CHLOROTHALONIL EXPLANATION Chlorothalonil was evaluated by the Joint Meetings of 1974, 1977, 1979, 1981 and 1983 (Annex 1, FAO/WHO, 1975a, 1978a, 1980a, 1982a, and 1984). A toxicological monograph was prepared by the Joint Meeting in 1974 (Annex 1, FAO/WHO, 1975b) and monograph addenda were prepared in 1977, 1979, 1981, and 1983 (Annex 1, FAO/WHO, 1978b, 1980b, 1982b, and 1985a). A corrigendum to the 1983 monograph addendum was published in 1985 (Annex 1, FAO/WHO, 1985c). The 1981 JMPR reduced the temporary acceptable daily intake (TADI) from 0.03 to 0.005 mg/kg b.w., which was endorsed by the 1983 Meeting because of insufficient metabolism data and inadequate data on the carcinogenic potential of chlorothalonil. These data, plus additional mutagenicity data, have been submitted and are reviewed in this monograph addendum. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA Biochemical aspects Absorption, distribution and excretion The absorption of 14C-chlorothalonil (purity 99.7%) through the skin was assessed in male Sprague-Dawley rats. A dose of 5 mg/kg was applied (46.7 µ/cm›) to the clipped back (25 cm›) of each rat. Twenty- seven animals were treated and groups of three rats were subsequently killed at 2, 4, 8, 12, 24, 48, 72, 96, and 120 hours after application. The treated skin, blood, kidneys, liver, intestinal contents, remaining carcass, urine, faeces and cage washes were analyzed for radioactivity. The rate of absorption from the skin was relatively constant (6.3% of the applied dose per day) from 24 to 120 hours after application. Animals exposed for 120 hours had absorbed 27.7% of the dose and excreted 18% of the dose in the faeces, 6% in the urine, with 20% lost at the time of application due to evaporation. Approximately 4% of the dose remained in the carcasses of animals exposed for 120 hours. Mean concentration of radioactivity in blood, liver and kidney appeared to plateau after 72 hours. Excretion of radioactivity in faeces appeared to be related to the blood concentrations, but urinary excretion appeared to be independent of blood concentrations. The urinary excretion pattern, attaining constancy of 1.2% of the applied dose per day, suggested that the renal excretory mechanism for chlorothalonil and/or its metabolites becomes saturated and is an active, rather than passive, form of excretion. Residues that remain on the skin surface, nonetheless, constituted the bulk of activity. Data suggest that the rate of absorption of chlorothalonil was constant and that the amount of the dose absorbed was dependent upon the exposure time (Marciniszyn et al., 1984a). Biliary excretion of ring labeled 14C-chlorothalonil (purity 99.7%) was examined in Sprague-Dawley rats orally gavaged with 5 mg/kg. Animals (8 males, 4 females) were fasted, except for water, 16 hours prior to bile duct cannulation. Fifty percent of the males and females had sodium taurocholate (a choleretic substance) infused at a rate of 25 mg/hour. Animals were restrained and bile samples collected at hourly intervals from 0 to 48 hours after dosing. Blood was sampled at 6 and 24 hours and at termination. Urine and faecal samples were also collected periodically. Levels of radioactivity were determined in each bile, blood, urine and faecal sample and in the G.I. tracts, carcasses and cage washings. Approximately 91% of the administered radioactivity was recovered. The presence of activity in the blood, urine and bile demonstrate that absorption via the gut occurs. The data indicate that approximately 33% of the administered dose was absorbed, with the non absorbed material (67%) found in the faeces and G.I. tract. Biliary excretion accounted for 17-21% of the administered dose, with maximum concentrations eliminated within 2 hours of dosing. Urinary excretion, of about 8-12% of the labeled dose, shows this to be a significant route of elimination, but not a major one. No appreciable tissue binding was demonstrated as evidenced by low residual carcass levels, approximately 2% of the administered dose. Absorption via blood was also minimal with the maximum concentration less than 0.4% of the labeled dose (Marciniszyn et al., 1985a). The fate of orally administered 14C-chlorothalonil (purity 99.7%) at three dose levels (5, 50 and 200 mg/kg) was investigated in Sprague-Dawley rats to determine the effects of increasing doses of the test material. Four animals per sex per dose were killed 2, 9, 24, 96 and 168 hours after dosing and urine, faeces, and selected tissues assayed for radioactivity. The average recovery of the radiolabel at each of the dose levels was approximately 89% for males and 96% for females. The major route of elimination was via the faeces (83-87%) and was essentially complete by 48 hours in low-dose females and low/mid-dose males, and by 72 hours in the mid/high-dose females and high-dose males. A delay in stomach emptying time was observed for mid- and high-dose males and females. Urinary excretion was 92-93% complete for low-dose rats within 24 hours, 48 hours at mid-dose and 95% complete for high-dose rats within 72 hours. Urinary excretion of the radiolabel at the three dose levels was 5-7% of the administered dose in males, and 5-11.5% in females. Urinary excretion was essentially saturated as the dose level was increased. The highest concentrations of radiolabelled material in non-G.I. tissues were found in the kidney, being approximately 0.7% of the dose per gram of kidney for males and 0.4% in females at peak concentration (2 hrs) for the 5 mg/kg dose level. Kidney concentrations were greatest at 2, 9 and 24 hours for low, mid and high dose, respectively (Marciniszyn et al., 1984b and 1985b). Male Sprague-Dawley rats were administered, via oral gavage, 14C-chlorothalonil (purity 99.7%) at a dose level of 200 mg/kg in order to isolate and identify the urinary metabolites. Urine was collected at 17, 24 and 48 hours after dosing. Urinary metabolites accounted for 2.4% of the administered dose and, except for 30% of the radiolabel which was nonextractable from the urine, were determined to be trimethylthiomonochloroisophthalonitrile and dimethylthiodichloroisophthalonitrile. This suggests the formation of glutathione (GSH) conjugates. These thiols were excreted in urine both as free thiols and as their methylated derivatives. The authors suggest a metabolic pathway such that hepatic metabolism proceeds through conjugation with GSH followed by enzymatic degradation. The smaller conjugates are then transported via the bloodstream to the kidney where they are converted to thiol metabolites and excreted in the urine (Marciniszyn et al., 1985c). In order to determine if chlorothalonil would react in vitro with GSH, chlorothalonil was incubated with GSH prior to isolation of biliary metabolites. In vitro studies have indicated that GSH forms mono, di, tri, and possibly tetra conjugates with chlorothalonil. On the other hand, data available on the isolation and identification of metabolites in bile of rats dosed with 14C-chlorothalonil suggest that GSH conjugates of chlorothalonil may be formed in the liver and eventually excreted in urine. Data thus far suggest that the major metabolite in bile is the di-GSH conjugate of chlorothalonil, while mono-GSH was not detected (Savides et al., 1985a). Data from a multiple dose study at 1.5, 5, 50 or 160 mg/kg, each administered five times at 24 hour intervals to male Sprague-Dawley rats, indicate that there were shifts in the times to peak blood concentrations with increasing single and multiple doses of chlorothalonil for both sexes. Significant depletion (> 50%) of the radiolabel from blood occurred by 24 hours post-dose for both sexes at dose levels less than or equal to 50 mg/kg. At 160 mg/kg, an apparent plateau in radiolabel concentration in blood was reached after a single dose, suggesting saturation of blood between 50 and 160 mg/kg. The concentrations of radiolabel in kidneys after single dose administration showed no apparent sex-related differences, but the times to peak kidney concentrations did appear to increase with increased dose level for both sexes. With multiple doses, the maximum kidney concentration was found 2 hours after the fifth dose at all dose levels. As with blood levels, peak kidney concentrations may have reached a plateau by the final 160 mg/kg dose. The maximum kidney concentration after five doses was proportional to the total administered dose at 1.5 mg/kg (3.12 µg equiv/g) and 5 mg/kg (8.03 µg equiv/g); at 50 mg/kg (31.5 µg equiv/g) and 160 mg/kg (105 µg equiv/g), kidney concentrations were proportional with one another, but kidney concentrations were not proportional between the two lower and two higher doses. In this multiple dose study, kidney concentrations at 1.5, 5 and 160 mg/kg decreased 50% by 24 hours, but decreased only 20% by 27 hours at 50 mg/kg. By 7 days after the fifth dose, kidneys contain 14, 16, 23 and 25% of their maximum concentrations at 1.5, 5, 50 and 160 mg/kg, respectively. The authors suggest that these data demonstrate apparent saturation of blood, plateau of radiolabel in kidneys, and a trend toward slower depletion (or greater retention) of radiolabel from kidney caused by increased and/or repeated doses of chlorothalonil. The authors further suggest that shifts in metabolism occur between doses of 5 and 50 mg/kg/day for some parameters and between 50 and 160 mg/kg/day in other parameters. (Savides et al., 1985b). The effect of a single administration of chlorothalonil (purity 97.8%) on liver and kidney GSH concentrations was assessed in male Sprague-Dawley rats, administered 5 mg/kg chlorothalonil i.p. or 5000 mg/kg via oral gavage. Concentrations of GSH in liver and kidney determined 2 hours after i.p., or 24 hours after oral gavage, demonstrated no differences between control and i.p. groups regarding GSH levels. However, chlorothalonil administered orally caused lower hepatic GSH and higher renal GSH concentrations. The authors suggest that this supports the proposed metabolic pathway, which includes a GSH conjugate formed in the liver which is subsequently metabolized in the kidney to a sulfur-containing, potentially nephrotoxic, compound (Sadler et al., 1985a) Groups of Sprague-Dawley male rats (5 per dose) were administered 5000 mg/kg chlorothalonil (purity 97.8%) via oral gavage to measure the time course of the acute effect of a single dose on body weight, liver and kidney weights and liver and kidney GSH concentrations. Rats were sacrificed at 1, 3, 9, 18, 24 or 48 hours post-dosing. The data demonstrated significantly-increased relative liver and kidney weights, reduced hepatic GSH concentration up to 24 hours post-dosing, and significantly-increased renal GSH concentration up to 48 hours after treatment. The authors suggest that the hepatic GSH changes were related to its conjugation with chlorothalonil, but that the results were inconclusive regarding the renal GSH changes (Sadler et al., 1985b). Toxicological studies Special Study on Carcinogenicity Rat Groups of Fischer 344 rats (60 males and 60 females per group) were administered chlorothalonil (98.1% pure) in the diet at dosage levels of 0, 800, 1600 or 3500 ppm (equal to 0, 40, 80 and 175 mg/kg b.w./day) for 116 weeks (males) or 129 weeks (females). Rats were examined daily for gross signs of toxicity, including mortality. Body weight and food consumption were recorded periodically throughout the study. Haematology, clinical chemistry and urinalysis parameters were examined routinely througout the study and at termination in 10 rats per sex per dose. All animals were necropsied, selected organs weighed and a complete list of tissues/organs examined microscopically (Wilson et al., 1985c). Survival was comparable in all groups, both sexes, for the first 24 months. Continuation on study decreased survival in high dose males resulting in all males sacrificed at 27 months. Females were terminated on schedule at 30 months. The major cageside clinical observation included dark yellow urine in high-dose males and females from weeks 27-91. An increased brown/red staining around the anogenital region of mid- and high-dose females was also observed. There was a significant body-weight decrease (10-29%) in high-dose males and females throughout the study, as well as a 5-12% body-weight decrease in both sexes at the mid dose. There was no body-weight reduction in low-dose animals. Food consumption was unaffected, except for an increase in high-dose animals, generally towards the last half of the study. Monuclear cell leukemia is a common finding (approx. 20%) in Fischer 344 rats at an average age of 2 years (so-called "Fischer rat leukemia"). In this particular study there was an inverse relationship with dose in that this finding was most pronounced in controls. This was supported by numerous haematological, clinical chemistry and micropathological findings. These effects were most noticeable in control males. They included: decreased red blood cells, haemoglobin, haematocrit, and platelet counts, with increased mean corpuscular volume, mean corpuscular haemoglobin, reticulocytes, nucleated red blood cells and segmented neutrophils. These changes were accompanied by enlarged spleen at 0 and 40 mg/kg, and are suggestive of a macrocytic normochromic regenerative anaemia. Also, in control males, there were increases in total bilirubin, aspartate amino transferase, alanine amino transferase and alkaline phosphatase levels, findings which are common in Fischer rats in later stages of this disease. Parameters measured which were compound related and associated with the effects on the kidneys included increased blood urea nitrogen and serum creatinine in high-dose males and females, decreased serum albumin and serum glucose in high-dose males and females, increased urine volume and decreased specific gravity in all treated males throughout the study, and in all treated females initially (first year), but in high-dose females only, after the first year. The relative kidney weights were significantly increased in all treated males and in mid-dose and high-dose females only. Relative liver weight was affected in the same groups, being significantly increased in all dosed males, and mid- and high-dose females. Gross necropsy of all animals demonstrated a compound-related effect on the kidneys and stomach. In all dosed male groups and the high-dose female group there were kidney masses and/or nodules as well as increased granularity of the surface of the kidneys (the latter observed in all dose groups). There were increased incidences of erosions and ulcerations in the non-glandular stomach of all dosed rats as well as a significant incrase in discoloration of the mucosa in high-dose males. Histologically there was evidence of compound-related effects on the kidneys, oesophagus, stomach and duodenum. Non-neoplastic changes in the kidney included: chronic glomerulonephritis which increased in severity in a dose-related manner in all groups; dose-related increase in cortical tubular hyperlasia in dosed rats; increased incidence of tubular cysts in dosed rats; and increased incidence in dosed males only of hyperplasia of the papillary/pelvic epithelium. Other changes included increased hyperplasia/hyperkeratosis of the squamous mucosa of the oesophagus (all dose groups); increased mucosal hypertrophy of the duodenum (all dose groups); hyperplasia of the parathyroid (all dosed-male and high-dose female groups, considered a secondary lesion as a result of severe chronic renal disease); increased hyperplasia/ hyperkeratosis of the squamous mucosa of the stomach in all dose groups; increased incidences of foci of necrosis or ulcers in the glandular stomach (all dose groups) increased incidence of suppurative prostatitis in all male dose-groups (considered associated with treatment-related renal lesions); complete involution of the thymus was increased in high-dose males and all female dose-groups. Interesting inverse dose-related changes included: chronic interstitial prostatitis (lower incidences in mid- and high-dose male groups); medullary tumours of the adrenal (lower incidence in mid- and high-dose female groups); osteoschlerosis of the femur and sternum (lower incidence in female dose-groups); and basophilic cell focus/foci of the liver (lower incidence in dosed females; this change is a common finding in aging Fischer 344 rats). There were also inverse dose-related changes for pituitary adenomas and fibromas of the skin. Neoplastic changes associated with treatment were observed in kidneys and stomach (forestomach). Tubular adenomas and carcinomas, anaplastic renal carcinomas, and transitional cell carcinomas were originally described in the kidney of treated rats only, being statistically significant in all dosed rats except low-dose females (Table 1). There was also a possible decrease in time to tumours in high-dose rats for renal adenomas and carcinomas. Re-examination of the renal tissues was performed by an independent pathologist, who did not identify transitional cell or anaplastic renal carcinomas (Busey, 1985d; Table 2). In the original pathological examination there was poor correlation between the incidence of cortical tubular hyperplasia and the observation of a tubular adenoma or carcinoma (Table 3). However, in the re-evaluation by an independent pathologist there was very good correlation between the incidence of epithelial hyperlasia (in the proximal convoluted tubules) and the observation of tubular adenoma and carcinoma (Tables 4 and 5). The incidence of papillomas and carcinomas of the stomach were dose-related, but statistically significant only in high-dose females (0/60, 1/60, 1/60, 2/60 for males and 0/60, 1/60, 2/60, and 6/60 for females at 0, 40, 80 and 175 mg/kg dose levels, respectively) (Table 6). Although there was no apparent correlation between forestomach tumours and the incidence of hyperplasia or hyperkeratosis, the non- neoplastic changes may have been obscured by the progression to tumour. Nonetheless, there was a dose-related increase in the severity of hyperplasia/hyperkeratosis in the forestomach. Results of this study demonstrate that chlorothalonil produced renal adenomas and carcinomas in Fischer 344 rats (both sexes) at > 40 mg/kg b.w. Secondary to this response was a dose-related increase in papillomas of the stomach (McGee & Brown, 1985). Table 1. Incidencea of renal tumours of epithelial origin, original report Tumour type Control 40 mg/kg/day 80 mg/kg/day 175 mg/kg/day M F M F M F M F Tubular adenoma 0 0 2 2 4 4 11b** 9** Tubular carcinoma 0 0 5 1 2 2 7* 11** Transitional-cell 0 0 0 0 0 0 2b 0 carcinoma Anaplastic renal 0 0 0 0 1 0 0 3 carcinoma Total animals with 0 0 7* 3 7* 6* 19** 23** these tumours a Kidneys from 60 animals of each sex were examined for all groups. b Includes one male with tubular adenoma and transitional cell carcinoma. * Statistically different from control - p< 0.05 (Fisher's exact test). ** Statistically different from control - p< 0.01 (Fisher's exact test). Table 2. Incidence of renal tumours of epithelial origin, independent evaluation. Tumour type Control 40 mg/kg/day 8mg/kg/day 175 mg/kg/day M F M F M F M F Tubular adenoma 0 0 2 3 5 10 7a 15b Tubular carcinoma 0 0 4 1 2 0 14a 12b Total animals with 0 0 7 4d 7 10e 19 24c these rumours a Includes 2 males with combined incidence of tubular adenoma and tubular carcinoma. b Includes 3 females with combined incidence of tubular adenoma and tubular carcinoma. c Includes 1 female with atubular carcinoma, originally diagnosed as invasive lipomatous tumour. d Includes 1 female with a tubular adenoma, originally diagnosed as negative. e Includes 4 females with a tubular adenoma, originally diagnosed as negative. Table 3. Correlation of renal hyperplasia with tubular adenoma and carcinoma, original report (males). Pathological Control 40 mg/kg/day 80 mg/kg/day 175 mg/kg/day finding Glomerulonephritis 39/60 56/60 56/60 60/60 Cortical tubular 0/60 7/60 9/60 22/60 hyperplasia Kidney adenoma 0/60 7/60 7/60 19/60 or carcinoma Number of tumour- 0/0 0/7 0/7 3/19 bearing rats with renal hyperplasia Table 4. Correlation of renal hyperplasia with tubular adenoma and carcinoma, independent evaluation (males). Pathological Control 40 mg/kg/day 80 mg/kg/day 175 mg/kg/day finding Chronic progressive 47/60 52/60 54/60 57/60 nephropathy Focal epithelial 0/60 6/60 20/60 6/60 hyperplasia (prox. conv. tub.) Epithelial hyperplasia 0/60 32/60 30/60 36/60 (prox. conv. tub.) Kidney adenoma or 0/60 7/60 7/60 19/60 carcinoma Number of tumour- 0/0 6/7 7/7 19/19 bearing rats with renal hyperplasia Special Studies on Mutagenicity Three recent chromosomal aberration studies, carried out on technical chlorothalonil, are summarized in Table 7. The rat and mouse bone marrow cytogenetic assays were negative. The Chinese hamster bone marrow cytogenetic assay was positive, but no dose-response relationship was established. A series of in vitro gene mutation assays were conducted in Salmonella typhimurium with and without a metabolic system obtained from rat kidney (Table 8). The compounds tested included technical chlorothalonil, four manufacturing impurities and eight known or potential metabolites. The results did not provide any evidence of a mutagenic potential of any of the tested compounds. Table 5. Correlation of renal hyperplasia with tubular adenoma and carcinomm, independent evaluation (females). Pathological Control 40 mg/kg/day 80 mg/kg/day 175 mg/kg/day finding Chronic 45/60 49/60 47/60 51/60 progressive nephropathy Focal epithelial 6/60 22/60 34/60 42/60 hyperplasia (prox. conv. tub.) Epithelial 5/60 35/60 39/60 48/60 hyperplasia (prox. conv. tub.) Kidney adenoma 0/60 4/60 10/60 24/60 or carcinoma Number of tumour- 0/0 4/4 10/10 21/24 bearing rats with renal hyperplasia Short-term Studies Additional electron and light microscopic evaluations wee conducted on kidneys from the short-term rat and mouse studies reviewed by the 1983 Joint Meeting in order to investigate and further clarify ultrastructural renal changes. Mouse Histopathological re-evaluation of the kidneys in the Shults (1983) study identified microscopic kidney changes in males at 750 ppm. These changes, which consisted of hyperplasia of the epithelium of the proximal convoluted tubules, were minimal to slight (severity), involved only 4/15 males, and were not considered to be clearly treatment-related effects (Busey 1985b & c). Table 6. Incidencea of tumours in the gastric mucosa Site/ Control 40 mg/kg/day 80 mg/kg/day 175 mg/kg/day Tumour type M F M F M F M F Forestomach: Papilloma 0 0 1 1 1 2 2 6* Squamous carcinoma 0 0 0 0 0 0 1 1 Total number of animals with fore-stomach rumours 0 0 1 1 1 2 3 7* Fundle stomach: Mucosal polyp 1 0 0 0 0 0 0 0 Adenocarcinoma 0 0 0 0 0 1 0 0 a Stomachs from 60 animals of each sex were examined. * Statistically different from control - p < 0.05 (Fisher's exact test). Rat EM and light microscopy of renal tissue from the Wilson et al. (1984) study confirmed the absence of a demonstrated microscopic change in female kidneys. In males there was evidence of an increased incidence of hyperplasia of the proximal convoluted tubules at 40 mg/kg b.w. Electron and light microscopy identified a compound- related increased number of irregular intracytoplasmic inclusion bodies in the proximal convoluted tubules of all males, including controls. The number of such inclusions increased with dose at > 1.5 mg/kg b.w. but showed a tendency to reversal at the low dose (105 mg/kg) only following a 13 week recovery period (Wilson et al., 1985a & 1985b). The exact toxicological significance of these inclusion bodies is unknown since there were no associated degenerative renal changes, a spontaneous occurence in controls was also observed, and there was a tendency to reversal after a 13 week recovery period. The NOEL is > 1.5 mg/kg b.w. The previous NOEL was 3 mg/kg b.w. (Busey, 1985a; Colley, 1983). Table 7. Results of mutagenicity assays of chlorothalonil Test system Test substance Dose level/ Results Reference concentration Mouse bone Chlorothalonil 250, 1250, & Negativea Mizens et marrow cytogenetics 2500 mg/kg, al., 1985a assay orally - in vivo Rat bone marrow Chlorothalonil 500, 2500, & Negativeb Mizens et cytogenetics 5000 mg/kg, al., 1985b assay - orally in vivo Chinese hamster Chlorothalonil 500, 2500, & Weak clastogen Mizens et bone marrow 5000 mg/kg positive al., 1985c cytogenetics given as in treated assay - single treatment; groups: 5 × in vivo 50, 125, & 250 50 & 5 × 250 mg/kg given mg/kg. No as 5 daily dose-response treatments relationship. orally a Positive control (MMS) gave expected positive response at 65 mg/kg. b Positive control (MMS) gave expected positive response at 130 mg/kg. Table 8. Results of mutagenicity assays of technical chlorothalonil, manufacturing impurities, and possible metabolites of technical chlorothalonil Test system Test Dose level/ (Ames test) substance concentration Results Reference S. typhimurium Chlorothalonil Non-activation Negative Jones et al., TA98, TA100, 0.16, 0.8, 4.0, 1984 TA1535, TA1537, 8.0, & 16.0 & TA1538 W/S9 µg/plate. and W/O S9* Activation 0.5, 2.5, 12.5, 25, & 50 µg/plate S. typhimurium 2,5,6- 20, 100, 500, Negative Jones et al., TA98, TA100, Trichloro-3- 1000, & 2000 1985j TA1535, TA1537, cyanobenzamide µg/plate & TA1538 W/S9 (for both and W/O S9* activation & non-activation) S. typhimurium 2,4,5,6- Non-activation Negative Jones et al., TA98, TA100, Tetrachloro-3- 6, 30, 150, 1985k TA1535, TA1537, cyanobenzamide 300, & 600 & TA1538 W/S9 µg/plate. and W/O S9* Activation 10, 50, 250, 500, & 1000 µg/plate Table 8. (Con't) Test system Test Dose level/ (Ames test) substance concentration Results Reference S. typhimurium 2,5,6- Non-activation Negative Jones et al., TA98, TA100, Trichloro-4- 20, 100, 400, 1985l TA1535, TA1537, hydroxy-3- 800, & 2000 & TA1538 W/S9 cyanobenzamide µg/plate. and W/O S9* Activation 40, 400, 1000, 3000, & 6000 µg/plate S. typhimurium 2,3,5,6- 20, 100, 500, Negative Jones et al., TA98, TA100, Tetrachlorobenzonitrile 1000, & 2000 1985d TA1535, TA1537, µg/plate & TA1538 W/S9 (for both and W/O S9* activation & non-activation) S. typhimurium 2,4,5,6- 100, 500, Negative Jones et al., TA98, TA100, Tetrachlorobenzamide 2500, 5000 & 1985e TA1535, TA1537, 10,000 µg/plate & TA1538 W/S9 (for both and W/O S9* activation & non-activation) S. typhimurium 2,4,5-Trichloro- 20, 100, 500, Negative Jones et al., TA98, TA100, 3-cyanobenzamide 1000, & 2000 1985f TA1535, TA1537, µg/plate & TA1538 W/S9 (for both and W/O S9* activation) & non-activation) Table 8. (Con't) Test system Test Dose level/ (Ames test) substance concentration Results Reference S. typhimurium 2,5,6-Trichloro Non-activation Negative Jones et al., TA98, TA100, 4-thioisophthalonitrile 250, 400, 630, 1985g TA1535, TA1537, 1000, 1600, & TA1538 W/S9 & 2500 µg/plate. and W/O S9* Activation 400, 630, 1000, 1600, 2000, 2500, 3000, 4000, & 5000 µg/plate S. typhimurium 2,5,6-Trichloro- 100, 500, 2500, Negative Jones et al., TA98, TA100, 3-carboxybenzamide 5000, & 10,000 1985h TA1535, TA1537, µg/plate & TA1538 W/S9 (for both and W/O S9* activation & non-activation) S. typhimurium 2,4,5- 0.5, 2.5, 10, Negative Jones et al., TA98, TA100, Trichloroisophthalonitrile 35, & 70 1985i TA1535, TA1537, µg/plate & TA1538 W/S9 (for both and W/O S9* activation & non-activation) S. typhimurium 2,3,5,6- 4, 20, 100, Negative Jones et al., TA98, TA100, Tetrachloroterphthalonitrile 200, & 400 1985a TA1535, TA1537, µg/plate & TA1538 W/S9 (for both and W/O S9* activation & non-activation) Table 8. (Con't) Test system Test Dose level/ (Ames test) substance concentration Results Reference S. typhimurium Isophthalonitrile 40, 200, 1000, Negative Jones et al., TA98, TA100, 2000, & 4000 1985b TA1535, TA1537, µg/plate & TA1538 W/S9 (for both and W/O S9* activation & non-activation) S. typhimurium Pentachlorobenzonitrile 10, 50, 250, Negative Jones et al., TA98, TA100, 500, & 1000 1985c TA1535, TA1537, µg/plate & TA1538 W/S9 (for both and W/O S9* activation & non-activation) * The S9 fraction was prepared from rat kidney homogenate Histopathological re-evaluation of the Wilson et al., (1981) rat study by two separate pathologists confirmed the finding of epithelial hyperplasia of the proximal convoluted tubule in males at all levels (e.g > 40 mg/kg) and in females at > 175 mg/kg b.w. Cytoplasmic inclusion bodies or "hyaline droplets" were also identified using neutral red stain in both sexes at all doses. The angular material (cytoplasmic inclusions), which represent an analogous finding to the E.M. evaluation, was seen only in males (Trump et al., 1985; Busey 1985a). COMMENTS Chlorothalonil was evaluated by the Joint Meetings in 1974, 1977, 1979, 1981 and 1983 and additional metabolism data and a rat carcinogenicity study were requested. Chlorothalonil has also been evaluated by IARC (1983) and classified as a compound with limited evidence of oncogenic potential to humans. The additional data provided to the 1985 Joint Meeting demonstrated preferential excretion via the bile and faeces, with secondary excretion in the urine. These metabolism data suggest metabolic pathways involving initial hepatic biotransformastions, conjugation with reduced glutathione (GSH) and enzymatic degradation. There is additional metabolism in the kidney, formation of the thiol metabolites, and excretion in the urine. There was a suggestion of the formation of a sulfur-containing, potentially nephrotoxic, compound in the G.I. tract or kidney. At higher doses there was a plateau of radioactivity in kidneys with subsequent slower removal. It was apparent that there is a shift in metabolism which occurs between doses of 50 and 160 mg/kg b.w. suggesting saturation of an active, rather than passive, uptake mechanism in the kidney. Rat and mouse bone-marrow cytogenetic assays were negative. A hamster bone-marrow cytogenetic assay was positive. In 1983 the Meeting expressed concern for the demonstrated nephrotoxicity and potential tumourigenicity which was evident from earlier studies in rats and mice. The mouse oncogenicity study reviewed in 1983 demonstrated compound-related effects on the kidney at > 750 ppm, with a compound-related increased incidence of renal cortical tubular adenomas and carcinomas in males. The rat oncogenicity study reviewed by this Meeting demonstrated compound-related neoplastic changes in kidneys of treated males (> 800 ppm) and females (> 1600 ppm). Renal tubular adenomas and carcinomas were significantly increased in all dosed rats except low-dose females. There was also a decrease in time to tumour formation which was evident at the high-dose level. Exposure of these rats to chlorothalonil in the diet produced a pronounced increase with dose in the occurrence and severity of renal tubular epithelial hyperplasia and chronic glomerulonephritis. In 1974 the Meeting evaluated five long-term studies in which rats were administered chlorothalonil at doses of 4 to 15,000 ppm with no evidence of a tumourigenic effect on the kidney, although there was clear evidence of epithelial degeneration and hyperplasia at doses > 60 ppm. The Meeting was informed of additional ongoing oncogenicity studies in mice and rats, exposed via the diet to chlorothalonil. In consideration of these new studies, which are being conducted at lower doses than previous rat and mouse oncogenicity studies, the recognized change in metabolism at doses approximating 50 mg/kg b.w., the absence of demonstrated mutagenic potential in a complete battery of such assays, the absence of tumourigenic response in several long-term studies reviewed in 1974, and the conflicting evidence from carcinogenicity studies conducted by the National Cancer Institute (USA), the Meeting extended the TADI and required the submission of these ongoing tests when completed. However, because of concern for the demonstrated oncogenicity in rodents the safety factor used in estimating the TADI was increased. TOXICOLOGICAL EVALUATION LEVEL CAUSING NO TOXICOLOGICAL EFFECT Rat: 10 ppm in the diet, equivalent to 0.5 mg/kg b.w. Dog; 120 ppm in the diet, equivalent to 3 mg/kg b.w. ESTIMATE OF TEMPORARY ACCEPTABLE DAILY INTAKE FOR MAN 0-0.0005 mg/kg b.w FURTHER WORK OR INFORMATION REQUIRED (by 1987) 1. Carcinogenicity studies in rats and mice are understood to be in progress. Although the Meeting recognized that these studies are not scheduled for completion until September of 1988 and June of 1987, the Meeting recommended any available data be submitted for evaluation when available. 2. Further metabolism data to identify the change in metabolic pattern with increasing dose as, well as further characterization of the GSH conjugation occurring in the G.I. tract and kidney. REFERENCES Busey, W.M. A subchronic toxicity study of T-117-11 in Rats (SDS (1985a) Biotech Study No. 5TX-81-0213), Revised Pathology Report, Experimental Pathology Laboratories, Inc., SDS Document No. 753-5TX-85-0056-002. Submitted to WHO by SDS Biotech. Busey, W.M. A 90-day feeding study in mice with T-117-11, Revised (1985b) Pathology Report, Experimental Pathology Laboratories, Inc., SDS Document No. 753-5TX-85-0053-002. Submitted to WHO by SDS Biotech. Busey, W.M. A chronic dietary study in mice with T-117-6 (SDS Biotech (1985c) Study No. 5TX-79-0102); Pathology report. Experimental Pathology Laboratories, Inc. Submitted to WHO by SDS Biotech. Busey, W.M. A two-year tumorigenicity study of T-117-11 in rats (SDS (1985d) Biotech Study No. 5TX-80-0234); Pathology report. Experimental Pathology Laboratories, Inc. Submitted to WHO by SDS Biotech. Colley, J., et al. A 13-week subchronic toxicity study of T-117-11 (1983) in rats (followed by a 13-week withdrawal period). Electron and Light Microscopy of Kidneys, Huntingdon Research Centre, SDS Document No. 5TX-81-0213. Submitted to WHO by SDS Biotech. IARC. Monographs on the evaluation of the carcinogenic risk of (1983) chemicals to humans. Vol 30. Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1984) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with technical chlorothalonil, report No. 694-5TX-84-0064-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985a) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,3,5,6- tetrachloroterphthalonitrile, report No. 694-5TX-84- 0090-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985b) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with isophthalonitrile, report No. 694-5TX-84-0094-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985c) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with pentachlorobenzonitrile (PCBN), report No. 694-5TX-84- 0095-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985d) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,3,5,6- tetrachlorobenzonitrile, report No. 694-5TX-84-0091-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985e) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,4,5,6- tetrachlorodibenzamide, report No. 694-5TX-84-0092-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985f) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,4,5-trichloro-3- cyano-benzamide, report No. 694-5TX-84-0093-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985g) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,5,6-trichloro-4- thio-isophthalonitrile, report No. 694-5TX-84-0124-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985h) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,5,6-trichloro-3- carboxybenzamide, report No. 694-5TX-84-0139-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985i) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,4,5- trichloroisophthalonitrile, report No. 694-5TX-84-0086-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr; & Ignatoski, J.A. Salmonella/ (1985j) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,5,6-trichloro-3- cyanobenzamide, report No. 694-5TX-84-0088-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (1985k) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,4,5,6-tetrachloro- 3-cyanobenzamide, report No. 694-5TX-84-0087-002. Submitted to WHO by SDS Biotech (unpublished). Jones, R.E., Killeen, J.C., Jr. & Ignatoski, J.A. Salmonella/ (19851) mammalian-microsome plate incorporation assay (Ames Test) with and without renal activation with 2,5,6-trichloro-4- hydroxy-3-cyanobenzamide, report No. 694-5TX-84-0089-002. Submitted to WHO by SDS Biotech (unpublished). McGee, D.H., & Brown. A tumorigenicity study of T-117-11 in rats (1985) (5TX-80-0234), International Research and Developmental Corporation, SDS Document No. 099-5TX-80-0234-006. Submitted to WHO by SDS Biotech. Marciniszyn, J.P., Savides, M.C., Killeen, J.C., Jr., & (1984a) Ignatoski, J.A. Study of the dermal absorption of 14C-chlorothalonil by male rats, report No. 649-4AM-84- 0010-001. Submitted to WHO by SDS Biotech (unpublished). Marciniszyn, J.P., Killeen, J.C., & Ignatoski, J.A. Study of the (1984b) distribution of radioactivity following oral administration of 14C-chlorothalonil to male Sprague-Dawley rats, report No. 631-4AM-83-0011-002. Submitted to WHO by SDS Biotech (unpublished). Marciniszyn, J.P., Killeen, J.C., & Ignatoski, J.A. Pilot study of the (1985a) biliary excretion of radioactivity following oral administration of 14C-chlorothalonil to Sprague-Dawley rats, report No. 633-4AM-83-0062-002. Submitted to WHO by SDS Biotech (unpublished). Marciniszyn, J.P., Killeen, J.C., & Ignatoski, J.A. Study of the (1985b) distribution of radioactivity following oral administration of 14C-chlorothalonil to female Sprague-Dawley rats, report No. 631-4AM-84-0078-002. Submitted to WHO by SDS Biotech (unpublished). Marciniszyn, J.P., Killeen, J.C., & Ignatoski, J.A. Identification of (1985c) metabolites in urine and blood following oral administration of 14C-chlorothalonil to male rats: the thiolmetabolites in urine, report No. 621-4AM-83-0061-001. Submitted to WHO by SDS Biotech (unpublished). Mizens, M., Killen, J.C., Jr., Claudio, G., & Ignatoski, J.A. In Vivo (1985a) bone marrow chromosomal aberration assay in mice with a single dose of technical chlorothalonil, report No. 625-5TX- 83-0029-002-001. Submitted to WHO by SDS Biotech (unpublished). Mizens, M., Killen, J.C., Jr., Claudio, G., & Ignatoski, J.A. In Vivo (1985b) bone marrow chromosomal aberration assay in rats with a single dose of technical chlorothalonil, report No. 625-5TX- 83-0028-002-001. Submitted to WHO by SDS Biotech (unpublished). Mizens, M., Killen, J.C., Jr., & Ignatoski, J.A. Acute and subchronic (1985c) in vivo bone marrow chromosomal aberration assay in Chinese hamsters with technical chlorothalonil, report No. 625-5TX-83-0014-003. Submitted to WHO by SDS Biotech (unpublished). Sadler, E.M., Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A. (1985a) Acute effect of technical chlorothalonil on hepatic and renal glutathione content in rats, report No. 732-5TX-84- 0006-001. Submitted to WHO by SDS Biotech (unpublished). Sadler, E.M., Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A. Time (1985b) course of the acute effect of technical chlorothalonil on hepatic and renal glutathione content in male rats, report No. 751-5TX-85-0032-001. Submitted to WHO by SDS Biotech (unpublished). Savides, M.C., Marcinsizyn, J.P., Killeen, J.C., Jr., & (1985a) Ignatoski, J.A. Isolation and identification of metabolites in the bile of rats orally administered 14C-chlorothalonil, I. Synthesis and characterization of glutathione conjugates of chlorothalonil, report No. 633-4AM-84-0104-001. Submitted to WHO by SDS Biotech (unpublished). Savides, M.C., Marcinsizyn, J.P., Killeen, J.C., Jr., & (1985b) Ignatoski, J.A. Study of the distribution of radioactivity following repeated oral administration of 14C- chlorothalonil to male Sprague-Dawley rats, interim report No. 631-4AM-84-0079-001. Submitted to WHO by SDS Biotech (unpublished). Shults, S.K. A 90-day feeding study in mice with technical (1983) chlorothalonil. Unpublished report from SDS Biotech Corp. Submitted to WHO by Diamond Shamrock Corp. Trump, B.F., et al. Re-evaluation of the renal tissue from the "90-day (1985) toxicity study of T-117-11 in rats", 5TX-80-0200, Warwick Institute for Science and the Environment, SDS Document No. 753-5TX-85-0055-002. Submitted to WHO by SDS Biotech. Wilson, N.H., et al. A 90-day toxicity study of technical (1981) chlorothalonil in rats. Report submitted to WHO by Diamond Shamrock Corp. (unpublished). Wilson, N.H., Killeen, J.C., Jr., Haley, B.L., & Ignatoski, J.A. (1984) A subchronic toxicity study of technical chlorothalonil in rats, report amendment No. 562-5TX-81-0213-004-001. Submitted to WHO by SDS Biotech (unpublished). Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A. Histopathologic (1985a) re-evaluation of renal tissue from a subchronic toxicity study of technical chlorothalonil in rats (5TX-81-0213). Document No. 753-5TX-85-0056-002. Submitted to WHO by SDS Biotech (unpublished). Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A. Histopathologic (1985b) re-evaluation of renal tissue from a 90-day toxicity study of technical chlorothalonil in rats (5TX-80-0200). Document No. 753-5TX-85-0055-002. Submitted to WHO by SDS Biotech (unpublished). Wilson, N.H., Killeen, J.C., Jr., & Ignatoski, J.A. A tumorigenicity (1985c) study of technical chlorothalonil in rats, document No. 099-5TX-80-0234-008. Submitted to WHO by SDS Biotech (unpublished).
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: 1987 evaluations Part II Toxicology) Chlorothalonil (Pesticide residues in food: 1990 evaluations 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)