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 ------- 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 categories were established to facilitate further development and application of environmental technology. Elimination of traditional grouping was consciously planned to foster technology transfer and 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 ECOLOGICAL RESEARCH series. This series describes research on the effects of pollution on humans,plant and animal species, and materials. Problems are assessed for their long-and short-term influences. Investiga- tions include formations, transport, and pathway studies to determine the fate of pollutants and their effects. This work provided the technical basis for setting standards to minimize undesirable changes in living organisms in the aquatic, terrestrial, and atmospheric environments. This document is available to the public through the National Technical Information Service, Springfield, Virginia 22161 ------- 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 ------- 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. ------- 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 ------- 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. ------- 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 ------- 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 ------- 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 ------- 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. ------- 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 ------- 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 ------- 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) ------- 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 ------- 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. ------- 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. ------- 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 ------- |