CHLOROTHALONIL EXPLANATION First draft prepared by Dr. E. Arnold, Health and Welfare Canada, Ottawa, Canada Chlorothalonil has been reviewed by seven previous Joint Meetings from 1974 to 1987 (Annex 1, 1975ab, 1978ab, 1980ab, 1982ab, 1984, 1985a, 1986ac, 1987b, and 1988b). Data considered on chlorothalonil itself include metabolism and biotransformation studies in mice, rats and dogs; pharmacological studies in mice and rats; acute toxicity in several species; short-term toxicity studies in mice, rats and dogs; in vivo and in vitro genotoxicity studies; teratology studies in rats and rabbits; and a number of long-term toxicity/oncogenicity studies in mice and rats. Data on the metabolite 4-hydroxy-2,5,6- trichloroisophthalonitrile have also been considered including genotoxicity, reproduction and carcinogenicity studies. In 1987, a temporary ADI of 0-0.003 mg/kg bw based on toxicity data for chlorothalonil, not the 4-hydroxy derivative, was extended. However, because of concern for the demonstrated oncogenicity in rats and pending completion of an ongoing oncogenicity study in rats, a high safety factor was used. The carcinogenicity study in rats together with further metabolism data have now been submitted. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA Biochemical aspects Metabolism Oral administration Rats A single oral dose of 14C-chlorothalonil at 50 mg/kg bw in aqueous (0.75% methyl cellulose) suspension was given to nine germ-free Sprague-Dawley male rats in a volume of 10 ml/kg bw. A tenth rat was given the vehicle alone as a control. Urine and faeces were collected at 24 hour intervals to 96 hours. At study termination at 96 hours, blood was collected and the kidneys and the rest of the carcass were saved. All samples were frozen pending analysis. Urinary excretion of radioactivity accounted for 2.36-4.02% (mean 3.12%) of the administered dose while faecal excretion accounted for 61.3-95.6% (mean 84.4%). Blood levels accounted for 0.0098-0.0229% (mean 0.0143%) of the dose in 8 of the 9 rats and 0.0517% in the remaining rat. The kidney contained 0.037-0.066% of the dose in all rats. Carcass radioactivity accounted for 0.177-0.285% of the dose in the 8 rats with similar blood levels and 1.78% in outlier rat. Urinary excretion of test material occurred mainly in the first 24 hours after dosing except in one rat which showed almost equal excretion at 0-24 and 24-48 hours. Faecal excretion also was predominantly in the first 24 hours except in the one rat in which peak excretion was observed in the period 24-48 hours. Eight 0-24 hour and four 24-48 hour urine samples were analyzed for methylated thiols. No methylated thiols were detected in the 0-24 hour urine of 6/8 rats or in 24-48 hour urine from 2/4 rats. No monothiol derivative was detected in any urine sample. The dithiol derivative was detected only in one 24-48 hour sample. The trithiol derivative was detected in two samples of 0-24 hour urine and two of 24-48 hour urine representing urine from three rats. The rat which excreted trithiols in both periods was also the rat which excreted the dithiol derivative. The highest concentration of thiols detected accounted for 0.053% of the administered dose (Magee et al., 1990). Dogs Two male beagle dogs were given 14C-chlorothalonil by gelatin capsule at an average dose of 49.9 mg/kg bw. Urine was collected for 24 hour periods for 10 days and faeces for 24 hour periods for 12 days. Recovery of test material during the study was virtually complete (mean 100.1% of the administered dose). Most of the material was excreted in the faeces (mean 99.6% of the administered dose) with only small amounts in urine and cage washings (0.2% and 0.3%, respectively). However, white particles were observed in the 0-24 hour faeces which may have been unabsorbed material. Urine samples were analyzed for mono-, di-, and trithiols but none were detected at 3 ng, the limit of sensitivity of the method (Savides et al., 1989a). In order to confirm the results of the above study, a second study in dogs was carried out. Three male beagle dogs were given 14C-chlorothalonil suspended in 0.75% aqueous methylcellulose by gavage at a nominal dose level of 50 mg/kg bw (estimate of actual dose level based on the recovery data: 28-63 mg/kg bw). The dog which received the highest amount (63 mg/kg) regurgitated about 16% of the dose one hour and fifteen minutes after dosing. Two of these dogs were the same ones dosed in the above study which had received control diet for approximately 5 months between the studies. For collection of urine the dogs were catheterized for 4, 10 and 24 hours, respectively. Subsequent urine samples were collected from the time of catheter removal to 24 hours and then at 24 hour intervals until 8 days after dosing. Faeces were collected every 24 hours. Faecal radioactivity in the first 24 hours after dosing accounted for 53.9-96.4% of the nominal administered dose or 76.2-98.1% of the estimated actual dose. A further amount of 0.53-8.6% of the nominal dose (0.96-6.8% of estimated actual dose) was recovered in faeces on days 2-3. Urinary excretion accounted for 0.66-1.17% of the nominal dose (0.73-1.86% of the estimated actual dose) and was predominantly recovered in the first 24 hours. Results from catheterization indicated that most of the excretion occurred during the first 10 hours after dosing. Aliquots of urine from two of the dogs were analyzed to generate chromatographic profiles. For both dogs the profiles of 4 to 10 hour samples were sufficiently similar to permit pooling. The 12 and 14 hour samples from one of the dogs were pooled and the sample collected from 10-24 hours in the other dog was also analyzed. No methylated thiols were detected in any of the samples. Less than half of the radiolabel in urine was extractable in acidified ethyl acetate suggesting that the metabolites were more polar than those observed in the rat (Savides et al., 1990a). Monkeys Four male Chinese rhesus monkeys (Macaca mulatta) were given uniformly benzene ring labelled 14C-chlorothalonil, suspended in 0.75% aqueous methylcellulose, orally by gavage at a dose of 50 mg/kg bw. An indwelling catheter was placed in the saphenous vein of the lower leg to permit periodic blood sample collection. Urine was collected using an external catheter device for 48 hours after dosing and then the catheters were removed and the monkeys were housed in metabolism cages for 96 hours after dosing and urine and faeces were collected at 72 and 96 hours. Blood concentration vs. time plots indicated considerable variability between animals. Peak blood levels were seen 3-18 hours after dosing. In two of the monkeys blood levels rose rapidly; in one of these the blood level declined rapidly while in the other blood levels declined rather slowly. The other two animals took longer to attain peak blood levels and the blood levels declined at an intermediate rate. In these latter two animals higher peak blood levels were reached than in the two which reached the peak rapidly. Blood elimination half life was 6.9, 7.6, 19.8 and 35.0 hours in the four animals. Area under the blood concentration curve was 52,000-158,000 ng-eq hr/ml for the 0-30 sampling interval which was compared to 94,000 ng-eq hr/ml following the same dose in the rat. Over 96 hours a total of 1.75-4.13% of the administered dose was excreted in urine and 52.3-91.6% in faeces. In 3/4 monkeys, urine excreted in the period 24-48 hours contained the highest amount of test material. The other animal excreted most of the radioactivity 12-48 hours after dosing. The same 3/4 monkeys had peak faecal excretion also in the 48 hour sample. The other monkey had high excretion in the first 12 hours and virtually complete faecal excretion by 48 hours after dosing. Extraction of urine with acidified ethyl acetate recovered 32- 65% (mean 49%) of the radiolabel in the urine. Under similar conditions, about 75% of the radiolabel was extractable in rat urine. This suggests that in monkeys the radiolabel is excreted as more polar metabolites than in the rat. Monothiols were not detected in any of the urine samples. Dithiol was detected in urine of only one of the monkeys while trithiols were detected in all urine samples. The total amount of thiols excreted by monkeys was 0.001 to 0.01% of the administered dose. This was compared to the excretion in the rat in which 1.63% of the administered dose was excreted in the form of di- and trithiols (Savides et al., 1990b). Dermal administration Rats Four groups of 5 male CD Sprague-Dawley (Charles River) rats were treated dermally with 14C-chlorothalonil in acetone at a dose of about 5 mg/kg bw. Urinary excretion in the first 48 hours after dosing accounted for about 3% of the administered dose (range 2.52-4.0%) with approximately equal amounts excreted after 0-24 and 24-48 hours. There was considerable variability between the four groups with respect to thiol excretion in urine. Total thiols represented 0.07, 0.01, 0.007 and 0.001% of the administered dose in the four groups. Monothiol was detected in the 0-24 hour sample of one group only. Dithiols were detected in urine from two of the four groups. Trithiol was detected in all urine samples. These data were compared to the results of oral studies. Urinary excretion was greater following oral than following dermal treatment (8% ± 3%) and there was at least a 20 fold difference between the amount of the administered dose excreted as thiols (Savides et al., 1989b). Special in vitro study on metabolism Liver and kidney mitochondrial preparations were prepared from male CD Sprague-Dawley (Charles River) rats. The preparations were incubated with one of the following sulfur analogs of chlorothalonil: the monothiol analog, the dithiol analog, the monoglutathione analog and the diglutathione analog. With the dithiol analog no increase in oxygen consumption was observed following the addition of adenosine diphosphate (ADP) to either liver or kidney mitochondrial preparations indicating complete inhibition of state 3 respiration. The monothiol analog also affected oxygen uptake by liver mitochondria but had no effect on kidney mitochondria. Neither of the glutathione analogs had an effect on oxygen uptake (Savides et al., 1988). Toxicological studies Short-term studies Dogs Renal pathology data from a two year dog study, evaluated by the JMPR in 1974, were re-examined. In this study 8 males and 8 females were given diets containing 0, 60 or 120 ppm of chlorothalonil. Four dogs/sex/group were sacrificed at one year and the remaining four dogs/sex/group at two years. Renal tubule vacuolation was observed in all of the females except one high dose dog at two years. In males the lesion was observed in 0/4, 0/4 and 3/4 dogs at one year and 2/4, 0/4 and 1/4 dogs at two years at 0, 60 and 120 ppm, respectively. A second pathologist examining these tissues concluded that the observed lesion was probably an artifact of fixation. The NOAEL of 120 ppm, originally established in 1974, was confirmed by this re-examination of the data. Long-term/Oncogenicity studies Rats Groups of 55 weanling Charles River Fischer 344 rats/sex/dose level were given diets containing chlorothalonil at dose levels to provide nominal intakes of 2, 4, 15 or 175 mg/kg bw/day for 99-125 weeks. Additionally, groups of 10 rats/sex/dose were given the same diets for one year to serve as interim sacrifice animals. The target lower dose levels for the study were 1.5 and 3 mg/kg bw/day but the possibility of reduced bioavailability in the diet due to binding dictated the use of the higher (2.0 and 4.0 mg/day) levels. The diet was presented twice a week and was frozen prior to use to minimize binding and increase bioavailability. Achieved concentrations provided dose levels of at least 1.5, 3.3, 14.7 and 173 mg/kg bw/day. At the 175 mg/kg bw/day dose level discolored urine was observed during the first year of the study, which occurred at a lower incidence during the second year. Labored breathing was observed in a few animals in each group, which was increased at the end of the study in the high dose group. Survival was reduced in males at 175 mg/kg bw/day and this group was sacrificed at 99 weeks. The other groups of males were sacrificed at 111 weeks at which time survival in the 15 mg/kg bw/day group was lower than in controls. All groups of females were sacrificed at 125 weeks. The females at 175 mg/kg bw/day had lower survival than controls at termination. Body weights were lower than the controls in both sexes at 175 mg/kg bw/day and in males at 15 mg/kg bw/day. A slight but consistent depression in body weight was also seen at the nominal 4 mg/kg bw/day level but the difference was not considered to be biologically significant. Food consumption was reduced in both sexes at 175 mg/kg bw/day during week 1 and increased on a "g/kg bw" basis for the remainder of the study. There were no treatment-related effects on haematological parameters or ophthalmology. At 175 mg/kg bw/day both sexes showed a number of changes in clinical chemistry: increased phosphorus and cholesterol levels and reduced alkaline phosphatase and alanine amino transferase levels. Males at 175 and 15 mg/kg bw/day had increased BUN levels. Males at 175 mg/kg bw/day showed increased urinary volume and reduced specific gravity at 18 and 23 months. Absolute kidney weights were increased in males and females at 175 mg/kg bw/day at 12 months and to a lesser extent in females given nominal levels of 4 or 15 mg/kg bw/day. At terminal sacrifice only the females at 175 mg/kg bw/day were affected. Chronic progressive nephropathy was observed in all groups but was more severe at 175 mg/kg bw/day and to a lesser extent at 15 mg/kg bw/day than in the low dose groups. Focal epithelial hyperplasia in kidney was observed in all groups but was significantly increased in incidence at 175 mg/kg bw/day and slightly increased in incidence in females at 4 and 15 mg/kg bw/day. Epithelial hyperplasia and hyperkeratosis of the non-glandular stomach were seen at doses of 4 mg/kg bw/day and higher and were dose-related in incidence and severity. In the kidney, tubular carcinomas were observed in both sexes at 175 mg/kg/ bw/day (7/55 males and 11/55 females) but in no other group. Tubular adenomas were seen in 1/55, 1/54, 1/54, 3/54 and 17/55 males at 0, 2, 4, 15 and 175 mg/kg bw/day, respectively, and in 24/55 females at 175 mg/kg bw/day. In the stomach, non-glandular papillomas were seen in 0, 0, 3, 2 and 5 males and 1, 1, 2, 4 and 7 females at 0, 2, 4, 15 and 175 mg/kg bw/day, respectively. No squamous cell carcinomas were observed in males but one was seen in each the control and 15 mg/kg bw/day groups and 3 in the 175 mg/kg bw/day females. The NOEL for non-neoplastic effects in this study was 1.5 mg/kg bw/day. There was a possible slight increase in incidence of kidney and stomach neoplasms at 15 mg/kg bw/day but no tumorigenicity was observed at the nominal 2 and 4 mg/kg bw/day levels (actual 1.5 and 3.3 mg/kg bw/day) (Wilson and Killeen, 1989). COMMENTS A study in germ-free rats was conducted to test the hypothesis that at least part of the metabolism of chlorothalonil in the rat is carried out by the microflora in the gastrointestinal tract. In germ- free rats, urinary excretion of radioactivity accounted for about 3% of the administered dose, about half the amount observed in urine of non-germ-free rats dosed at the same level. Thiols in urine accounted for about 0.05% of the administered dose in germ-free rats compared to 1.6% of the administered dose in non-germ-free rats. These data support the theory that the microflora of the gastrointestinal tract are involved in the metabolism of chlorothalonil in the rat. Studies in dogs and monkeys also indicated lower urinary excretion of radioactivity than with non-germ-free rats: about 1% of the administered dose in the dog and 1.8-4.1% of the administered dose in the monkey. No thiols were detected in dog urine and only small amounts in monkey urine (0.001-0.01% of the administered dose). An in vitro study with rat liver and kidney mitochondria demonstrated a toxic effect on mitochondria from both tissues with the dithiol metabolites of chlorothalonil, but no effects with glutathione conjugates. In an oncogenicity study in rats, kidney and stomach tumours were observed at 175 mg/kg bw/day. There were slight increases in kidney tubule adenomas and nonglandular stomach papillomas at 15 mg/kg bw/day but there was no evidence of tumorigenicity at lower dose levels. Focal epithelial hyperplasia in kidney and epithelial hyperplasia and hyperkeratosis of the nonglandular stomach were observed in a dose- related incidence. The NOAEL for non-neoplastic lesions in the kidney and stomach was 1.5 mg/kg bw/day. The NOAEL for neoplastic lesions was 3.3 mg/kg bw/day. The metabolism of chlorothalonil in the rat differs from that in the monkey and dog. This difference is related to a considerable degree to the gut flora in the rat. This fact suggests that the dog or the monkey may be more suitable models than the rat for predicting the metabolism of chlorothalonil by man. The existing data on reproduction were considered to be unsatisfactory. However, a new rat reproduction study is presently underway. A two-year study in dogs was evaluated in 1974. No treatment- related effects on the kidney were observed at 24 months in four males and four females given 120 ppm chlorothalonil in the diet. Since the present rat oncogenicity study demonstrates a NOAEL and the metabolism data assist in elucidating the mechanism, concern regarding carcinogenicity has been alleviated. Since the dog is now considered to be a more relevant model than the rat, the dog study has been selected as the most suitable basis for estimating an ADI. Therefore, a safety factor 100 was applied to the NOAEL in the study (3.0 mg/kg bw/day) to estimate of the ADI. TOXICOLOGICAL EVALUATION Level causing no toxicological effect Mouse: 15 ppm in the diet, equal to 1.6 mg/kg bw/day Rat: 1.5 mg/kg bw/day Dog: 120 ppm in the diet, equivalent to 3.0 mg/kg bw/day Estimate of acceptable daily intake for humans 0-0.03 mg/kg bw Studies which will provide information valuable to the continued evaluation of the compound - Reproduction study in rats known to be in progress - Observations in humans REFERENCES Magee, T.A., Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1990). Study to evaluate the metabolic pathway of chlorothalonil (14C-ASC-2787) in germ-free rats. Unpublished report no. 3060-88-0219-AM-001 from Ricerca Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA. Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1988). A study to evaluate the effects of sulfur-containing analogs of chlorothalonil on mitochondrial function. Unpublished report no. 1479-87-0037-AM-001 from Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA. Savides, M.C., Marciniszyn, J.P.,and Killeen, J.C.,Jr., (1989a). Study to compare the metabolism of chlorothalonil in dogs with its metabolism in rats following oral administration of 14C-chlorothalonil. Unpublished report no. 1626-88-0008-AM-001 from Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA. Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1989b). Study to determine the metabolic pathway for chlorothalonil following dermal application to rats. Unpublished report no. 1625-87-0057-AM-001 from Ricerca Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA. Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1990a). Study of the urinary excretion of radiolabel by catheterized dogs following oral administration of 14C-chlorothalonil by gavage. Unpublished report no. 3086-89-0041-AM-001 from Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA. Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr. (1990b). Study to evaluate the urinary metabolites of chlorothalonil from male rhesus monkeys. Unpublished report no. 3349-89-0179-AM-001 from Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA. Wilson, N.H., and Killeen, J.C., Jr. (1989). A tumorigenicity study of technical chlorothalonil in rats. Unpublished report no. 1102-84-0103-TX-007 from Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA.
See Also: Toxicological Abbreviations Chlorothalonil (EHC 183, 1996) Chlorothalonil (HSG 98, 1995) Chlorothalonil (ICSC) Chlorothalonil (WHO Pesticide Residues Series 4) Chlorothalonil (Pesticide residues in food: 1977 evaluations) Chlorothalonil (Pesticide residues in food: 1981 evaluations) Chlorothalonil (Pesticide residues in food: 1983 evaluations) Chlorothalonil (Pesticide residues in food: 1985 evaluations Part II Toxicology) Chlorothalonil (Pesticide residues in food: 1987 evaluations Part II Toxicology) Chlorothalonil (Pesticide residues in food: 1992 evaluations Part II Toxicology) Chlorothalonil (IARC Summary & Evaluation, Volume 30, 1983) Chlorothalonil (IARC Summary & Evaluation, Volume 73, 1999)