* Charles & Conn, LLC, Durham, North Carolina;
BASF Corporation, Research Triangle Park, North Carolina; and
BASF Aktiengesellschaft, Ludwigshafen/Rhein, Germany
Received September 15, 1999; accepted December 2, 1999
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ABSTRACT |
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Key Words: metiram; rat; mouse; rodent; subchronic toxicity; chronic toxicity; carcinogenicity; fungicide; dietary; T3; T4; ETU.
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INTRODUCTION |
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Ethylenethiourea (ETU) is a metabolite and a minor technical impurity of metiram and other EBDC fungicides, a class of non-systemic fungicide used worldwide for the control of a variety of diseases on a wide range of fruits, vegetables, cereals, and ornamental crops. The rate of formation in vivo of ETU from metiram was determined to be 7.5% (U.S. EPA, 1992). Oral doses of ETU are rapidly absorbed and excreted, primarily in the urine and more quickly in mice than in rats (U.S. EPA, 1992). Concentrations in blood and tissues are generally similar with the exception of somewhat higher levels in the thyroid, with half-lives for elimination from maternal blood of 5.5 and 9.4 h in mice and rats, respectively. Unchanged ETU was the primary metabolite in rats with small quantities of ethylene urea. In mice, the principal identified metabolites were ETU and imidazolinyl sulfenate.
Lifetime feeding studies were performed on rats and mice to assess the possible long-term toxicity and carcinogenicity of metiram. After these studies, subchronic toxicity studies in rats and mice were performed to further investigate the thyroid as a target organ for metiram and its metabolite ETU, and to establish the no-observed-adverse-effect level (NOAEL) for thyroid toxicity.
All studies were conducted in accordance with Good Laboratory Practice regulations and applicable toxicology guidelines for pesticide testing (EEC, 1987EEC, 1988; U.S. EPA, 1984; U.S. EPA-FIFRA, 1990; MAFF, 1985). The two chronic studies were conducted at Huntingdon Research Centre, Huntingdon, England and the two subchronic studies were conducted at the laboratories of BASF Aktiengesellschaft.
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MATERIALS AND METHODS |
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Homogeneity and stability of the test chemical in the diets were verified analytically by gas chromatography. Concentration analyses of the dietary formulations for each group in the rat chronic/carcinogenicity study were determined at study initiation and quarterly thereafter until termination. In the mouse carcinogenicity study, concentration analyses were performed at study initiation and at 6-month intervals thereafter and at study termination. In the subchronic rat and mouse studies, concentration analyses were determined at study initiation and at week 13. These analyses indicated that all diets were homogeneous. Concentration analyses confirmed that the ppm levels in the diets in all studies were generally within ±15% of targeted levels.
In the chronic/carcinogenicity study in rats, diets were administered dietary dose levels of 0, 5, 20, 80, or 320 ppm (approximately 0, 0.2, 0.8, 3.1, or 12.3 mg metiram/kg/day for males and approximately 0, 0.2, 1.0, 3.8, or 15.5 mg metiram/kg/day for females). The doses were selected based on the results from a 13-week subchronic range-finding study (with a 6-week recovery phase) that was performed at doses of 0, 50, 100, 300, or 900 ppm. Reversible abnormal movements, unphysiological (abnormal) reflexes, and muscle atrophy were observed in females at 300 and 900-ppm dose, and in males at 900-ppm, in this range-finding study. Eighty rats per sex per group were utilized for the chronic/carcinogenicity study, with 30 animals per sex per group of these animals intended for blood sampling and the thyroid function tests described below. These animals were not included in the carcinogenicity evaluation. The remaining fifty animals per sex per group were treated with metiram for up to 119 weeks in the case of males and 112 weeks in the case of females.
In the carcinogenicity study in mice, diets were administered for 89 weeks (females) or 95 weeks (males) at doses of 0, 100, 300, or 1000 ppm (approximately 0, 8, 24, or 79 mg metiram/kg/day for males and approximately 0, 9, 29, or 95 mg metiram/kg/day for females). Dose levels were selected based on the results of a 4-week range-finding study performed at doses of 0, 100, 300, 1000, or 3000 ppm, where significant liver toxicity was observed in the 3000-ppm dose group. Fifty-two mice per sex per group were utilized.
For the subchronic toxicity study in rats, 13 animals per sex per group were dosed for 13 weeks at dietary concentrations of 0, 5, 80, 320, or 960 ppm (approximately 0, 0.4, 5.8, 23.5, or 73.9 mg metiram/kg/day for males and approximately 0, 0.4, 6.7, 27.3, or 88.8 mg metiram/kg/day for females). In the subchronic mouse study, 10 mice per sex per group were given diets containing 0, 300, 1000, 3000, or 7500 ppm (approximately 0, 84, 302, 853, or 2367 mg metiram/kg/day for males and approximately 0, 133, 465, 1448, or 3565 mg metiram/kg/day for females) for 13 weeks.
Laboratory animals and care.
In the lifetime feeding studies, male and female Charles River CD rats (28 days old) and CFLP mice (23 days old) were obtained from Charles River, France S.A., St. Aubin-les-Elbeuf, France and Anglia Laboratory Animals, Alconbury, England, respectively. In the subchronic studies, male and female Wistar rats and B6C3F1 mice were obtained from Dr. Karl Thomae GmbH, Biberach/Riss, Germany and Charles River Wiga GmbH, Sulzfeld, Germany, respectively. Rats and mice were selected for the studies based on examination by a veterinarian after an acclimation period of at least 5 days. Rats were housed either in groups of 5 (chronic/carcinogenicity study) or individually (subchronic study) in elevated stainless steel, wire-mesh cages. Mice were housed either in groups of 4 (carcinogenicity study) or individually (subchronic study) in polypropylene cages with bedding. A 12-h light/dark cycle was maintained in the room, and food and water were available ad libitum.
For all studies, animals were observed for overt toxicity, moribundity, and mortality at least twice daily. Animal weights, detailed clinical observations, and food consumption were determined weekly until study termination. Water consumption was measured daily for 5 days/week during study weeks 12 and 24 in the chronic/carcinogenicity study in rats in the control and high-dose groups. Ophthalmoscopic examinations were conducted prior to treatment and at weeks 7, 13, 26, 52, 78, 104, and 119 in the chronic/carcinogenicity study in rats and prior to treatment and at study termination in the subchronic rat study. No ophthalmoscopic examinations were performed in the mouse studies.
Clinical pathology.
In the chronic/carcinogenicity rat study, prior to treatment and during weeks 5, 12, 24, 50, 76, and 102, we performed, on rats from both the control and high dose groups, clinical chemistry and hematology (10 rats/sex/group), and urinalyses (5 rats/sex/group). For the subchronic rat and mouse studies, clinical chemistry and hematology were performed on 13 rats and 10 mice/sex/group and urinalyses on 10 rats and mice/sex/group. These tests were carried out on study days 29 and 84 in rats and study days 96 or 97 (during necropsy) in mice. Clinical pathology was not performed in the mouse carcinogenicity study. However, blood smears were evaluated histologically. Blood samples were collected by orbital sinus venipuncture under light ether anesthesia (chronic/carcinogenicity study in rats) or under no anesthesia (subchronic rat and mouse studies). Urine samples were collected overnight.
The parameters evaluated in the chronic/carcinogenicity study in rats are identified by (*). Hematology parameters evaluated included cell morphology, red blood-cell count, hematocrit, hemoglobin, leukocyte count, leukocyte differential, thromboplastin time (subchronic rat study only), and platelet count. Serum chemistry parameters investigated in the subchronic toxicity study in rats and mice included assays for alanine aminotransferase (ALT), albumin, aspartate aminotransferase* (AST), blood urea nitrogen* (BUN), lactate dehydrogenase (LDH), calcium, chloride, creatinine, gamma-glutamyl transferase, globulin, glucose*, inorganic phosphorus, potassium*, magnesium, serum alkaline phosphatase*, sodium*, total bilirubin, total cholesterol, total protein*, thyroid stimulating hormone (TSH), triiodothyronine* (T3), and thyroxine* (T4).
In addition, in the chronic/carcinogenicity study in rats, 3 rats/sex/group were evaluated during weeks 4, 12, 26, 52, and 104 for thyroid function by measuring the clearance of an intravenous dose of 131I from the plasma and by measuring the incorporation of radioiodine into the thyroid and the protein fraction of the thyroid. Urinalysis measurements in both rat studies and the subchronic mouse study included bilirubin, glucose, ketones, pH, protein, specific gravity, volume, and urobilinogen.
Anatomic pathology.
All animals in the chronic studies surviving to the scheduled study termination were euthanized by carbon monoxide asphyxiation, exsanguinated, and subjected to gross and microscopic examinations. In the subchronic studies, microscopic examinations were performed on all animals in the control and high-dose groups, and on selected major organs and gross lesions in animals from the low and mid-dose groups. A complete necropsy was performed on all animals. The following organs were weighed in the chronic/carcinogenicity study in rats: adrenals*, brain, heart, kidneys*, liver*, ovaries, pituitary, spleen, testes*, thymus, thyroid/parathyroids*, and uterus. The organ weights taken in the subchronic study in rats and mice are identified by (*), with the exception that thyroids/parathyroids were not weighed from mice. No organ weights were taken in the carcinogenicity study in mice. The following tissues from each animal were preserved in either 4% or 10% neutral-buffered formalin: adrenals, aorta, bone, brain (medullary, cerebellar and cortical sections), cecum, esophagus, eyes, gallbladder (mice only), heart, kidneys, duodenum, jejunum, ileum, colon, rectum, lacrimal glands, larynx, liver, lung, mammary gland, cervical and mesenteric lymph nodes, ovaries, pancreas, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, mid thoracic, and lumbar), spleen, sternum (for bone marrow), stomach (glandular and non-glandular), testes, thymus, thyroid/parathyroids, trachea, urinary bladder, uterus, and any other tissues with gross lesions. Preserved tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.
Statistics.
Analysis of variance followed by Student's t-test were used to assess the significance of inter-group differences in the food consumption, water consumption, body weight, clinical laboratory data, and thyroid function test data. Analysis of organ weights was performed using analysis of covariance. Organ weights were adjusted for final body weight as covariate, where this was the more efficient method of analysis. Where appropriate, organ weights were log-transformed to stabilize variance. Group means were compared using both Students t-test (Winer, 1971) and Williams test (Peto and Pike, 1973
).
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RESULTS |
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Thyroid hormone determinations revealed the following statistically significant changes: serum T3 was increased (males) at 320 and 960 ppm; T4 was decreased at 960 ppm (both sexes); and TSH was increased in all treated animals when compared to the controls. However, with respect to the T3 and TSH values there was no dose-response relationship, although the doses were in a range of 5 to 960 ppm (0.4 to 81 mg/kg/day). Thus, the statistically significant results might be a result of the somewhat low values of the control groups, and without any biological significance. In support of this lack of biological significance at the lower dose levels, increased thyroid weights were observed only in males of the highest-dose group. Moreover, there were no histological changes observed in the thyroid at any dose level in either sex. Therefore, a biologically relevant, treatment-related effect was only seen at 960 ppm (81 mg/kg/day).
Neurological examinations revealed, in the highest-dose groups, signs of muscle weakness (hind-limb weakness, ataxia, reduced grip strength); female animals were more affected than males. However, at this dose, body weight was also impaired in the animals. Moreover, no morphological changes in the peripheral or central nervous system were observed, although thorough neuropathological examinations were performed using perfusion-fixed tissues of the animals. Taking into account all of the above findings, the NOAEL under the test conditions was 5.8 mg/kg/day for males and 6.7 mg/kg/day for females.
Chronic/Carcinogenicity Study in Rats
Salient results in rats are presented in Tables 2 and 3. No treatment-related clinical findings were obtained in the animals. Mortality, food consumption, food utilization, body weight and water consumption were also unaffected. The hematological and clinical chemistry parameters did not show any adverse effect due to treatment in any of the parameters evaluated, including the functional parameters of the thyroid (T3, T4, and thyroid function tests with 131I).
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Subchronic Study in Mice
Selected results from the subchronic toxicity study in mice are presented in Table 4. There were no changes, during the clinical examinations, that were treatment-related. Moreover, clinical chemistry and hematology evaluations were unaffected by treatment. Total serum T4 concentrations were reduced in both sexes at dose levels equal to or greater than 1000 ppm. In addition, an increase in T3 was found in high-dose males. A minimal or slight hypertrophy and vacuolization of the thyroid follicular epithelium were found at dose levels equal to or greater than 3000 ppm in both sexes.
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Carcinogenicity Study in Mice
Selected results from the carcinogenicity study in mice are presented in Tables 5 and 6. Among treated females, the statistical assessment of mortality rate revealed no significant differences from the controls. However, a marginally higher mortality incidence in the 1000-ppm group from week 40 until study termination resulted in their termination at week 88 while the remaining doses were terminated at week 94. In the 1000-ppm group, retarded body weight gain was observed during the first 14 weeks of treatment in both sexes. The food consumption of the males was reduced by about 5% during the first 52 test weeks and that of the females was increased by about 10% from the 53rd test week until study termination.
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DISCUSSION |
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Similar to the thionamide drugs, the primary toxicological finding with ETU in laboratory animals is inhibition of the synthesis of thyroid hormones T4 and T3, leading to elevated serum levels of TSH via feedback stimulation of the hypothalamus and pituitary (Hill et al., 1989). Prolonged and continuous elevation of serum TSH levels results in hypertrophy and hyperplasia of the thyroid follicular cells in rats, mice, monkeys, and dogs, and ultimately, in the development of nodular hyperplasia, adenoma, and/or carcinoma in rats and mice (Chhabra et al., 1992
; Graham and Hansen, 1972
; Innes et al., 1969
; Ullman, 1972
), but not hamsters (Gak et al., 1996). There is evidence for reversibility (Arnold et al., 1982
, 1983
). Direct evidence for inhibition of thyroid hormone synthesis by ETU has been obtained in rats in vivo (Arnold et al., 1982
, 1983
; O'Neill and Marshall, 1984). ETU also reversibly inhibited thyroid peroxidase-catalyzed iodination reactions in vitro (Doerge and Takazama, 1990
). Similarly, the long-term stimulation of the pituitary via hypothalamic thyrotropin-releasing hormone (TRH) also results in morphologic changes in the pituitary of rats in all 4 species tested, culminating in adenomas of the pars distalis after a 2-year exposure in mice (Arnold et al., 1982
, 1983
; Chhabra et al., 1992
).
The sequence of events relating thyroid hormone inhibition via hormonal imbalance to the onset of pituitary and thyroid follicular neoplasia in rodents is well-characterized, with the inference that the threshold for the early steps in the sequence, particularly the key elevation of TSH levels, is necessarily a threshold for the remaining steps in the process, including carcinogenesis. Thus, for purposes of human carcinogenic risk assessment, the principle of the existence of a threshold for thyroid and pituitary neoplasia resulting from thyroid inhibition has been accepted (U.S. EPA, 1992; Paynter, 1988). In addition, in comparison to laboratory animals humans are expected to exhibit a lesser degree of sensitivity to thyroid inhibitors (Costigan, 1998). The reasons for this are 2-fold: first, humans have a substantial reserve supply of thyroid hormone, much of which is carried in thyroxine-binding globulin, a serum protein that is missing in laboratory rodents (Odell, 1967). Secondly, under conditions of prolonged thyroid insufficiency, caused for example by dietary iodine deficiency, the primary human response is goiter rather than neoplasia (Martindale, 1972
). Thus, a large uncertainty factor is not needed to insure adequate protection of the human population from effects of dietary exposure to ETU.
The rat has generally been the most sensitive species to ETU-induced thyroid effects, followed by the dog and monkey, with the mouse being relatively insensitive. A NOEL for the induction of thyroid tumors was demonstrated in combined perinatal and adult exposure 2-year feeding studies in rats at 25 ppm, equivalent to 1.1 to 1.2 mg/kg/day, and an overall NOEL for the effects of ETU on the thyroid-pituitary axis has been demonstrated in 2-year chronic feeding studies in the rat at 5 ppm equivalent to 0.37 to 0.49 mg/kg/day in males and females, respectively (Hunter et al., 1979a).
Induction of liver tumors by ETU has been observed only in the mouse, and only at dietary levels of 330 ppm, equivalent to 55 to 61 mg/kg/day, or higher (Hunter et al., 1979b). These dose levels were also associated with centrilobular hepatocellular cytomegaly and other typical indications of generalized work-related stress to the liver, in addition to thyroid inhibition and TSH-induced follicular hyperplasia and neoplasia. Hepatocellular tumors have not been seen in rats or hamsters. In the mice, no increase was noted in the incidence of liver tumors after 2 years of dietary feeding at 100 ppm, equivalent to 17 to 18 mg/kg/day.
There is a wide variability in the incidence of liver tumors among various strains of mice, partly dependent on hormonal and/or nutritional, as well as genetic, factors. Genetic factors are particularly operative in the case of the B6C3F1 and other C3H-derived strains. Their high and variable background tumor incidence indicates the presence of a significant population of "initiated" or latent tumor cells whose potential is readily expressed under various stressed conditions. It has been acknowledged by numerous regulatory authorities (IARC, 1987; IPCS, 1990) that the induction of these types of tumors is of questionable relevance for assessment of oncogenic potential in human populations, where the background incidence of liver cancer is extremely low.
In an overview of all ETU-related studies, the U.N. FAO/WHO (1994) concluded that the thyroid effects of ETU are due to the interference with the synthesis of thyroid hormone and that the liver effects are due to stress-related liver growth from increased functional demand. These changes are reversible when exposures are brief or intermittent. Only prolonged exposure may lead to tumor formation by a secondary mechanism. Available mechanistic data indicate that a threshold for the mechanisms involved can be established.
As previously mentioned, ETU is an impurity, as well as a metabolite, of metiram. To calculate the total systemic exposure to ETU, one must consider the intentionally added ETU content of 2% and the endogenous rate of formation of 7.5% from metiram. Therefore, the animals had a total systemic exposure of 9.5%. At the NOAEL of 3.5 mg/kg/day, there was an ETU systemic exposure of 0.33 mg/kg/day. There were no morphological or functional changes in the thyroid, even at the highest dose level of 320 ppm, equivalent to a mean intake of metiram of 13.9 mg/kg/day in both sexes (ETU intake of 1.32 mg/kg/day). Metiram was not found to be carcinogenic in rats. In the subchronic rat study, the NOAEL based on thyroid toxicity was 80 ppm, equal to an average intake of 25 mg/kg/day. The intake values in the studies presented here are consistent with the reported NOEL for ETU thyroid toxicity in the rat of 0.37 to 0.49 mg/kg/day in males and females, respectively (Hunter et al., 1979a).
In the carcinogenicity study in mice reported here, the histopathological parameters were unaffected at even the highest dose level of 1000 ppm. Assuming a similar rate of endogenous formation of ETU from metiram in mice, as has been determined in rats, this corresponds to a mean daily ETU intake of 8.4 mg/kg/day. No thyroid or liver hyperplasia or tumors were observed. The test substance was found non-carcinogenic in mice. Again these levels of ETU exposure are consistent with the NOEL of 17 to 18 mg/kg/day for liver and thyroid effects of ETU in mice (Hunter et al., 1979b).
In conclusion, metiram has been tested in rodents over a wide range of dose levels, including high doses at and above the MTD. The findings of these studies indicate metiram's general low toxicity and absence of carcinogenicity following chronic dietary exposure in the rat and mouse.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Arnold, D. L., Krewski, D. R., Junkins, D. B., McGuire, P. F., Moodie, C. A., and Munro, I. C. (1983). Reversibility of ethylenethiourea thyroid-induced lesions. Toxicol. Appl. Pharmcol. 67, 264273.[ISI][Medline]
Chhabra, R. S., Eustis, S., Haseman, J. K., Kurtz, P. J., and Carlton, B. D. (1992). Comparative carcinogenicity of ethylene thiourea with or without perinatal exposure in rats and mice. Fundam. Appl. Toxicol. 18, 405417.[ISI][Medline]
Costigan, M. (1998). The relevance of rat thyroid gland tumours to humans. United Kingdom Health and Safety Executive, Toxicology Unit, Bootle.
Doerge, D. R., and Takazama, R. F. (1990). Mechanism of thyroid peroxidase inhibition by ethylenethiourea. Chem. Res. Toxicol. 3, 98101.[ISI][Medline]
Dunnett, C. W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 10961121.[ISI]
Dunnett, C. W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482491.[ISI]
Elia, M. C., Arce, G., Hurt, S. S., O'Neill, P. J., and Scribner, H. E. (1994). The genetic toxicology of ethylenethiourea: A case study concerning the evaluation of a chemical's genotoxic potential. Mutat. Res. 341, 141149.[ISI]
European Economic Community (EEC) (1987). Good Laboratory Practice, Official Journal of the European Communities, L15: January 17, 1987.
European Economic Community (EEC) (1988). Methods for the Determination of Toxicity, Official Journal of the European Communities, L133: May 20, 1988.
Gak, J. C., Graillot, C., and Truhaut, R. (1976). Differences in hamster and rat sensitivity to the effects of long term administration of ethylenethiourea. Eur. J. Toxicol. Environ. Hyg. 9, 303312.[ISI][Medline]
Graham, S. L., and Hansen, W. H. (1972). Effects of short-term administration of ethylenethiourea upon thyroid function of the rat. Bull. Environ. Contam. Toxicol. 7, 1925[ISI][Medline]
Hill, R. N., Erdreich, L. S., Paynter, O. E., Roberts, P. A., Rosenthal, S. L., and Wilkinson, C. F. (1989). Thyroid follicular cell carcingenesis, A review. Fundam. Appl. Toxicol. 12, 629697.[ISI][Medline]
Hunter, B., Barnard, A. V., Prentice, D. E., and Gregson, R. (1979a). Metiram: Tumorigenicity to mice in long-term Ddietary administration. Study performed by Huntingdon Research Centre for BASF Aktiengesellschaft, Ludwigshafen/Rhein, Germany, BSF 198/78265.
Hunter, B., Barnard, A. V., and Street, A. E. (1979b) Metiram: Toxicity and tumorigenicity in prolonged dietary administration to the rat. Study performed by Huntingdon Research Centre for BASF Aktiengesellschaft, Ludwigshafen/Rhein, Germany; BSF 199/7915.
Innes, J. R. M., Ulland, B. M., Valerio, M. G., Petrucelli, L., Fishbein, L., Hart, E. R., Pallotta, A. J., Bates, R. R., Falk, H. L., Gart, J. J., Klein, M., Mitchell, I., and Peters, J. (1969). Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note. J. Natl. Cancer Inst. 42, 11011114.[ISI][Medline]
Martindale, W. B. (1972). Carbimazole and other antithyroid agents. In The Extra Parmacopieia, 26th ed. (N. W. Blacow, Ed.), pp 379385. Pharmaceutical Press, London.
Ministry of Agriculture, Forestry and Fisheries (MAFF) (1985). Agricultural Production Bureau, Japan. Good Laboratory Practice Standards, 59 NohSan No. 3850, January, 1985. In Agricultural Chemical Laws and Regulations. Japan (II), 1985. Society of Agricultural Chemical Industry, pp. 5671. Tokyo.
Odell, W. D., Rayford P. L., and Ross, G. T. (1967). Studies of Thyrotropin Physiology by Means of Radioimmunoassays. Proceedings of the 1966 Laurentian Hormone Conference. (G. Pincus, Ed.), Vol. 23, pp. 4785. Academic Press.
O'Neil, W. M., and Marshal, W. D. (1984). Goitrogenic effects of ETU on rat thyroid. Pest. Biochem. Physiol. 21, 92101.[ISI]
Paynter, O. E., Burin, G. J., Jaeger, R. B., and Gregorio, C. A. (1988). Goitrogens and thyroid follicular cell neoplasia: Evidence for a threshold process. Reg. Toxicol. Pharmacol. 8, 102119.[ISI][Medline]
Peto, R., and Pike, M. (1973). Conservatism of the approximation sigma (O-E) 2-E in the log-rank test for survival data or tumor incidence data. Biometrics 29, 579.[ISI][Medline]
Ullman, B. M. (1972). Brief communication: Thyroid cancer in rats from ETU intake. J. Natl Cancer Inst. 49, 583584.[ISI][Medline]
United Kingdom Advisory Committee on Pesticides (1990). Position document on ethylene bisdithiocarbamates and ethylene thiourea. ACP 32 (208/90).
U.N. Food and Agricultural Organization and World Health Organization (U.N. FAO/WHO) (1994). Plant production and protection paper 122. Ethylenethiourea, ethylenebisdithiocarbamates, mancozeb, maneb, metiram, and zineb. Report of the Joint Meeting of the FAO panel of experts on pesticide residues in food and the environment and the WHO expert group on pesticide residues, Geneva, September 2029, 1993.
U.S. Environmental Protection Agency (U.S. EPA) (1984). Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Series 821, 831, and 832. U.S. Department of Commerce, National Technical Information Service PB86108958, Revised Ed., November, 1984.
U.S. Environmental Protection Agency (U.S. EPA) (1992). Position Document 4 for the EBDCs and ETU. 57 Federal Register 7484, March 2, 1992.
U.S. Environmental Protection Agency-FIFRA (U.S. EPA-FIFRA) (1990). Environmental Protection Agency-Federal Insecticide, Fungicide and Rodenticide Act; Good Laboratory Practice Standard. Federal Register. 40 CFR Part 160, July 1, 1990.
Winer, B. J. (1971). Statistical principals in experimental design, 2nd ed. McGraw-Hill, New York.