
3. Effect of Dimilin on Organ Weights of Male Rats
Following Daily Administration of Dimilin 5
4. Effect of Dimilin on Plasma Testosterone Level in Male
Rats Following Daily Administration of Dimilin 6
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INTRODUCTION
Insect growth regulators are a new group of compounds that disrupt growth
and normal development of the insect in various immature stages (1). One of
these compounds, the insecticide Dimilin (Diflubenzuron; TH 6040; 1(4-
chlorophenyl )-3-(2,6-difIuorobenzyl)urea), has emerged as one of the
promising urea-based larvicides. This compound prevents the incorporation of
glucose into endocuticle through the inhibition of chitin synthesis (2).
Dimilin has been successfully used in controlling a variety of arthropod
pests injurious to plants and animals (3,4). This compound, when spread
directly onto cattle, effectively reduces the egg hatching of stable flies
and horn flies. Secretion of Dimilin into the egg damages the developing
embryo which accounts for the "sterility" observed in these insects. This
compound has also shown excellent potential for controlling the larval stages
of mosquitos (5,6), Diptera (7,8), certain Lepidoptera (9), and Coleoptera
(10).
Dimilin's biotransformation in lactating cows, castrated sheep, and rats
has been reported. It appears to be absorbed, metabolized, and excreted in
the urine. The major metabolites of Dimilin excreted by the cow, sheep, and
rats are the result of the hydroxylation of difluorobenzyol and chlorophenyl
rings and the cleavage between the carbonyl and amide groups. The mutagenic
potential of the diflubenzuron metabolites has also been investigated using
the Salmonella mutagenicity test (11).
On account of Dimilin's high toxicity toward many destructive insects and
its extremely low toxicity in rats (Oral LDso >10 g/kg) and mice
>4.64 g/kg), Dimilin has been proposed for use as an insecticide on
commercial crops for control of such pests as cornborers. However, it has
been reported that when Dimilin was given in feed to baby chicks for 13
weeks, the males failed to mature—the combs, wattles, feathers, and voice
remained undeveloped. These adverse effects with Dimilin were accompanied by
decreases in plasma testosterone proportional to Dimilin dosage (12).
Thus, in view of these adverse findings, and the lack of information
concerning other possible effects of Dimilin on the reproductive system
of weanling male rats (in terms of plasma testosterone, development of
reproductive organs), the present study was undertaken.
CONCLUSION
When weanling male rats were given daily doses of 15, 150, and 300
mg/kg body weight of Dimilin suspended in oil over a period of 14, 28, 42,
and 96 days, Dimilin produced a transient depression in plasma levels of
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testosterone in the prepubertal period without causing any noticeable
delaying effect on the development of the reproductive organs.
EXPERIMENTAL PROCEDURES
ANIMALS AND DOSAGE
Male Long-Evans rats were obtained as weanlings from Blue Spruce Farms,
Inc., Altamont, New York 12009. They were acclimated to a controlled animal
room environment with a light cycle of 14 hours of light per 10 hours of
darkness for a period of 4 days. The rats were approximately 25 days old
(55 g mean body wt) at the initiation of the study. Dimilin (U.S. EPA Lot
no. PP312; 99% purity) was administered daily by intragastric intubation as a
suspension in vegetable oil to groups of rats throughout the duration of the
experiment. The control animals were given only vegetable oil. The dosage
levels of Dimilin were 0, 15, 150, and 300 milligrams of Dimilin per kilogram
of body weight.
EXPERIMENTAL DESIGN AND CONDUCT
On the day before the start of pesticide administration, weanling male
rats were weighed and randomly assigned to control and treatment groups.
Those rats in the lower and upper ranges of body weights were first
eliminated. The control group contained 15 animals and each test group
consisted of 8 animals to provide optimal observations and contrast for
statistical evaluation. The experimental design is presented in Table 1.
TABLE 1. EXPERIMENTAL DESIGN
No. of
Group Controls No. of Low No. of Medium No. of High Day of Sampling
(oil only) Dose Rats Dose Rats Dose Rats and/or Sacrifice
I
II
III
IV
V
15*
15
15
15
15
-
8
8
8
8
-
8
8
8
8
-
8
8
8
8
1
14
28
42
96
* Controls for Group I received no treatment; controls for other groups
received vegetable oil, without Dimilin, by intragastric intubation.
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All animals were housed individually in galvanized metal cages with food and
water supplied ad lib. Body weights were recorded weekly and the dosage was
adjusted according to the mean body weight to attain the specified dosage
levels.
On the day treatment began, blood samples from 15 non-treated animals
(Group I, Table 1) were collected for testosterone analysis. The animals
were sacrificed; organ weights of testes, prostate, seminal vesicle, and
adrenal glands were obtained and the tissues preserved in 10 percent neutral
buffered formalin. The remaining groups of animals were sacrificed at the
same time of day throughout the experiment to minimize variation due to
diurnal rhythms of circulating testosterone. Handling, ambient environment,
food, water, etc., were consistent for all animals. Wet weights of the above
mentioned organs were recorded and the organs were fixed in 10 percent
neutral buffered formalin.
The testosterone concentrations in plasma for the control animals and the
test groups were determined by a radioimmunoassay procedure and the analyses
were carried out by Smith Kline Clinical Laboratories, Inc., Burbank,
California. Testes from all groups (control and treatment groups) and the
adrenal glands from animals of Group III (Table 1) were submittted for
histopathological evaluation.
All histology and pathology examinations were performed on 5-micron
sections stained with hemotoxylin and eosin by Dr. B. D. Ward, Veterinary
Pathologist, Mississippi State University, Starkville, Mississippi.
The statistical analysis was performed on arithmetic means from log-
transformed data and employed analysis of variance procedures.
RESULTS
The mean body weights of the control and treatment groups following daily
administration of Dimilin are given in Table 2. The results show that after
14, 28, 42, and 96 days of Dimilin treatment there are no significant
differences in body weights between the control and DimiTin-treated rats.
Organ weights for the various treatment groups are given in Table 3.
One-way analysis of variance did not reveal any dose-related changes in the
various organ weights among the different groups. There are some minor
differences observed, however. The only consistent observation is a
significant decrease in the relative adrenal gland weights of all animals on
Dimilin for 28 days.
The values for the plasma testosterone levels are given in Table 4. The
animals in Group II, which had received Dimilin for 14 days revealed a
decrease in plasma testosterone at all three dosage levels. Lower levels of
testosterone were also observed in animals receiving Dimilin for 28 days at
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TABLE 2. EFFECT OF DIMILIN ON BODY WEIGHT OF MALE RATS
FOLLOWING DAILY ADMINISTRATION OF DIMILIN
Weights (g) are expressed as mean ± S.D.
Group Control9 15 mg/kg/dayb 150 nig/kg/day15 300 mg/kg/dayb Remarks
I 54.3 ± 4.7
II 123.8 ± 10.5
124.3 ± 10.4 125.5 ± 8.7
III 212.3 ± 19.1 214.1 ± 23.6 221.5 ± 16.5
IV 298.1 ± 35.2 291.8 ± 26.5 307.3 ± 25.9
V 481.3 ± 34.3 488.6 ± 28.5 479.6 ± 21.0
25 days of
age
122.8 ± 14.5 14 days on
Dimilin
39 days of
age
209.0 ± 14.4 28 days on
Dimilin
53 days of
age
301.1 ± 33.3 42 days on
Dimilin
67 days of
age
465.1 ± 35.2 96 days on
Dimilin
121 days of
age
a = 15 animals each
b = 8 animals each
150 mg/kg/day, and at 300 mg/kg/day, and in animals receiving Dimilin for 42
days at 150 mg/kg/day. However, it is interesting to note that by day 96,
the levels of plasma.testosterone in the treated animals had increased. At
day 96, the plasma testosterone values of Dimilin treated animals were not
significantly different from the plasma testosterone values of the controls.
The histological evaluation of the testicular tissues of the control and
treated groups and the adrenal glands of Group III (Table 1) animals,
revealed no histopathological changes attributable to Dimilin.
Eighteen plasma samples (12 experimental plasma samples and 6 samples
from pooled plasma of untreated animals) were submitted for an inter-
laboratory cross-check comparison to Endocrine Sciences, Tarzana, California.
The results of the entire 18 samples were subjected to the t-test for
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TABLE 3. EFFECT OF DIMILIN ON ORGAN WEIGHTS OF MALE RATS FOLLOWING DAILY ADMINISTRATION OF DIMILIN
Orqan Weights (mg)
Group
I
Control a
II
Control3
15 mg/kg/dayb
150 mg/kg/dayb
300 mg/kg/dayb
HI
Control3
15 mg/kg/dayb
150 mg/kg/dayb
300 mg/kg/dayb
IV
Control3
15 mg/kg/dayb
150 mg/kg/dayb
300 mg/kg/dayb
V
Control3
15 mg/kg/dayb
150 mg/kg/dayb
300 mg/fcg/dayb
Testes
337.3
1156.3
1209.0
1252.0
1178.8
2341.4
2246.3
2283.0
2277.5
2965.5
2931.6
2915.3
2918.8
3448.1
3451.5
3510.6
3644.5*
± 42.3
± 257.5
± 150.2
± 118.4
± 211.9
± 197.0
± 298.4
± 270.6
± 308.4
± 361.5
± 239.9
± 204.3
± 257.2
± 239.5
± 131.1
± 232.9
± 200.1
Prostate
11.7 ±4.3
43.3 ± 9.2
47.9 ± 7.1
48.0 ± 6.0
44.6 ± 14.1
116.8 t 34.9
94.0 ± 26.5
102.4 ± 16.4
103.4 t 11.6
165.1 ± 51.2
155.6 ± 35.4
176.1 t 64.8
163.5 ± 52.4
345.7 ± 33.8
356.4 ± 43.5
344.1 t 59.5
347.6 ± 64.6
are expressed as mean ± S.D.
Seminal Vesicles
With Fluid
Intact Without Fluid
23.6 ± 7.0
40.3 ± 7.4
45.3 ±11.7
41.9 ± 8.5
44.6 ± 8.3
210.1 ± 54.4
193.3 ± 49.4
196.6 ± 38.4
211.3 ± 61.0
661.0 ±
607.6 ±
699.3 ±
632.8 ±
1244.5 ±
1264.1 ±
1259.9 ±
1336.5 ±
187.7
53.5
169.6
154.5
187.5
186.6
158.1
116.8
.
267.3
249.3
287.8
239.4
360.3
358.3
353.3
369.9
± 57.5
± 28.3
± 75.4
± 51.7
± 46.4
± 77.9
± 41.1
± 46.9
Adrenal
21.0
40.5
35.6
37.8
41.5
69.9
60.8*
59.8*
60.3*
53.3
47.4
47.9
46.1
58.3
61.8
58.6
56.3
±3.5
± 8.4
± 5.7
± 2.4
± 7.6
± 11.6
± 7.8
± 4.8
± 3.8
± 10.8
±9.9
± 4.3
t 4.2
± 13.0
± 15.3
± 10.4
± 8.8
Remarks
25 days
14 days
39 days
28 days
53 days
42 days
67 days
96 days
121 days
of age
on Dimilin
of age
on Dimilin
of age
on Dimil in
of age
on Dimilin
of age
a = 15 animals each
b = 8 animals each
* Significantly different from Control P<.05 (one-way analysis of variance)
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TABLE 4. EFFECT OF DIMILIN ON PLASMA TESTOSTERONE LEVEL IN MALE RATS
FOLLOWING DAILY ADMINISTRATION OF DIMILIN
Plasma Testosterone Values are expressed as
Mean ± S.D. (ng/D)
Group Control9 15 mg/kg/dayb 150 mg/kg/dayb 300 mg/kg/dayb Remarks
I 13.3 ±8.6 - - - 25 days of
age
II 74.7 ± 81.3 21.4 ± 57.2* 16.7 ± 34.2* 4.2 ± 4.3* 14 days on
Dimilin
39 days of
age
III 213.3 ± 163.4 217.8 ± 96.9 115.5 ± 77.5* 113.6 ± 72.3* 28 days on
Dimilin
53 days of
age
IV 480.0 ± 264.2 319.8 ± 328.5 145.0 ± 120.2* 353.3 ± 200.2 42 days on
Dimilin
67 days of
age
V 177.9 ± 112.5 251.0 ± 194.8 269.4 ± 173.7 439.9 ± 297.0 96 days on
Dimilin
121 days of
age
a = 15 animals each
b = 8 animals each
*Significantly different from control P<.05 (one-way analysis of variance)
differences; there was no statistically significant difference between the
testosterone values reported by the two clinical laboratories.
DISCUSSION
The data presented in this report indicate that Dimilin had no adverse
effects on body weight or organ weights of weanling rats; however, a decrease
in levels of testosterone in plasma was noted. Testosterone values were
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significantly lower in the prepubertal period, but this effect of Dimilin
began to disappear with the onset of puberty. Histological examination of
the testicular tissues of animals having lower levels of testosterone in
plasma did not reveal any Dimilin-induced changes in interstitial or germinal
cells. Similarly, no changes were observed in the weights of seminal
vesicles or prostates of these animals.
In conclusion, the results of this study show that Dimilin given to male
weanling rats at dosage levels of up to 300 milligrams per kilogram per day
transiently depresses the testosterone levels in plasma during the
prepubertal period, yet has no delaying effect on the development of the
reproductive organs.
REFERENCES
1. Marx, J. L. Chitin Synthesis Inhibitors: New Class of Insecticides.
Science 197:1170, 1977.
2. Post, L. C., B. J. Dejong, and W. R. Vincent. l-(2,6-disubstituted
benzoyl)-3 Phenylurea Insecticides: Inhibitors of Chitin Synthesis,
Pestic. Biochem. Physiol. 4:473, 1974.
3. Ivie, G. W., and J. E. Wright. Fate of Diflubenzuron in the Stable Fly
and House Fly. J. Agric. Food Chem. 26:90, 1978.
4. Ivie, G. W. Fate of Diflubenzuron in Cattle and Sheep. J. Agric. Food
Chem. 26:81, 1978.
5. Mulla, M. S., H. A. Darwazeh, and R. L. Norland. Insect Growth
Regulators: Evaluation Procedures and Activity Against Mosquitos.
J. Econ. Entomol. 67:329, 1974.
6. Schaefer, C. H., H. W. Wilder, and F. S. Mulligan. A Practical
Evaluation of TH 6040 as a Mosquito Control Agent in California. J.
Econ. Entomol. 68:183, 1974.
7. Miller, R. W. TH 6040 as a Feed Additive for Control of the Face Fly
and House Fly. J. Econ. Entomol. 67:697, 1974.
8. Miller, R. W., C. Corley, and K. R. Hill. Feeding TH 6040 to Chickens:
Effect on Larval House Flies in Manure and Determination of Residues in
Eggs. J. Econ. Entomol. 68:181, 1975.
9. Granett, J. and D. M. Dunbar. TH 6040: Laboratory and Field Trials for
Control of Gypsy Moths. J. Econ. Entomol. 68:99, 1975.
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10. Moore, R. F. and H. M. Taft. Boll Weevils: Chemosterilization of Both
Sexes with Busulfan plus Thompson-Hayward TH 6040. J. Econ. Entomol.
68:96, 1975.
11. Seuferer, S. L., H. D. Braymer, and J. J. Dunn. Metabolism of
diflubenzuron by Soil Microorganisms and Mutagenicity of the
Metabolities. Pestic. Biochem. Physio!. 10:174, 1979.
12. Smalley, H. E. Comparative Toxicology of Some Insect Growth Regulators.
Clin. Toxicol. 9:27, 1976.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-80-031
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
SELECTED TOXICOLOGICAL STUDIES OF DIMILIN
IN WEANLING MALE RATS
5. REPORT DATE
February 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Yogendra M. Patel and John A. Santolucito
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, Nevada 89114
10. PROGRAM ELEMENT NO.
A2AL1D
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency—Las Vegas, NV
Office of Research and Development
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89114
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/07
15. SUPPLEMENTARY NOTES
This work was performed for EPA's Office of Toxic Substances
Washington, D.C. 20460
16. ABSTRACT
The effects of the subacute doses of Dimilin [l(4-chlorophenyl)-3-(2,6-
difluoropbenzyl)urea] on the reproductive system of weanling male rats were examined
over a period of 96 days. The parameters evaluated were: plasma testosterone level,
growth of reproductive organs (testes, prostate, seminal vesicles) and adrenal glands,
and histological examination of tissues for pathological changes associated with the
administration of Dimilin. The animals, 25 days old, were given 0, 15, 150, and 300
milligrams/kilogram/day of Dimilin suspension in vegetable oil by intragastric
intubation for a period of 0, 14, 28, 42, and 96 days. The data indicate that Dimilin
lad no adverse effects on body weight or organ weights of weanling rats, but a
decrease in circulating testosterone in the plasma of animals of prepubertal age was
noted. However, this effect of Dimilin began to disappear with the onset of puberty.
The histological examination of the test animals with lower circulating testosterone
in plasma failed to reveal any Dimilin-induced changes in interstitial or germinal
cells. On the basis of these observations, it is concluded that Dimilin, at 15, 150,
and 300 mg/kg/day dosage levels, transiently depresses the testosterone in plasma in
the prepubertal period, yet has no delaying effects on the development of the
reproductive organs.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
urea
boxicology
ats
Dimilin
99A
57S
57Y
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
IINH ASSTFTFH
21. NO. OF PAGES
16
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
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