PESTICIDE RESIDUES IN FOOD - 1983 Sponsored jointly by FAO and WHO EVALUATIONS 1983 Data and recommendations 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, 5 - 14 December 1983 Food and Agriculture Organization of the United Nations Rome 1985 CHLOROTHALONIL TOXICOLOGY Explanation Chlorothalonil was evaluated by the Joint Meetings of 1974, 1977, 1979 and 1981 (FAO/WHO, 1975, 1978, 1980 and 1982). The 1981 Meeting reduced the temporary acceptable daily intake (ADI) from 0.03 to 0.005 mg/kg b.w. inadequate metabolism data and a low no-effect level (NOEL) in a rat reproduction study with the 4-hydroxy-2, 5, 6-trichloro-isophthalonitrile metabolite. Additional data were required by 1983 in order to clarify the metabolism, nephrotoxicity and mutagenicity of the parent compound and the 4-hydroxy metabolite. These data are reviewed in this monograph addendum. Temporary maximum residue limits (MRLs) were estimated during previous meetings for a variety of commodities. Certain studies on residues were required by 1983 and others considered desirable. Desirable data and information have been provided in response to several questions raised at the 1981 Meeting. One such question concerned the efficiency of tumble extraction for field-incurred residues of chlorothalonil, hexachlorobenzene (HCB) and pentachlorobenzonitrile (PCBN). Another concerned apparent discrepancies between two studies on residues resulting from the cooking of chlorothalonil-treated foods. A revised report has been provided, based on the original data. In response to the suggestion of the 13th Session of the Codex Committee on Pesticide Residues (CCPR) that the recommended limit for clorothalonil on grapes did not represent good agricultural practice (GAP), information on GAP and additional residue data were provided to the 1981 Meeting, but were received too late for evaluation. These data are also reviewed in this addendum, together with new or additional residue data on other commodities, information on national tolerances, and monitoring data on selected commodities from one country. The Meeting re-examined the definition of the residue in response to a request of the 14th Session of the Codex Committee on Pesticide Residues (CCPR). EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOCHEMICAL ASPECTS Absorption, Distribution and Excretion Chlorothalonil (2, 4, 5, 6-tetrachloroisophthalonitrile, DS-2787), labelled in the benzene ring, was orally administered as a water suspension to male Sprague-Dawley rats (five per group) housed in metabolism cages. Urine and faeces from rats administered a single dose of 0, 5 or 200 mg/kg were analysed by high performance liquid chromatography (HPLC) to determine the extent to which chlorothalonil was metabolized to 4-hydroxy- 2 3, 5-trichloroisophthalonitrile (DS-3701) and/or other metabolites, at 2, 9, 24, 36, 48, 60, 72, 84 and 96 hours after dosing. Blood and other tissue samples were collected at necropsy, but not analysed. HPLC analyses of high dose faecal extracts showed that 28 percent of the administered dose was eliminated as DS-2787 and 5 percent as DS-3701. A third peak of radioactivity eluted from the HPLC in the same time frame as the trichloroacid amide (DS-46851) and accounted for 1.4 percent of the administered dose. Analyses of faecal extracts from the low dose level showed that chlorothalonil, DS-3701 and DS-46851 (tentative) accounted for 1.6, 6.2 and 2.6 percent of the administered dose, respectively. HPLC analyses of urine samples showed that, at the 200 mg/kg dose level, chlorothalonil, DS-3701 and DS-46851 represented 0.05, 0.29 and 3.53 percent, respectively, of the administered dose. At the 5 mg/kg dose level, DS-2787, DS-3701 and DS-46851 accounted for 0.08, 0.58 and 4.48 percent, respectively, of the administered dose (Ignatoski et al 1983a). Chlorothalonil, labelled in the benzene ring, was orally administered to male Sprague-Dawley rats at dose levels of 0, 5, 50 and 200 mg/kg b.w. to determine the distribution of radioactivity in rats at 2, 9 and 24 hours after dosing. Approximately 80 percent of the administered dose was found in the contents of the small intestine at 2 hours, 90 percent in the large intestine at 9 hours and 95 percent in the large intestine at 24 hours after dosing. Accumulation of radioactivity in all tissues except the gastro-intestinal tract was less than 1 percent of the dose. The total µg equivalents (expressed as chlorothalonil) found in the gastro-intestinal tract contents were directly proportional to the dose administered. Profiles of the concentration of labelled material in blood and other tissues as a function of time showed that only at the 5 mg/kg dose was the concentration essentially zero 24 hours after dosing. At the 50 and 200 mg/kg doses, blood and tissue concentrations of the labelled material (s) were persistent in significant quantities 24 hours after dosing when compared to the 5 mg/kg dose (Ignatoski et al 1982). 14C-Chlorothalonil was administered orally as a microparticulate suspension in 0.5 percent methylcellulose to male Charles River CD-1 mice (16 per group) at dose levels of 0, 1.5, 15 or 105 mg/kg b.w. Urine, faeces, blood and other selected tissues were assayed for radioactivity. The total recoveries of radioactivity ranged from 93 to 99 percent for the low dose, 78 to 85 percent for the mid dose and 73 to 77 percent for the high dose. The major route of elimination of radiolabelled material was via the faeces. Faecal elimination was complete by 24 hours for the low and mid doses and by 96 hours for the high dose. Delayed faecal elimination at the high dose resulted from large amounts of radioactivity remaining in the stomach contents at 9 and 24 hours. Approximately 20 percent and 9 percent of the high dose was found in the stomach contents at 9 and 24 hours, respectively. There was also a high amount of radio activity in the large intestine (13 percent) at 9 hours. The high dose of chlorothalonil may have had an effect on stomach emptying time, which delayed faecal elimination of radioactivity. Furthermore, the soft consistency of the faeces suggested that there was increased motility or water retention in the lower gastro-intestinal tract but no increased faecal elimination at the high dose. Urinary excretion at all doses generally varied between 5 and 10 percent of the administered dose. The total amount of radioactivity found in nine tissues and blood was less than 3 percent of the administered dose (Ignatoski et al 1983b). 14C-Chlorothalonil was administered intraduodenally to male rats (donor animals) at a dose level of 5 mg/kg b.w. and bile was collected for 24 hours after dosing. The bile collected during the first 6 hours post-dosing was pooled from individual animals and administered intraduodenally to recipient rats. The recipient rats received 0.2 percent to 2.4 percent of the total radioactivity administered to the donor rats. Bile from the recipient animals was also collected for 24 hours after dosing. Data from the donor animals indicated that 1 to 6 percent of the administered radioactivity was excreted in bile within 24 hours after dosing. Approximately 19 percent of the radioactivity administered to recipient rats was excreted within 24 hours after dosing. Characterization of the radioactivity with respect to the parent compound or metabolites was not performed (Ignatoski et al 1983c). 14C-chlorothalonil was administered intraduodenally in maize oil to Sprague-Dawley rats (six per group) at dose levels of 0.5, 5 or 50 mg/kg b.w. or in methyl-cellulose at a dose level of 5 mg/kg b.w. The percent of the administered dose excreted in bile from animals receiving 5 mg/kg b.w. in maize oil (31.2 percent) was essentially identical to those animals receiving 0.5 mg/kg b.w. in maize oil (35.8 percent). At 50 mg/kg b.w. in maize oil, only about 11 percent of the administered dose was excreted in bile. When methylcellulose was the vehicle at 5 mg/kg b.w., about 7 percent of the administered dose was absorbed and excreted in bile within 24 hours, suggesting that absorption was facilitated by maize oil as compared with methylcellulose (Ignatoski et al 1983d). 14C-Chlorothalonil was administered intraduodenally in maize oil to male Sprague-Dawley rats (five per dose) at six dose levels (0.5, 5, 10, 50, 100 and 200 mg per kg). Data suggested that when chlorothalonil is administered intraduodenally to rats, the excretion profile in bile is dependent upon the dose and further that there is possibly a pharmacokinetic overload in response to increasing doses of chlorothalonil (Ignatoski et al 1981). Two male Sprague-Dawley rats were administered orally 5 mg 14C-chlorothalonil/kg b.w. as a microparticulate suspension in 0.5 percent methylcellulose. Blood samples, obtained at periodic intervals by orbital sinus puncture, showed peak blood levels between 4 and 7 hours post-dosing, which rapidly declined thereafter. The total amount of radioactivity in the blood never exceeded one percent (1 percent) of the administered dose, with the majority of the activity in plasma (70 percent) and the remainder in the red blood cells (packed cell volume) (Ignatoski et al 1983e). In a separate study there were dose related changes in the mean blood concentrations (ng equivalents/ml), versus time. The kinetics were non-linear at 200 mg/kg with a shift in the time of peak concentration at 50 and 200 mg/kg caused possibly by affecting stomach emptying and a resultant lag in absorption (Ignatoski et al 1983f). TOXICOLOGICAL STUDIES Short-Term Studies Mouse Groups of CD-1 Charles River mice (15 males and 15 females/group) were administered chlorothalonil (98.9 percent pure, containing 0.03 percent HCB or less) in the diet at dosage levels of 0, 7.5, 15, 50, 275 and 750 ppm for 13 weeks. Five males and five females/group were subjected to interim necropsies after six weeks of compound administration. Routine examinations were performed for gross signs of toxicity, physical examinations, body weight changes, food consumption and selected clinical chemistry parameters. At terminal sacrifice all surviving animals were subjected to complete gross necropsy, brain and kidneys were weighed and a histological evaluation made of stomach and kidneys. There were no compound-related deaths during the study or any other untoward signs of toxicity, mood consumption and body weight gains were comparable among groups. Mean compound consumption ranged between 1.2 and 141.2 mg/kg/day for the respective doses. There were no compound related effects on the clinical chemistry parameters, including blood urea nitrogen (BUN) and creatinine. Alkaline phosphatase levels were increased in high-dose females. There were no significant differences in brain weight; however, absolute and relative kidney weights were increased in females, but not in males, at 275 and 750 ppm. Gross necropsy was unremarkable except for proteinaceous plugs in the lumen of the bladder in males, generally distributed among all groups, with slight increased incidence in high- dose males. Microscopic examination of the stomach revealed an increased finding of hyperplasia and hyperkeratosis of the squamous epithelial cells in both sexes at 50, 275 and 750 ppm. Histologic evaluation of the kidney did not reveal any compound-related changes. Generalized vacuolation of the cortical tubular epithelial cells and hyperplasia of the epithelial cells of the proximal convoluted tubules in high-dose males at the interim sacrifice were not evident at terminal sacrifice. Other changes were sporadic and not considered compound-related. Chlorothalonil was demonstrated to be without adverse effects on mice at levels up to and including 15 ppm (2.5-3.0 mg/kg/day) when incorporated in the diet for 90 days (Shults 1983). Rat Groups of Charles River CD rats (20 sex/dose) were administered chlorothalonil (98 percent pure, with 0.03 percent HCB) in the diet at dosage levels of 0, 40, 80, 175, 375, 750 and 1 500 mg/kg b.w./day for 90 days. Animals were examined routinely for mortality and gross signs of toxicity; body weights and food consumption were measured periodically and haematological, clinical chemistry and urinalysis parameters examined pre-test, at 30 days and at the termination of the study. All surviving animals were necropsied, selected organs weighed and a complete list of tissues/organs examined microscopically. Survival was comparable among all groups. Indication of cathartic action related to compound ingestion was evident in both sexes at 750 and 1 500 mg/kg/day. Evidence of soft stools, reduced faecal output, mucus in stools, swelling and irritation of the anus occurred with greater frequency and severity in the two highest dose groups. There were significant dose-related body weight reductions in both sexes at dose levels of 375 mg/kg/day end greater. Food consumption comparisons indicated compound-related increases throughout the study. There were significant but spurious increases in haemoglobin, haematocrit and erythrocyte counts in males, which are not considered to be compound- related. Mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration were unaffected by treatment. Red blood cell morphology was normal in all groups. There were compound-related decreases in glucose levels at > 375 and > 750 mg/kg in males and females, respectively. There were similar reductions in BUN at > 80 and > 375 for males and females, respectively. All other clinical chemistry determinations were normal except for depressed SGPT activity, present in both sexes in all treatment groups. This effect was generally dose-related. A special evaluation of serum thyroxine (T-4) and triiodothyronine (T-3) demonstrated depressed T-4 levels at > 175 and 1 500 mg/kg in males and females, respectively. This depression correlates with the decreased rate of body weight gain at those levels. There were dose-related increases in specific gravity and decreased urine volume for males given > 375 mg/kg of chlorothalonil. There was also an increased incidence of dark urine as the dose was increased from 375 to 1 500 mg/kg. There were no similar findings in females. Although several absolute and relative organ weight changes were determined, only the kidney weight changes are considered to be compound-related. Relative kidney weights were increased in both sexes at all treatment levels, but gross and histopathological evaluations revealed no correlative compound-related effects. Gross necropsy findings were unremarkable among all groups. The only dose-related histologic effect of treatment, which was inversely related to dose, was a finding of acute gastritis in the non-glandular portion of the stomach in all treatment groups. Based on the relative kidney weight changes at all levels, with compound-related effects on specific gravity and urine volume at > 375 mg/kg, a clear no-adverse-effect level has not been demonstrated. The depressed SGPT activity at all treatment levels in both sexes, considered to be compound-related, is difficult to interpret, particularly since relative liver weights were increased at > 750 mg/kg for both sexes (Wilson et al 1981). Technical grade chlorothalonil (98.2 percent pure) was administered in the diet to groups of Sprague-Dawley rats (25/sex/dose) at dose levels of 0, 1.5, 3, 10 and 40 mg/kg for 13 weeks. Selected animals from each group were continued on a control diet for an additional 13-week recovery period. Serial sacrifices were conducted at 6 weeks (5/sex/group), 13 weeks (10/sex/group) and 26 weeks (all remaining animals). Mortality was monitored daily and physical examinations conducted periodically. Body weight gain and food/water consumption were recorded weekly. Haematological, clinical chemistry and urinalysis parameters were determined throughout the study. Complete necropsies were performed and selected organs weighed; microscopic examination was done on all animals. In addition, EM examination of kidneys was also performed. There was no mortality associated with treatment. Physical examinations were unremarkable as were body weight, food consumption, urinalysis and haematological determinations. The only differences between control and treatment groups regarding clinical chemistry evaluations were reduced alkaline phosphatase and glutamic pyruvic transaminase activities in both sexes at 10 and 40 mg/kg. The differences were more pronounced in males and remained depressed thru week 13, while females tended to recover by week 13. Gross necropsies did not reflect treatment-related effects, except for increased kidney weights in males and females at 3, 10 and 40 mg/kg/day and increased liver weights in males at 40 mg/kg/day. Microscopic examination revealed an increased incidence of epithelial hyperplasia and hyperkeratosis in the non-glandular portion of the stomach in both sexes given 10 and 40 mg chlorothalonil/kg b.w./day. Examination of the kidney revealed marginal increases in the incidence of dilated medullary tubules but no effects on cortical tubules at 10 and 40 mg/kg/day. There were no other adverse effects noted in other organs/tissues. After the 13-week recovery period, these apparent compound-related effects had reversed. Microscopic examinations did not provide evidence for the cause of organ weight changes observed in the liver and kidneys. Chlorothalonil was without adverse effects on the Sprague-Dawley rat at doses up to and including 3 mg/kg/day (Wilson et al 1983a, b). Groups of Sprague-Dawley CD rats (10 males and 10 females/group) were fed 4-hydroxy-2, 5, 6-trichloroisophthalonitrile (99.6 percent pure) in the diet at dosage levels of 0, 10, 20, 40, 75, 125, 150, 500 and 750 mg/kg b.w./day for 61 to 69 days. Complete physical examinations were conducted routinely and body weight and food consumption measured throughout the study. Haematology, clinical chemistry and urinalysis parameters were routinely determined. Gross necropsy, organ weights and histologic examinations were performed at the termination of the study. Mortality was significantly increased in males at > 125 mg/kg and in females at > 75 mg/kg. There were no mortalities in dose groups of 0 to 40 mg/kg. Gross observation included dose-related occurrence of piloerection in all dose groups and pale skin and eyes in males and females at > 20 and > 75 mg/kg respectively. Body weights were depressed by treatment at levels > 40 mg/kg in both sexes. Food consumption was similarly decreased at the same dietary levels. Haematological evaluations presented evidence of anaemia in treated groups. There were significant decreases in the red blood cell count, haematocrit, haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin at > 75 and > 40 mg/kg in males and females, respectively. Reticulocytes were similarly increased in males and females at > 75 and > 40 mg/kg, respectively. Nucleated red blood cells were increased in both males and females at > 40 mg/kg. Clinical chemistry parameters demonstrated similar compound- related effects with increases in alkaline phosphatase, BUN, SGPT and SGOT at > 125 and > 75 mg/kg in males and females, respectively. Both males and females at > 75 mg/kg had reduced total protein, albumin and globulin levels, with an increased albumin globulin (A/G) ratio at the same dosage level. Urinalyses were unremarkable at doses up to and including 75 mg/kg. There were no evaluations conducted at higher doses. Gross necropsies presented occurrences of pale liver, pale/discoloured kidney, gastric ulceration, distended urinary bladder and flaccid heart at 75 mg/kg in both sexes. Since body weight decreased at 40 mg/kg, brain weight was used for comparison of relative organ weights. Accordingly, males and females at 40 mg/kg had reduced heart and kidney-to-brain weights. Females at that level also had reduced ovarian weights. At 75 mg/kg males, had reduced liver, spleen and testes weights, while females had reduced liver weights. Histopathological examination demonstrated compound-related effects in males and females at > 40 mg/kg in the form of erythroid hyperplasia and depressed granulopoiesis in bone marrow and spleen. There were also increased incidences of ovarian follicular degeneration and necrosis and renal cortical atrophy in females (the latter evident in males at > 75 mg/kg). At > 75 mg/kg, males and females had increases of hepatic haemosiderosis, toxic centrilobular hepatitis and myocardial degeneration. In females, there was also evidence of increased focal adrenal cortical necrosis, vacuolative degeneration and renal cortical tubular degeneration. These became evident in males at > 125 mg/kg, along with an increased incidence of testicular degeneration. Females were more sensitive to the toxic effects of the metabolite than were males. There were no adverse effects demonstrated at dose levels up to and including 20 mg/kg (Murchison et al 1979). Special Studies on Reproduction Rat A one-generation reproduction study was performed using Charles River CD rats, wherein 4-hydroxy-2, 5, 6-trichloroisophthalonitrile was administered in the diet. Five groups containing 12 males and 24 females each were given dosage levels of 0, 10, 20, 30, 60 and 120 ppm for 18 weeks before mating and continuously through two successive reproduction cycles. F1a and F1b offspring were necropsied at weaning with tissues preserved from 5 males and 5 females in each group from the Flb pups. These tissues were not examined microscopically. Litters were culled at day 4 to 10 per litter (sexes equal) and the litter weights were determined at days 0, 4, 7, 10, 14 and 21. Indices of reproductive performance were determined. Mortality, body weight and food consumption of F0 parents were unaffected by treatment, with the exception of increased food consumption in high-dose males. The overall mating index in F1a and F1b litters was poor, being 62.5 percent and 54.2 percent, respectively. Gestation index for both control litters was similarly reduced, being 73.3 percent and 69.2 percent, respectively. Overall mating and gestation indices in the dose groups were greater than control values and, therefore, unaffected by treatment. There were no compound-related effects on maternal body weights during gestation and lactation. There were no compound-related effects observed on the mean number of live pups in either F1a or F1b litters from birth through lactation. Live born and stillborn indices were comparable among all groups in both litters. Pup viability in the high dose group at day 4 was significantly lower than controls in the F1a and F1b litters. There were no compound-related effects on the lactation or litter viability indices, although slightly reduced litter viability indices were reported for both litters (86.7 and 84.6 percent, respectively) at the high dose. No statistically significant differences in mean live pup weights between control and treatment groups were reported at birth or day 4, although there was a negative trend in the F1a high dose group at day 4. This was not evident in the F1b litter. There were, however, statistically significant differences for the high dose group in both litters at days 7, 10, 14 and 21 and at 60 ppm on days 10, 14 and 21 in the F1b litter. There were no compound-related gross findings in any of the animals necropsied. The no-effect level in this study was demonstrated at 30 ppm (Ford 1982). Special Studies on Teratogenicity Rat Groups of Sprague-Dawley rats (25 females/groups) were administered chlorothalonil orally, via gavage, doses of 0, 25, 100 and 400 mg/kg/day from day 6 through 15 of gestation. Surviving females were necropsied on day 20 and foetuses delivered by hysterotomy. The number and position of viable/non-viable foetuses, early/later resorptions, mean number of corpora lutea and total number of implantations were recorded. External, internal and skeletal examinations of foetuses were performed for evidence of abnormalities and anomalies. Half of the foetuses were evaluated for soft tissue anomalies and the other half for skeletal effects. There was no dose related mortality in the 25 and 100 mg/kg/day groups. However, the deaths during treatment of three dams in the 400 mg/kg group were considered related to compound ingestion. There were no abortions in any group. General appearance and behaviour were unremarkable, except for evidence of cathartic action at 400 mg/kg (e.g. loose faeces, matting of urogenital fur). Mean maternal body weights were significantly different (less) than control at the high dose level. Food consumption was significantly reduced in all treatment groups initially (days 6 to 9) and in the high-dose group throughout the dosing period (days 6 to 15). There were no differences compared to controls for mean number of viable foetuses, implantation sites, corpora lutea or foetal weights. There was a significant increase in the number of early resorptions in the high-dose group, as well as post-implantation losses, when compared to controls. There were no reported effects on number or percentage of foetuses/litters with external, internal or skeletal malformations or developmental variations at any dose level administered. Chlorothalonil was considered maternally toxic to rats at 400 mg/kg but there was no evidence of teratogenicity at any level tested (Rodwell et al 1983). Rabbit Groups of pregnant Japanese White rabbits (9/group) were administered orally, via gastric intubation, doses of 0, 5 and 50 mg/kg of chlorothalonil from day 6 through 18 of gestation. All does were sacrificed on day 29, pups delivered by caesarean section, and the number of implants, live and dead foetuses recorded. Pups were examined for gross malformation, visceral organs removed and examined, and carcases stained with Alizarin Red S for skeletal evaluation. Body weights were reduced in high-dose females and 4/9 does aborted. Foetal resorptions were increased at 5 and 50 mg/kg compared to controls. The number of implants and number of live foetuses were reduced in the high-dose group when compared to controls. External examination revealed 1/74 foetuses at 5 mg/kg with hydrocephaly and 1/36 foetuses at 50 mg/kg with cleft palate. There were no visceral or skeletal anomalies reported. Chlorothalonil is considered maternally toxic at 5 and 50 mg/kg, but there was no evidence presented of teratogenic effects (Shirasu & Teramoto 1975). In a pilot teratology study, groups of rabbits were orally dosed with 4-hydroxy-2, 5, 6-trichloroisophthalonitrile at 1, 5, 10, 25 and 50 mg/kg/day from day 6 through 18 of gestation. Maternal death and toxicity occurred at doses of 10, 25 and 50 mg/kg with no adverse effects observed at 1 mg/kg. In the 5 mg/kg dose group, one female aborted, one was not pregnant and one female presented a litter of runts. The other females in this group had normal pregnancies and pups (Wazeter & Goldenthal 1976). Groups of Dutch Belted rabbits (10 per/group) were orally intubated with 4-hydroxy-2, 5, 6-trichloroisophthalonitrile at dose levels of 0, 1, 2.5 and 5 mg/kg/day from day 6 through 18 of gestation. Pups were delivered by Caesarean section on day 28 and the number, location and distribution of live/dead foetuses, corpora lutea, implantations and early/late resorptions recorded. All foetuses were examined grossly, sectioned for visceral anomalies and stained for skeletal anomalies. There were three deaths, one at 2.5 and two at 5 mg/kg. There were no other gross signs of toxicity reported and body weight gains were uniform among all groups. There were no compound-related effects on the number of implantation sites, foetal weights, sex ratios or foetal anomalies. The number of females with early resorbed foetuses and number of females aborting was increased compared to controls at the high dose. However, most of these early resorptions in the high- dose group occurred in one female which had 10 implantation sites and 10 resorptions. Otherwise, resorptions were comparable among treatment and control groups. There were no visceral or skeletal anomalies or abnormalities directly attributable to compound ingestion at doses up to and including 5 mg/kg/day. However, this dose did present marginal effects of maternal toxicity, which were not apparent at 2.5 mg/kg (Wazeter & Goldenthal 1976). Special Studies on Carcinogenicity Mouse Groups of CD-1 mice (60 males and 60 females/group) were administered technical chlorothalonil in the diet at dosage levels of 0, 750, 1 500 and 3 000 ppm for 24 months. Mice were six weeks old at the initiation of the study. Animals were observed daily for mortality along, with other cage-side observations for signs of toxicity. Individual body weights, food consumption, physical examinations and haematological parameters were evaluated regularly throughout the study. All animals were necropsied, organ weights determined and selected tissues/organs examined histologically. There were no apparent compound-related effects on body weight gain, food consumption or general toxic response. Mortality was increased in high-dose males (40/60) compared to control males (29/60). Mortality in control females was 42/60. There was abdominal swelling in mid-and high-dose males, which was evident for the first six months, but no differences between control and treatment groups after six months. Distended abdomens were noted generally among all groups. Compound consumption resulted in 0, 119, 251 and 517 mg chlorothalonil/kg b.w./day for males; and 0, 134, 278, and 585 mg chlorothalonil/kg b.w./day for females in the 0, 750, 1 500 and 3 000 ppm dose groups, respectively. Haematology measurements revealed an increased incidence of abnormal erythrocyte morphology, which was unrelated to other normal findings and not considered significant. Decreased haemoglobin, haematocrit and red blood cell values were evident in high-dose males at 24 months and in high-dose females at 18 and 24 months. There was an increase of hyperplastic bone marrow (sternum) for males and females of all treatment groups, in relation to corresponding controls, as well as hyperplasia of the red pulp in the spleen of treated males. The spleens of all female groups were comparable with control females, which had the most significant increase in hyperplastic red pulp. Haemosiderosis was unremarkable or not evident. Examination of organ weights demonstrated an increase in absolute and relative spleen weights for high-dose females but no significant pathological changes. Gross necropsy indicated the spleen was enlarged for mid- and high-dose males. Absolute and relative ovary weights in all females were decreased, with no significant dose-related or histopathologic changes. Absolute and relative testes weights for high-dose males were decreased but no compound related histomorphic changes were observed. Absolute and relative liver weights were increased in mid- and high-dose males and all females but were not statistically significant and were not accompanied by histopathologic changes. Absolute and relative kidney weights were increased in all treatment groups (p < 0.01) and were considered dose-related. Gross necropsy of all organs revealed compound-related effects in the kidney, described as renal enlargement, discolouration, surface irregularities, pelvic dilation, cysts, nodules and masses in treated groups only. There were no other significant compound-related effects on other organs or tissues reported. Histopathological data demonstrated compound-related effects on the stomach, esophagus and kidneys. The incidence and severity of hyperplasia and hyperkeratosis of the squamous mucosa in the oesophagus of treated males and females was significantly increased in a dose- and compound-related manner. Microscopic examination of the stomach demonstrated a more significant dose-related increase in the incidences of hyperplasia and hyperkeratosis of the squamous mucosa in male and female mice in the treated groups. This finding was virtually non-existent in control groups. There was a significant increase in squamous cell tumours in the 1 500 ppm females but the incidence was not dose-related. The incidence of squamous cell tumours in males was 0/60, 1/60, 5/60 and 2/60 in control, 750, 1 500 and 3 000 ppm groups, respectively. In females the incidence was 0/60, 2/60, 6/60 and 5/59 in control, 750, 1 500 and 3 000 ppm groups, respectively. Glandular epithelial tumours were increased in the treated groups but were not significant or dose-related. The incidence of glandular epithelial tumours in males was 0/60, 1/60, 2/60 and 0/60 in control, 750, 1 500 and 3 000 ppm groups, respectively. In females the incidence was 0/60, 1/60, 1/60 and 2/59 in control, 750, 1 500 and 3 000 ppm groups, respectively. Microscopic examination of the kidney revealed a significant finding of chronic glomerulonephritis present in all control and treatment groups, which was increased predominantly in males at 3 000 ppm. Tubular degeneration was increased in males at 750 and 1 500 ppm and in females at 1 500 ppm. The incidence of cortical cysts was increased in all treated males and in high-dose females. The neoplastic changes, reported as adenomas and carcinomas of the renal cortical tubules, were increased in the males in all treated groups but not in females. The incidence was 0/60, 6/60, 4/60 and 4/60 for control, 750, 1 500 and 3 000 ppm male groups, respectively. There were no neoplastic changes in the female groups, except for one low-dose female with a kidney haemangiosarcoma. Chlorothalonil has presented evidence of causing nephrotoxicity in earlier studies in rats, mice and dogs, predominantly in males. In an NCI rat study there was presumptive evidence of adenomas and carcinomas of the renal tubular epithelium. The NCI mouse study was negative for evidence of kidney tumours. Although primary renal tumours in rodents are rare, there was no positive trend for adenomas and carcinomas in the renal cortical tubules of male mice in this study, and therefore the evidence for tumourigenicity of chlorothalonil in the kidney remains elusive. The effects on the kidney, in this study, are nonetheless considered compound-related (Wilson et al 1983a; Tierney 1983). Groups of six-week old CD-1 mice (60 males and 60 females per group) were administered the chlorothalonil metabolite 4-hydroxy-2, 5, 6-trichloroisophthalonitrile in the diet at dosage levels of 0, 375, 750 and 1 500 ppm for 105 consecutive weeks. Animals were observed daily for mortality and other gross signs of toxicity. Individual body weights, food consumption, physical examinations and haematological parameters were evaluated regularly throughout the study. All animals dying or sacrificed during and at the termination of the study were necropsied; organ weights were determined, and selected tissues/organs were examined histologically. The mortality in the low- and mid-dose females reached 75 percent during month 20 and, therefore, all surviving low- and mid-dose females, as well as 10 control females (to serve as concurrent controls) were terminated at month 20. Mortality in the high-dose females reached 75 percent at month 22 at which time all females in that group and in the control group were sacrificed. Survival of the male mice was comparable between control and treated groups. There were no significant differences between control and treatment group animals on the basis of cage-side observations for signs of toxicity. Body weights were significantly reduced in both males and females given 1 500 ppm throughout the study. There were also sporadic decreases in males and females at 750 ppm that were evident during the first year of study and were comparable to controls during the second year. There were significant increases in food consumption for high-dose females compared to controls throughout the study and for high-dose males during the first year. Average compound intake throughout the study, on a mg/kg/day basis, for males was 50-88 (at 375 ppm), 90-180 (at 750 ppm) and 190-425 (at 1 500 ppm); for females it was 58-103 (at 375 ppm), 100-275 (at 750 ppm) and 232-624 (at 1 500 ppm). Haematological examinations conducted at 12, 18 and 20-24 months revealed compound-related effects on red blood cells with decreases in all treatment groups at 12 months, decreases in males at 750 ppm and females at 750 and 1 500 ppm at 18 months, and decreases in males given 1 500 ppm at 24 months. Reticulocyte counts were increased in males given 1 500 ppm at 24 months and in females given 750 and 1 500 ppm at 20 months. Evaluation of differential leukocyte counts demonstrated moderate to marked increases in morphological changes in erythrocytes and leukocytes at 18 months in all treated females and 750 ppm males. At terminal sacrifice, all treatment and control groups presented evidence of changes, but they were more noticeable in the high-dose groups. Changes included anisocytosis, poikilocytosis, polychromic red cells, target cells, large platelets, crenated red cells and nucleated red cells. Bone marrow differential counts, including M/E ratios, demonstrated a decreasing trend of 2:1 for control females to approximately 1:1 for treatment groups. Similarly, a ratio of 1:2 for control males was approximately 0.9:1 for treated males. Organ weights and organ-to-body weight measurements demonstrated significant increases in spleen and liver weights and respective organ-to-body weight increases at 20 months in 750 ppm females. Absolute liver weights and liver-to-body weight ratios were also increased in 1 500 ppm females at 22 months. Absolute kidney weights and heart weights were also decreased at 22 months in the 1 500 ppm females. Male mice given 375-1 500 ppm revealed significant increases in liver weight and liver-to-body weight ratios and a positive trend in the spleen-to-body weight ratio. Absolute kidney weights were reduced in males at 1 500 ppm but were not considered significant, based on decreased body weight. Histopathological evaluations revealed an increase in bronchioalveolar adenomas in treated males. However, the differences between the control incidences were not dose-related or significant and, therefore, were not considered compound-related. The reported incidence in males was 2/59 (0 ppm), 6/57 (375 ppm), 6/57 (750 ppm) and 4/59 (1 500 ppm). The incidence of benign hepatocellular neoplasms and hepatocellular carcinomas was higher in control mice, with a negative trend in the treatment groups. There was one tubular adenoma and one tubular carcinoma of the kidney in the low-dose males but no incidences in any other groups. Non-neoplastic changes consisted of a dose-related increase of amyloidosis in the thyroid of both males and females. There was an overall marginal dose-related increase in the incidences of amyloidosis in treated males and females reported for the stomach, duodenum, ilex, liver, adrenal and urinary bladder. There was also an overall increase in amyloidosis reported for all female groups with respect to the spleen, kidney, jejunum and ovary. There was an increase in pigmentation (haemosiderin) of the spleen, which was compound-related and evident at all doses in both sexes. Chronic interstitial nephritis of the kidney was also considered compound- related and was increased in all treated females compared to controls. The test material was not oncogenic in CD-1 mice under the conditions of this study at doses up to and including 1 500 ppm. However, marginal and significant non-neoplastic changes, including amyloidosis, haemosiderin in the spleen, morphological changes in leukocytes and erythrocytes, increase in reticulocyte count as well as evidence of red cell haemolysis, failed to demonstrate a clear no- effect level for these effects (Hozan & Auletta 1981). Rat Groups of Sprague-Dawley CD rats (75 males and 75 females/group) were administered 4-hydroxy-2, 5, 6-trichloroisophthalonitrile in the diet at dosage levels of 0, 0.5 and 3 mg/kg/day for 104 weeks. Original dosage levels of 15 and 30 mg/kg/day were reduced at week 30 to 10 and 20 mg/kg/day, respectively, because of poor survival and anaemia. Animals were observed daily for mortality, gross signs of toxicity and general appearance. Individual body weights and food consumption were measured regularly during the study. Clinical laboratory studies were performed periodically throughout the study on 10 rats/sex/group at six-month intervals. Ophthalmological examinations and urinalyses were performed routinely, and faeces were collected and examined to evaluate the observed anaemia. Interim sacrifices were performed after one year on 10 rats/sex in all groups, except for the high-dose animals, which were all necropsied. Terminal necropsies were performed on all surviving animals after two years, selected organs weighed and complete histopathological examinations conducted. Pale skin and eyes were evident for the first 30 weeks in high- dose males and females, with similar but less marked findings observed in the 15 mg/kg group. Mortality was significantly increased in the 30 mg/kg males and females and in the 15 mg/kg females. The high-dose group was sacrificed at 12 months after the dose level had been reduced to 20 mg/kg at week 30. Decreasing the 15 mg/kg/day dose level at week 30 to 10 mg/kg similarly improved the survival, which was comparable to controls for the remainder of the study. Body weight was reduced in the 10/15 and 20/30 mg/kg males and females throughout the study, even after reduction of doses. Food consumption was unremarkable except for decreases in 10/15 and 20/30 mg/kg females and 20/30 mg/kg males, which was consistent with decreased body weights and increased mortality during the first 30 weeks. There were similar decreases in total serum protein, albumin, globulin and cholesterol in 20/30 mg/kg males and females and 10/15 mg/kg females after six months. These returned to control levels for the remainder of the study after doses were reduced to 20 and 10 mg/kg, respectively. There were significant haemopoietic effects in the 10/15 and 20/30 mg/kg animals, particularly females, during the first six months. Evidence of microcytic anaemia was provided by reduced red cell counts, haematocrit, haemoglobin, MCV and MCH, with accompanying increases in MCHC, reticulocytes and metarubricytes. Segmented neutrophiles were increased with a corresponding decrease in the percentage of lymphocytes. Specially stained bone marrow presented evidence of hypocellularity. Mallory's stain of liver tissue revealed an increased iron content (haemosiderin). After 18 and 24 months exposure, the 10/15 mg/kg group females continued to present evidence of anaemia (decreased Hct, Hgb, MCV, MCH and increased MCHC) with a positive bone marrow response (increased cellularity with a shift to increasing number of immature erythyroid cell types and increased number of animals with 1:1 M/E ratio). Prussian Blue staining demonstrated the presence of haemosiderin in the 10 mg/kg males and females which was not considered significant at 3 mg/kg. After 24 months exposure, there were decreased serum potassium levels in all treated females. Urinalyses, and examination for faecal occult blood were unremarkable, except for increased urine volume at six months in the high-dose animals. Ophthalmological examination at six months revealed increased pale ocular structures and spontaneous hemorrhage in high-dose males and females. At 24 months there were increased numbers of dilated pupils (not responding to light) and increased bilateral cataract disease in high-dose males. Comparison of selected organ weights demonstrated decreased absolute organ weights for kidney, heart and brain in high-dose males with no significant relative organ-to-body weight changes. High-dose females had decreased absolute kidney and heart weights with no relative weight changes, except for spleen and brain. Microscopic examination failed to confirm any compound-related effects on these organs. There were no significant compound-related non-neoplastic organ changes, except for haemosiderin in the liver of high-dose females and haemorrhage in CNS tissues, hypocellular bone marrow and post-mortem congestion of lymph in high-dose males and females. Examination of tissues/organs for neoplastic changes did not indicate any compound-related effects at any level tested. Data presented in this study demonstrate that the metabolite 4-hydroxy-2, 5, 6-trichloroisophthalonitrile is without adverse effects on male and female rats at levels up to and including 3 mg/kg/day for two years (McGee et al 1983). Special Studies on Mutagenicity Bacteria Chlorothalonil, dissolved in DMSO, was tested for mutagenic activity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 according to the plate incorporation procedure of Ames, at concentrations between 0.33 and 6.6 µg/plate with and without metabolic activation. Under the test conditions reported chlorothalonil (97.8 percent pure) did net induce point mutations in the five histidine-requiring strains of S. typhimurium at the concentrations tested (Banzer 1977a). Chlorothalonil was examined for mutagenic activity in S. typhimurium strains G-46, TA1530, C207, TA1531, C3076, TA1700, D3056 and TA1724 implanted in male albino mice. Host mediated assay at the dose level of 4 mg/kg (single IP administration) was carried out. No increase in mutation frequency of chlorothalonil treated group over the control was observed and the test compound (99 percent pure) was not mutagenic in the host mediated assay at the dose level tested (Legator 1974a). Chlorothalonil was tested for its mutagenic activity in S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, according to the plate incorporation procedure of Ames et al. (1975), at concentration between 1 and 10 µg/plate without metabolic activation and between 2 and 20 µg/plate with metabolic activation. In addition, Escherichia coli B/r WP2 hcr+4 and its uv-sensitive derivative WP2 hcr -5 were also employed in the plate incorporation procedure with specific selective medium at concentrations between 10 and 500-µg/plate without metabolic activation and between 10 and 100 µg/plate with metabolic activation. Under the given experimental test conditions, chlorothalonil (99.3 percent pure) failed to induce any increase in the number of revertant colonies above that of the control and, thus, was not mutagenic at the concentrations tested (Shirasu et al 1977). Cultured mammalian cells Chinese hamster cells (V-79) and mouse fibroblast cells (Balb/3T3) in culture, with and without metabolic activation, were exposed to chlorothalonil at a concentration of 0.3 µg/ml and 0.03 µg/ml, respectively, to detect mutations at the Ouabain resistant gene locus, according to the method of Schechtman. Acetone was the vehicle control without activation. S-9 alone was the negative control with activation. No evidence of mutagenic effect of chlorothalonil was observed in these two in vitro mammalian cell mutagenesis assay systems. The test compound (97.8 percent pure) was not mutagenic under the experimental conditions tested (Banzer 1977b). Cytogenic assay Chlorothalonil, suspended in 0.5% (w/v) Methocel E 15 premium (hydroxy-propyl methyl cellulose), was tested in rats, mice and Chinese hamsters for its ability to induce chromosomal aberrations in bone marrow cells. Prior to the chromosomal aberration test, a preliminary study was conducted in animals to determine appropriate dosage levels. In the cytogenetic assay, animals were orally dosed with the test compound with a 24-hour interval between doses. Six hours after the second dose, animals were killed, bone marrow removed from the femurs and slides prepared for the cells. One hundred metaphase cells per animal were evaluated for chromosomal aberrations. Ten male rats (Wistar) per group were orally dosed with 0, 8, 40, 200, 1 000 and 5 000 mg/kg chlorothalonil twice, with a 24-hour interval between doses. Ten male mice (Swiss C.F.L.P>) per group were orally dosed with 0, 4, 20, 100, 500 and 2 500 mg/kg chlorothalonil twice, with a 24-hour interval between doses. Ten male Chinese hamsters, per group were orally dosed with 0, 8, 40, 200, 1 000 and 5 000 mg/kg chlorothalonil twice, with a 24-hour interval between doses. No significant differences in the induced chromosomal aberrations were observed for the test animals between the treated and control groups at the dose levels tested. Under the given experimental test conditions, chlorothalonil (98.2 percent pure) was not considered to be a clastogenic agent (Siou et al 1981a). Micronucleus test Chlorothalonil, suspended in 0.5 percent (w/v) Methocel E 15 premium (hydroxy-propyl methyl cellulose), was tested in rats, mice and Chinese hamsters for its ability to induce the formation of micronuclei in polychromatic erythrocytes. Prior to the micronucleus test, a preliminary study was conducted in animals to determine appropriate dosage levels. In the micronucleus test, animals were orally dosed with the test compound, with a 24-hour interval between doses. Six hours after the second dose, animals were killed, bone marrow removed from the femurs and slides prepared from the cells. Two thousand polychromatic cells per treatment were examined for the presence of micronuclei. The number of rats and mice per group and the dosage levels of test compound used in this study were identical with that used in the previous cytogenetic assay (Siou et al 1981b). Ten male Chinese hamsters per group were orally dosed with 0, 4, 20, 100, 500 and 2 500 mg/kg chlorothalonil twice, with a 24-hour interval between doses. No significant differences in the mean value of polychromatic erythrocytes with micronuclei were observed between the treated and control groups at the dose levels tested. Under the test conditions reported, chlorothalonil (98.2 percentage pure) exhibited no clastogenic activity in the polychromatic stem cells of treated animals (Siou et al 1981b). Chlorothalonil was tested for its ability to induce clastogenic activity in the polychromatic erythrocytes of treated animals. Swiss albino male mice were given an oral dose of 6.5 mg/kg/day for five consecutive days. Three hours after the final administration, animals were killed, bone marrow removed from the femurs and slides prepared from cells. Two thousand polychromatic cells were examined for the presence of micronuclei. No significant differences in the frequency of micronuclei from the polychromatic erythrocytes were noted between the treated and control groups at the dose level tested. Under the test conditions of this experiment, chlorothalonil (98 percent pure) was not a clastogenic agent in the micronucleus test (Legator 1974a). Dominant lethal test Ten male mice were orally gavaged with 6.5 mg/kg/day chlorothalonil for 5 days. Following the treatment, the males were sequentially mated to two females per week for 8 weeks. The number of corpora lutea, total implantations and resorptions were counted for each pregnant female. The same number of untreated mice served as controls. No increase in the resorptions in the treated group was observed when compared to the control group. Chlorothalonil was not considered to induce dominant lethals in treated male mice at the dose level tested (Legator 1974a). DNA damage and repair The DNA damaging capacity of chlorothalonil was investigated by the bacterial system (PolA-/PolA+) using S. typhimurium strains TA1538 (repair deficient) and TA1978 (repair competent) in a spot test. The test compound was tested with and without metabolic activation at concentrations of 2, 10, and 20 µl of a 1.0 mg/ml stock solution. Chlorothalonil (97.8 percent pure) exhibited significantly different preferential cell killing between the two strains of S. typhimurium, with and without metabolic activation, at the concentrations tested. The test compound appears to have reacted with the DNA molecules in a manner causing preferential cell killing of strains, which lack an excision repair enzyme system. It is considered a DNA-modifying agent in this test (Banzer 1977c). Chlorothalonil was tested in Rec Assay using Bacillus subtilis H17 (recombination wild) and M44 (repair deficient) in a spot test. The compound was tested at concentrations of 2, 5, 10, 20, 100 and 200 µg/disc without metabolic activation. Chlorothalonil (99.3 percent pure) exhibited no marked difference in the growth inhibition zones of the strain H17 and M44. It is not considered a DNA-modifying agent at the concentrations tested (Shirasu et al 1977). Cell transformation Chlorothalonil, diluted in acetone and saline, was tested to determine its ability to induce malignant transformation of Fischer rat embryo (F1706 and H4536) cells in culture. Prior to the transformation assay, a preliminary cytotoxicity test was conducted to determine the maximum non-toxic dose level of the test compound. In the transformation assay, three concentrations of the test compound (0.001, 0.0001 and 0.00001 µg/ml) were incubated with each of the described cell lines for seven days. At the end of incubation, each culture was washed, refed with a complete medium and held for two additional weeks to look for foci of transformed cells. The cell cultures treated with the test compound were inoculated subcutaneously in the back of a newborn Fischer rat and examined for the development of tumours within three months. There was no increased number of transformed foci over the control in either of the treated cell cultures. The cultures that had been exposed to the test compound were not tumourigenic when injected into newborn Fischer rats. Chlorothalonil (96 percent pure) is not considered a transforming agent in the Fischer rat embryo cell lines at the concentrations tested (Price 1978a). The results of mutagenicity assays carried out with chlorothalonil are summarized in Table 1. Special Studies on Mutagenicity of Metabolites Bacteria 4-hydroxy-2, 5, 6-trichloroisophthalonitrils (DS-3701), dissolved in DMSO, was tested for mutagenic activity in S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 according to the plate incorporation procedure of Ames et al. (1975) at concentrations between 1 and 100 µg/plate, with and without metabolic activation. Under the conditions of this test, DS-3701 (99 percent pure) did not induce point mutations in the five histidine-requiring strains of S. typhimurium at the concentrations tested (Banzer 1977d). Table 1. Results of Mutagenicity Assays of Chlorothalonil Test Organism Test Substance Results Reference (% purity) GENE MUTATION STUDIES Bacteria S. typhimurium 97.8 No mutagenic activity was Banzer 1977a reported in TA98, TA100, TA1535, TA1537 and TA1538 with or without metabolic activation. 99.0 Negative response in G46, C207, Legator 1974a TA1530, TA1531, C 3076, TA1700, D3056 and TA1724 according to host mediated assay. 99.3 No mutagenic activity was Shirasu et al reported in TA98,TA100, TA1535, 1977 TA1537 and TA1538 without metabolic activation. E. coli 99.3 No mutagenic activity was Shirasu et al reported in WP2 hcr+4 and WP2 1977 hcr-5 with or without metabolic activation. Cultured Mammalian Cells Table 1. (con't) Test Organism Test Substance Results Reference (% purity) Chinese hamster cells 97.8 No mutagenic effect was reported Banzer 1977b (V79) and Mouse Fibroblast in these two mammalian cell lines Cells (Balb/3T3) with or without metabolic activation. CHROMOSOMAL EFFECTS Cytogenetics - In Vivo 98.2 No induced chromosomal aberrations Siou et al Rat, Mouse, Chinese were reported in bone 1981a hamster marrow cells of rat, mouse and Chinese hamster. Micronucleus - In Vivo 98.0 Negative responses were reported Legator 1974a Rat, Mouse, Chinese in the polychromatic erythrocytes & Siou et al hamster of treated animals. 1981b Dominant Lethal - Mice No mutagenic activity to induce Legator 1974a dominant lethals in male mice were reported. DNA DAMAGE AND REPAIR - Bacteria S. typhimurium 97.8 Positive response in demonstrating Banzer 1977c significantly preferential cell killing between the TA1538 and TA1978 with or without metabolic activation. Table 1. (con't) Test Organism Test Substance Results Reference (% purity) S. subtilis 99.3 Negative response. No marked Shirasu et al difference in the inhibition zones 1977 of the strain H17 and M44 was reported. Cell Transformation - 96.0 Negative responses were reported Price 1978a In Vitro in both treated cell lines. Fischer Rat Embryo Cell Lines (F1706 and H4536) 4-hydroxy-2, 5, 6-trichloroisophthalonitrile was examined for mutagenic activity in S. typhimurium strains G46, TA1530, C207, TA1531, C3706, TA1700, D3056 and TA1724 which was implanted in male Swiss albino mice according to the method of host mediated assay at the dose level of 6.5 mg/kg (single IP administration). No increase in mutation frequency of the treated group over the control was observed and the metabolite (99 percent pure) was not mutagenic in the host mediated assay at the dose level tested (Legator 1974b). Mammalian cells Chinese hamster cells (V-79) and mouse fibroblast cells (Balb/3T3) in culture were exposed to 4-hydroxy-2, 5, 6-trichloroisophthalonitrile (DS-3701) at a concentration of 30 µg/ml, with and without metabolic activation, to detect mutations at the Ouabain-resistant gene locus, according to the method of Schechtman et al. Acetone was the vehicle control. No evidence of mutagenic effect of DS-3701 was observed in these two in vitro mammalian cell mutagenesis assay systems. The test compound (99 percent pure) did not induce Ouabain-resistant mutations at the concentration tested (Banzer 1977e). Micronucleus test 4-hydroxy-2, 5, 6-trichloroisophthalonitrile (DS-3701) was tested in Swiss albino male mice at 6.5 mg/kg/day for five days for its ability to induce the formation of micronuclei in polychromatic erythrocytes. At the concentration tested, this compound (99 percent pure) did not increase the number of polychromatic cells with micronuclei (Legator 1974b). Dominant lethal test Ten male albino rats per group received a single oral dose or five daily doses of 0, 2, 4 or 8 mg/kg 4-hydroxy-2, 5, 6-trichloroisophthalonitrile (DS-3701). Following treatment, the male rats were sequentially mated with two virgin female rats per week for eight weeks. After 12 days, the females were sacrificed and their uteri were removed, implantation sites examined, resorption sites and total foetuses counted and foetal deaths noted. No significant differences in body weights of males, percent fertility, foetal deaths or resorptions were observed between the treated and the respective untreated groups either on a weekly or total basis. The test compound, DS-3701 (99 percent pure), did not induce dominant lethals at the dose levels tested (Hastings & Clifford 1975). 4-hydroxy-2, 5, 6-trichloroisophthalonitrile was tested for its ability to induce dominant lethals in treated male mice at 1, 3 and 6.5 mg/kg/day for five days. The compound (99 percent pure) was not considered as a mutagenic agent and did not induce dominant lethals at the dose tested (Legator 1974b, 1975). DNA damage and repair DNA damaging capacity of 4-hydroxy-2, 5, 6-trichloroisophthalonitrile (DS-3701) was evaluated by the bacterial system (PolA-/PolA+) using S. typhimurium strains TA1538 (repair deficient) and TA1978 (repair competent) in a spot test. The compound was tested with and without metabolic activation at concentrations of 2, 10 and 20 µl of a 1.0 mg/ml stock solution. Under these test conditions, DS-3701 (99 percent pure) did not interfere with the DNA mechanisms of S. typhimurium and, thus, was not considered a DNA-modifying agent (Banzer 1977f). Cell transformation 4-hydroxy-2, 5, 6-trichloroisophthalonitrile (DS-3701), diluted in acetone and saline, was tested to determine its ability to induce malignant transformation of Fischer rat embryo (F1706 and H4536) cells in culture. Prior to the transformation assay, a preliminary cytotoxicity test was conducted to determine the maximum nontoxic dose level. The test compound (0.1, 1 and 10 µg/ml) was incubated with each of the described cell lines for seven days. There was no increased number of transformed foci over control in either of the treated cell cultures. The cultures exposed to the test compound were not tumourigenic when injected into newborn Fischer rats. However, the F1706 cells exposed to DS-3701 did produce late tumours in the rats. The production of these late tumours was considered to be due to spontaneous transformation and not due to transformation by the test compound. DS-3701 (99 percent pure) is not considered a transforming agent in the Fischer rat embryo cell lines at the concentrations tested (Price 1978b). The results of mutagenicity assays of 4-hydroxy-2, 5, 6-trichloroisophthalonitrile are summarized in Table 2. COMMENTS Excretion and elimination studies in rats demonstrated that chlorothalonil is preferentially excreted in the faeces, with minor elimination of the parent compound or its metabolites in the urine. 4-hydoxy-2, 3,5-trichloroisophthalonitrile was identified as a major metabolite. Faecal elimination was complete (95 percent) within 24 hours on low doses, but was noticeably delayed (96 hours) at high doses. The total amount of radioactivity in the blood following the oral administration of labelled chlorothalonil was less than one percent of the administered dose, with most of the activity being found in the plasma. This information failed to clarify the general metabolism of chlorothalonil, particularly regarding the 4-hydroxy metabolite. Table 2. Results of Mutagenicity Assays of 4-hydroxy-2,5,6-trichloroisophthalonitrile Test Organism Test Substance Results Reference (% purity) GENE MUTATION STUDIES Bacteria Salmonella 99.0 No mutagenic activity was reported Banzer 1977d typhimurium in TA98, TA100, TA1535,TA1536 and TA1538 with or without metabolic activation. S. typhimurium 99.0 Negative response in G46, TA1530, Legator 1974b C270, TA1531, C3076, TA1700, D3056 and TA1724 according to host mediated assay. Cultured Mammalian Cells Chinese hamster Cells 99.0 No mutagenic activity was reported Banzer 1977e (V79) and Mouse Fibroblast in TA98,TA100, TA1535, TA1537 and Cells (Balb/3T3) TA1538 without metabolic activation. CHROMOSOME EFFECTS Micronucleus - In Vivo 99.0 Negative response was reported in Legator 1974b Mouse the polychromatic erythrocytes of treated mice. Table 2 (con't) Test Organism Test Substance Results Reference (% purity) Dominant Lethal - Rodent Rat 99.0 No induced dominant lethals were Hastings et reported in treated rats. al 1975 Mouse 99.0 No induced dominant lethals were Legator 1974b, reported in treated mice. 1975 DNA DAMAGE AND REPAIR - Bacteria S. typhimurium 99.0 Negative response. No marked Banzer 1977f difference in the inhibition zones of the strains TA1538 and TA1978 were reported. CELL TRANSFORMATION - IN VITRO Fischer Rat Embryo Cell 99.0 Negative responses were reported Price 1978b Lines (P1706 and H4536) in both treated cell lines. Short-term dietary studies in rats and mice exposed to chlorothalonil demonstrated increased incidences of hyperplasia and hyperkeratosis in the non-glandular portion of the stomach and hyperplasia of the epithelial cells of the proximal convoluted tubules in the kidney. Kidney and liver weights were also increased, but there was no microscopic evidence of morphological change. The no-observed- effect level (NOEL) was 3 mg/kg b.w. in both rats and mice. A short-term dietary study in rats using the 4-hydroxy metabolite at levels of 10-750 mg/kg presented evidence of anaemia and compound- related liver effects at dietary levels greater than 20 mg/kg b.w. A one-generation study in rats using the 4-hydroxy metabolite demonstrated adverse reproductive effects at doses greater than 30 ppm. These included poor pup viability and reduced mean live pup weights. Teratology studies with rabbits and rats gavaged with chlorothalonil produced no teratogenic responses; however, maternal toxicity was evident at >5 mg/kg and 400 mg/kg in rabbits and rats, respectively. Teratogenicity studies by gavage in the rabbit at maternally toxic doses of 5 mg 4-hydroxy metabolite/kg failed to induce terata. A wide range of mutagenicity studies, using both chlorothalonil and the 4-hydroxy metabolite, did not indicate mutagenic activity for either compound. A carcinogenicity study in mice exposed to chlorothalonil at dietary levels of 150-3 000 ppm produced glandular epithelial tumours in treated groups, which were compound-related but not dose-related. The neoplastic changes in the kidney, including adenomas and carcinomas of the renal cortical tubules were increased in males in all treated groups, but not in females. There was no positive trend for these kidney adenomas and carcinomas and, therefore, the evidence for the tumourigenicity of chlorothalonil in the kidney remains elusive. A NOEL was not determined. Carcinogenicity studies in rats and mice using the 4-hydroxy metabolite did not demonstrate oncogenic potential at doses up to and including 1 500 ppm in the diet (22.5 mg/kg b.w.) and 20 mg/kg b.w. in mice and rats, respectively. There was, however, clear evidence of anaemia at doses greater than 3 mg/kg b.w. in rats. A NOEL for non- oncogenic effects was not demonstrated in the mouse study. The Meeting expressed concern about the lack of adequate metabolism data for chlorothalonil and the 4-hydroxy metabolite and the need to clarify the incidence of kidney adenomas and carcinomas in rodents. The Meeting was informed that a long-term oncogenicity feeding study with chlorothalonil in the rat is currently being conducted, but the results will not be available until 1985. The Meeting, therefore, agreed that the temporary ADI should remain at the present level until further data are made available to clarify remaining questions on metabolism and carcinogenicity of chlorothalonil. 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 0-0.005 mg/kg b.w. FURTHER WORK OR INFORMATION Required (by 1985) 1. Further work to elucidate the metabolism of chlorothalonil and the 4-hydroxy metabolite. 2. Submission of the on-going long-term oncogenicity feeding study in the rat. Desirable Information on data which may clarify the type of anemia which has been demonstrated in mammals exposed to the 4-hydroxy metabolite. REFERENCES - TOXICOLOGY Ames, B.N., McCann, J. & Yamasake, E. Methods for detecting 1975 carcinogens and mutagens with the Salmonella mammalian- microsome mutagenicity test. Mutat. Res., 31: 347-364. Banzer, C.B. Activity of DTX-77-0035 in the Salmonella/microsomal 1977a assay for bacterial mutagenicity. Report from Microbiological Associates submitted to WHO by Diamond Shamrock Corp. (Unpublished) Banzer, C.B. Activity of DTX-77-0034 in an in vitro mammalian cell 1977b point mutation assay. Report from Microbiological Associates submitted to WHO by Diamond Shamrock Corp. (Unpublished) Banzer, C.B. Activity of DTX-77-0033 in a test for differential 1977c inhibition of repair deficient and repair competent strains of Salmonella typhimurium: repair test. Report from Microbiological Associates submitted to WHO by Diamond Shamrock Corp. (Unpublished) Banzer, C.B. Activity of DTX-0038 in the Salmonella/microsomal assay 1977d for bacterial mutagenicity. Report from Microbiological Associates submitted to WHO by Diamond Shamrock Corp. (Unpublished) Banzer, C.B. Activity of DTX-77-0040 in an in vitro mammalian cell 1977e point mututation assay. Report from Microbiological Associates submitted to WHO by Diamond Shamrock Corp. (Unpublished) Banzer, C.B. Activity of DTX-77-0039 in a test for differential 1977f inhibition of repair deficient and repair competent strains of Salmonella typhimurium: repair test. Report from Microbiological Associates submitted to WHO by Diamond Shamrock Corp. (Unpublished) Ford, W.H. A one-generation reproduction study in rats with DS-3701. 1982 Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Hastings, T.F, & Clifford, D. 8-week dominant lethal study of DAC-3701 1975 in rats. Report from Bio/tox Research Lab. Inc. submitted to WHO by Diamond Shamrock Corp. (Unpublished) Hozan, G.K. & Auletta, C.S. A chronic dietary study in mice with T-114 1981 (DS-3701). Report from Bio/dynamics, Inc. submitted to WHO by Diamond Shamrock Corp. (unpublished) Ignatoski et al. Dose-response determination of the excretion of 1981 radioactivity in rat bile following intraduodenal administration of 14C-chlorothalonil (14C-DS-2787). Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Ignatoski et al. Balance study of the distribution of radioactivity 1982 following oral administration of 14C-chlorothalonil (14C-DS-2787) to rats. Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Ignatoski et al. Distribution of radioactivity following oral 1983a administration of 14C-chlorothalonil to rats: extraction and analysis of 14C-materials in excreta. Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Ignatoski et al. Balance study of the distribution of radioactivity 1983b following oral administration of 14C-chlorothalonil to male mice. Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Ignatoski et al. Recirculation of radioactivity in rat bile following 1983c intraduodenal administration of bile containing 14C-chlorothalonil label. Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Ignatoski et al. Dose-response determination of the excretion of 1983d radioactivity in rat bile following intraduodenal administration of 14C-chlorothalonil. Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Ignatoski et al. Method development for the determination of 14C-DS- 1983e 2787 in blood. Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Ignatoski et al. Levels of radioactivity in blood following oral 1983f administration of 14C-chlorothalonil to male rats. Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Legator, M.S. Mutagenic testing with DAC 2787. Report submitted to 1974a WHO by Diamond Shamrock Corp. (Unpublished) Legator, M.S. Mutagenic testing with DAC 3701. Report submitted to WHO 1974b from Diamond Shamrock Corp. (Unpublished) Legator, M.S. Dominant lethal evaluation of DAC 3701 at 1.0 and 3.0 1975 mg/kg for five days. Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) McGee, D.H. et al. A two-year toxicity and tumourigenicity study of 1983 T-114 in rats. Report from International Research and Development Corp. submitted to WHO by Diamond Shamrock Corp. (Unpublished) Murchison, T.E. et al. A short-term dietary study in rats with 1979 T-114-2 (DS-3701). Report from Dawson Research Corp. submitted to WHO by Diamond Shamrock Corp. (Unpublished) Price, P. The activity of compound DTX-77-0037 in the Fischer rat 1978a embryo transformation assay system. Report from Microbiological Associates submitted to WHO by Diamond Shamrock Corp. (Unpublished) Price, P. The activity of compound DTX-77-0041 in the Fischer rat 1978b embryo transformation assay system. Report from Microbiological Associates submitted to WHO by Diamond Shamrock Corp. (Unpublished) Rodwell, D.E. et al. A teratology study in rats with T-117-11. Report 1983 from Wil Research Labs. Inc. submitted to WHO by Diamond Shamrock Corp. (Unpublished) Shirasu, Y. & Teramoto, S. Teratogenicity study of Daconil in rabbits. 1975 Report from Institute of Environmental Toxicology submitted to WHO by Diamond Shamrock Corp. (Unpublished) Shirasu, Y. et al. Mutagenicity testing on Daconil in microbial 1977 systems. Report from the Institute of Environmental Toxicology submitted to WHO by Diamond Shamrock Corp. (Unpublished) Shults, K. A 90-day feeding study in mice with technical 1983 chlorothalonil. Report from SDS Biotech Corp. submitted to WHO by Diamond Shamrock Corp. (Unpublished) Siou, et al. The chromosomal aberration test in the rat, mouse and 1981 hamster using chlorothalonil. Report from C.E.R.T.I. submitted to WHO by Diamond Shamrock Corp. (Unpublished) Siou, G. et al. The micronucleus test in the rat, mouse and hamster 1981b using chlorothalonil. Report from C.E.R.T.I. submitted to WHO by Diamond Shamrock Corp. (Unpublished) Tierney, W.J. A chronic dietary study in mice with technical 1983 chlorothalonil. Report from Bio/dynamics, Inc. submitted to WHO by Diamond Shamrock Corp. (Unpublished) Wazeter, F.X. & Goldenthal, E.I. Teratology study in rabbits. Report 1976 from International Research and Development Corp. submitted to WHO by Diamond Shamrock Corp. (Unpublished) 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. et al. A subchronic toxicity study of technical 1983a chlorothalonil in rats. Report submitted to WHO by Diamond Shamrock Corp. (Unpublished) Wilson, N.H. et al. Progress report for the electron microscopic 1983b evaluation of renal tissue from a subchronic toxicity study of technical chlorothalonil in rats. Report from Huntingdon Research Centre submitted to WHO by Diamond Shamrock Corp. (Unpublished) RESIDUES USE PATTERN Use patterns have been reviewed previously. Additional summary information indicated that uses on grapes are registered or recommended in Argentina, Australia (confirmed at the 13th Session of the CCPR), Austria, Brazil, Greece, Italy, Spain, The Philippines, Sri Lanka and Yugoslavia. Studies on the potential for chlorothalonil use on grapes are or have been in progress in Canada, Chile, Colombia, South Africa and the United States. The most common good agricultural practice is said to be three to six applications of either a 75 percent WP or a 500 g a.i./1 flowable (water dispersible) formulation at 1.5-2.0 kg a.i./ha in a spray volume up to 4 000 l/ha. The most common last-treatment-to-harvest interval is said to be 7 to 14 days, but occasionally it is 21 to 28 days. Diseases for which chlorothalonil is used are Plasmopara (downy mildew), Uncinula necator (powdery mildew) and Botrytis cinerea (grey mould). Good agricultural practice information from individual countries was not provided except for Spain, in which the following formulations and doses are recommended for fruit tree seeds, potatoes, vines, strawberries and winter cereals: Type of formulation Application rate LC 75% 0.15 - 0.2% LC 50% 0.25 - 0.3% P 5% 20 kg/ha RESIDUES RESULTING FROM SUPERVISED TRIALS Residue trials have been conducted in The Netherlands on onions and strawberries (Netherlands 1983) and on grapes in Australia, Canada, The Federal Republic of Germany and South Africa (Tables 1-5; Diamond Shamrock 1981). Grapes The current temporary maximum residue limit (MRL) of 5 mg/kg is based on a 46-day last-treatment-to-harvest interval and on residue trials in Canada, where the Meeting is informed that there are no recognized uses reflecting good agricultural practices (GAP). The only country in which the use of chlorothalonil on grapes is regarded as good agricultural practice and for which residue trials data are Table 1. Residues of chlorothalonil in grapes treated with Chlorothalonil-Australia 1973-741 Application Location Rate Number Pre-harvest Residue (a,i.) interval (days) (mg/kg) Hunter Valley 113 g/100 l2 7 -1 last spray 3.9 0-post-spray 6.1, 7.1 10 5.6 (8.6)4 225g/100 l3 7 -1 last spray 6.8 0-post-spray 10.7 10 8.7 (13.4)4 South Australia 0.5 kg/378 l2 6 1 1.4 7 0.6 18 1.6 (2.9)4 26 0.6, 0.3 1 kg/378 13 6 1 2.3 7 3.1 18 2.7 (4.9)4 26 0.8 1 BRAVO 75W wettable powder containing 75 percent active ingredient was used for treatment. Information on spray volume was not available. 2 Recommended rate. 3 2X recommended rate. 4 Figures in parentheses are corrected for recovery (64 percent Hunter Valley; 55 percent South Australia). Table 2. Residues of Chlorothalonil and D-3701 (4-hydroxy-2, 5,6-trichloroisophthalonitrile) in Grapes Treated With Chlorothalonil - Canada 1979 Application1 Residues (mg/kg)2 Pre-Harvest Location Rate Number Interval Chlorothalonil DS-3701 (kg a.i./ha) (days) Vineland, Ont. 1.65 5 40 0.28 0.02 0.26 0.01 <0.01, <0.01 0.01, 01 - - - ND 0.02 St. Catherines, 1.65 4 30 1.94 0.13 Ont. 1.62 0.03 - - - <0.01 0.01 Jordan, Ont. 1.65 3 30 3.80 0.04 4.07 0.06 - - - ND(<0.005) 0.01 1.65 2 30 0.54 0.01 1.65 1 30 0.63 0.01 1.04, 1.21 0.03, 0.1 - - - ND 0.01 1 BRAVO 75W wettable powder containing 75 percent active ingredient was used for treatment. Information spray volume was not available. 2 Uncorrected for 76 and 80 percent recoveries of chlorothalonil and DS-3701, respectively. Table 3. Residues of Chlorothalonil in Grapes Treated With Chlorothalonil - Federal Republic of Germany Application Pre- Residues (mg/kg) Spray Harvest Location Year Rate Volume Number Interval Treated Untreated (kg a.i.ha) (l/ha) (days) Bad Münster 1972 4.5 3 000 6 1 12.0 0.5 (Trial 129) (0.2% DaconilR 15 6.2 2787, 75% WP) 22 6.0 29 7.0 71 3 4 Welhausen 1972 4.5 3 000 6 0 13.0 0.05 (Trial 128) (0.2% DaconilR 14 8.0 2787, 75% WP) 21 6.8 28 8.0 70 5.8 0.06 Rhodt 1972 1.5-3.75 1 000- 7 1 26.2 0.05 (Trial 47) (0.2% DaconilR 2 5001 14 12.4 0.03 2787) 21 5.0 0.02 28 5.8 68 2.0 Volkach/Main 1973 1.46/spray 1 000 8 2 2.71 0.03 51 0.38 0.04 Grossbottwar 1974 2.19 × 6; 1 000 8 0 26.5 0.06 2.92 × 2 21 17.13 0.22 (6×0.3% soln 28 8.0 0.06 2×0.4% soln) 35 7.0 0.06 42 4.25 0.03 Table 3. (con't) Application Pre- Residues (mg/kg) Spray Harvest Location Year Rate Volume Number Interval Treated Untreated (kg a.i.ha) (l/ha) (days) 2.92/spray 1 000 8 0 28.0 (0.4% soln) 21 13.5 28 8.0 35 4.75 42 6.7 Mettenheim 1974 1.75/spray 800 6 0 1.45 0.05 (0.3% soln) 21 0.23 0.01 28 0.64 0.01 35 0.16 <0.01 42 0.13 <0.01 2.33/spray 800 6 0 1.9 (0.4% soln) 21 0.45 28 0.82 35 0.59 42 0.39 Deidesheim 1974 1.75/spray 800 6 0 3.7 <0.01 (0.3% soln) 21 1.07 0.02 28 0.55 <0.01 35 0.4 <0.01 42 0.23 <0.01 Table 3. (con't) Application Pre- Residues (mg/kg) Spray Harvest Location Year Rate Volume Number Interval Treated Untreated (kg a.i.ha) (l/ha) (days) 2.33/spray 800 6 0 5.15 (0.4% soln) 21 2.15 28 1.07 35 0.72 42 0.21 Würzburg 1975 24.4 total 1/800 10 0 3.83 0.01 (8 × 0.15% 1/1 000 21 0.27 0.02 2 × 0.2%) 1/1 300 28 0.62 <0.01 1/2 000 35 0.63 0.01 2/1 600 42 0.59 0.01 4/2 500 Ebernburg 1975 28 total 2/1 500 10 0 1.87 <0.01 (8×0.15% soln) 8/2 500 21 0.49 <0.01 2×0.18% sola) 27 0.76 0.26 35 0.52 0.01 42 0.76 <0.01 Deidesheim 1975 31.9 total 2 500 10 0 2.38 <0.01 (6×0.15% soln 21 1.14 0.04 4×0.2% soln) 35 1.85 0.04 43 2.0 0.09 1 Spray volume varied from 1 000 to 2 500 l/ha, apparently increasing during the season from early to mature growth stages. Table 4. Residues of Chlorothalonil in Wine From Grapes Treated With Chlorothalonil Federal Republic of Germany Application Pre- Residues (mg/kg) Spray Harvest Location Year Rate Volume Number Interval Treated Untreated (kg a.i./ha) (1/ha) (days) Hattenheim 1973 2.92 1 000 4 98 <0.01 <0.01 (0.4% DaconilR <0.01 <0.01 75% WP) Hattenheim 1972 NA1 NA NA NA 0.003 0.00 0.004 0.003 1 NA = Information not available. Table 5. Residues of Chlorothalonil and DS-3701 (4-hydroxy-2,5,6-trichloroisophthalonitrile) in Grapes Treated With Chlorothalonil - South Africa 1979-80 Application1 Pre- Residues (mg/kg)2 Rate Number Harvest (kg a.i./ha) Interval Chlorothalonil DS-3701 (days) 1.5/spray 8 1 18.4; 20.9 0.14; 0.14 2 12.4; 13.4 0.10; 0.11 4 3.6; 3.5 <0.05; <0.05 16 2.7; 2.5 0.06; 0.06 32 2.6; 2.3 0.07; 0.07 1 BRAVO 500, a water dispersible flowable formulation containing 500 g chlorothalonil/1, was used for treatment. Information on spray volume was not available. 2 Limit of determination for both chlorothalonil and DS-3701 was 0.05 mg/kg. Uncorrected for recoveries of 89 percent and 94 percent respectively. available is Australia (see Table 1), although specific information on use patterns was not available. Data from residue trials were however provided from The Federal Republic of Germany and South Africa together with additional data from Canada. The trials conducted in Australia therefore, warrant special consideration. Data were available from recommended and twice the recommended application rates, although the approved Australian pre-harvest interval (PHI) was not known. At the recommended rate and 7-18 days, the most common PHI, maximum residues were 5.6 mg/kg (or 8.6 mg/kg when corrected for 65 percent recoveries). These data represent chlorothalonil only, whereas previously recommended limits also include the 4-hydroxy metabolite, which can contribute approximately 5 percent of the total residue in grapes. Previously reviewed Canadian data at a 46-day PHI support the current 5 mg/kg limit based on that PHI. So do Canadian data at 30-40 day PHI submitted to the Meeting (Table 2), although neither PHI is commonly used. In Table 3, data from The Federal Republic of Germany include residues of 5-12-4 mg/kg at recommended pre-harvest intervals which significantly exceed the current 5 mg/kg limit, but result from exaggerated (twice the common recommended) application rates. Residues at commonly recommended application rates and pre-harvest intervals range from 4 to 17 mg/kg. Although these residues result from eight applications as opposed to the six recommended, the data appear to be atypically high when compared to other West German trials with six to ten applications in which residues do not exceed the current limit. Table 4 shows residues of 0.01 mg/kg in wine from grapes harvested 98 days after treatment. South African data (Table 5) are consistent with the current limit. Onions Additional residue data provided to the Meeting (Netherlands 1983) reflect good agricultural practices of the Netherlands. In eight trials conducted in 1981 represented by over 60 samples, residues in mature silver onions and set onions ranged from <0.05 to 0.34 mg/kg (0.57 mg/kg unwashed) at the 7-day last-application-to-harvest interval that is the basis of the current 5 mg/kg limit, and were <0.05 mg/kg (0.28 ppm unwashed) at the 28-day interval, which is regarded as good agricultural practice in The Netherlands. Strawberries In a 1981 trial (Netherlands 1983) understood to represent good agricultural practice in The Netherlands. residues in four samples of mature berries ranged from 1.5 to 3 mg/kg (2.1 mg/kg mean) 14 days after the last of five applications of a 75 percent WP formulation at 1.5 kg a.i./ha. Residues of 4-hydroxy-2,5,6-trichloroisophthalonitrile (DAC 3701) At the request of the 14th session of the Codex Committee on Pesticide Residues (CCPR), the Meeting re-examined the definition of the residue for chlorothalonil. Past evaluations of chlorothalonil by the Meeting as well as new data were considered in order to estimate the proportion of the major metabolite DAC 3701, in the total residue. The results of supervised trials carried out at various locations in the United States between 1965 and 1973 are summarized in FAO/WHO 1975, p.126-128. The concentration of DAC 3701 was lower than the 0.1 mg/kg limit of determination in all crops for which it was analysed except lima beans (in pod) where it accounted for 10 percent of the total residue, cantaloupe and grapes (5 percent) peaches and onions (2 percent), celery (1 percent) and peanuts (0.1 percent). No DAC 3701 was detected in crops grown in glasshouses. Similarly low residues were reported in peaches and no residues of DAC 3701 in whole banana or banana pulp, regardless of the rate of application (FAO/WHO 1978). DAC 3701 residues of less than 2 percent and 5 percent, respectively, in onions and grapes were reported in later supervised trials (FAO/WHO 1980). Data submitted to the Meeting more or less confirm the estimate for grapes and indicate that DAC 3701 does not exceed 5 percent of the parent compound residue in oranges. Lima beans In conducting the above review, the Meeting observed that the current 0.5 mg/kg limit for lima beans was based on data for beans without pod. The temporary MRL should be amended accordingly. FATE OF RESIDUES In Storage and Processing The 1981 Meeting listed as desirable an explanation of apparent inconsistencies between studies carried out in 1979 and 1980 on the nature of residues resulting from the cooking of chlorothalonil- treated foods. The main point of concern was the statement in the 1979 study that 14C-chlorothalonil was largely lost by volatilization during cooking under open conditions while of the 14C in the organic phase approximately 57 percent was characterized as the 4-hydroxy metabolite whereas 43 percent was unidentified and was near the thin- layer chromatography (TLC) origin. This was confirmed by a TLC autoradiogram in the 1979 study. In apparent conflict was the statement in the 1980 study that chlorothalonil was the major residue in organic extracts of chlorothalonil-fortified tomatoes after cooking with and without a pot cover and that there were negligible quantities of the metabolite. Again this was confirmed by an autoradiogram. It was further stated that the effect of cooking chlorothalonil with green beans was similar and this was again confirmed by a TLC autoradiogram. In response to the questions raised by the 1981 Meeting, the manufacturer submitted a redrafted report to present the data from the 1979 and 1980 studies in a more consistent manner (SDS 1983a). No additional studies were provided. The new report confirms previous conclusions that 85-98 percent of added chlorothalonil is lost through cooking under open conditions. It emphasizes that the residue of the hydroxy metabolite formed is only 2.4 percent of the chlorothalonil originally present and that the percentages of degradation products identified after cooking refer to the composition of the small residue remaining. EVIDENCE OF RESIDUES IN FOOD IN COMMERCE OR AT CONSUMPTION Data were provided from Sweden on chlorothalonil residues found in domestic and imported commodities for the period 1981 - 30 April 1983 (Sweden 1983). Of 1 085 samples analysed, residues in 1 070 were below 0.21 mg/kg. Maximum residues with the number of samples having residues over 0.21 mg/kg/total analysed in brackets were: cauliflower 0.41 mg/kg (1/165), celery 1.9 mg/kg (7/50), cucumbers 0.23 mg/kg (1/580), gherkins 0.66 mg/kg (2/49, melons 0.71 mg/kg (2/98) and strawberries 2.9 mg/kg (2/143). The highest levels found are well below current Codex temporary MRLs except gherkins and strawberries for which there are no Codex limits. METHODS OF RESIDUE ANALYSIS To compare the relative extraction efficiencies of tumble extraction and maceration techniques, Bravo 500R was field-applied to oranges at 12.9 l/ha and sampled at 0, 14 and 28 days after the last of three applications (SDS 1983b). The formulation contained 40.6 percent chlorothalonil, 0.33 percent pentachlorobenzonitrile (PCBN) and 0.21 percent hexachlorobenzene (HCB). For surface extraction, residues were extracted with methylene chloride by rotating the sample at 22 rpm for 2 h. One portion was analysed for chlorothalonil by electron capture gas chromatography after elution from a florisil column with a 50 percent methylene chloride:48.5 percent hexane: 1.5 percent acetonitrile eluant after a pre-wash with 20 percent methylene chloride:80 percent hexane. A separate portion was analysed for HCB and PCBN after separation from a florisil column by eluting successively with 20 percent methylene chloride:80 percent hexane and 0.05 percent acetonitrile in 1:1 hexane:methylene chloride. For maceration extraction, samples were blended with 385 ml acetone and 15 ml 1:1 (w/w) H2SO4:water, filtered, concentrated and after pH adjustment in aqueous solvent, partitioned with hexane. Clean-up and analysis for chlorothalonil, HCB and PCBN was as described above. The DS-3701 metabolite was extracted from the aqueous phase with 1:1 petroleum ether:diethyl ether after pH adjustment and methylated for gas chromatographic analysis. Analyses of samples extracted by both procedures were validated by fortifying oranges in the extraction vessel with organic solutions of chlorothalonil, HCB, PCBN and DS-3701 before analysis. Fortification levels and mean recoveries are given in Table 6. Determinations of field-incurred residues by surface extraction and maceration techniques are summarized in Table 7. Table 7 demonstrates that surface and maceration extraction techniques give comparable results for chlorothalonil, HCB. Table 6. Validation of Analytical Procedures for Determining Residues of Chlorothalonil and its Metabolites in Oranges. Chlorothalonil HCB PCBN DS-3701 Added Mean Added Mean Added Mean Added Mean mg/kg % mg/kg % mg/kg % mg/kg % Recovery Recovery Recovery Recovery Surface extraction 0.5-2.1 103 0.01 88 0.01-0.02 95 - - Maceration 0.5-2 85 0.01 77 0.01-0.02 82 0.03-0.05 69 Table 7. Comparison of Maceration and Surface Extraction Techniques for Measuring Field-lncurred Residues of Chlorothalonil and Metabolites in Oranges. Residue (mg/kg)1 Chlorothalonil D S-3701 HCB PCBN Days after Last Treatment Surface Macerated Macerated Surface Macerated Surface Macerated 0 11.2 10.1 0.04 0.006 0.004 0.1 0.07 (0.03) (0.05) (ND) (ND) (ND) (ND) (ND) 14 4.7 6.1 0.3 0.006 0.003 0.06 0.05 (0.03) (0.06) (ND) (ND) (ND) (ND) (ND) 28 5.3 4.1 0.014 0.003 ND 0.04 0.04 (ND) (0.13) (ND) (ND) (ND) (ND) (ND) 1 Each value is the mean of three replicates, each of which consists of four samples. Numbers in parentheses are the means of two untreated controls where ND = <0.01 mg/kg chlorothalonil and DS-3701, <0.003 mg/kg HCB and (0.005 mg/kg PCBN. NATIONAL MAXIMUM RESIDUE LIMITS REPORTED TO THE MEETING Country Commodity MRL (mg/kg) The Netherlands banana 0.2 tomato 1 Sweden fruit and vegetables 1 potatoes 0.1 banana (without peel) 0.05 APPRAISAL Limited information on good agricultural practice in the use of chlorothalonil on grapes was made available to the Meeting. Data on residue trials from several countries were also provided but from only one country in which uses on grapes are known to be approved or registered. The limited data reflecting approved uses, and some of the other data suggest that the 5 mg/kg limit may be too low. In the absence of data from residue trials and information on registered or approved uses from other countries, there is no firm basis on which to change the current limit. However, it should remain temporary irrespective of the ADI status until this information is provided. Additional residue data for onions do not warrant a revision in the current limit, and data provided for strawberries were not adequate to estimate a maximum residue level. Monitoring data from Sweden indicated that previous estimates for cauliflower, celery, cucumbers and melons are adequate. The meeting reviewed a revised report on the effects of cooking on chlorothalonil residues in food. In one of two previously reviewed reports, the major residue remaining in beans after cooking under open conditions was said to be unchanged chlorothalonil with little degradation. In the other report, the major residue under similar conditions was said to be the 4-hydroxy metabolite and unidentified material (57 percent and 43 percent of the remaining residue, respectively) with no chlorothalonil remaining. The redrafted report, based on the original two reports, still does not fully clarify the apparent inconsistencies noted by the 1981 Meeting, although the results are more clearly presented. Whether the residue is chlorothalonil, its hydroxy metabolite, a combination of those, or 57:43 hydroxy metabolite: unknown, the total residues remaining after cooking chlorothalonil-fortified green beans under open conditions in both studies are <7 percent of fortification levels. The 57:43 percent hydroxy metabolite: unknown is shown in the 1983 submission to represent 2.4 and 1.8 percent, respectively of the originally added chlorothalonil. For this reason, the Meeting concluded that the matter had been adequately considered. The Meeting also reviewed a study comparing surface and maceration extraction techniques for determining field-incurred residues of chlorothalonil, HCB and PCBN in oranges and data on levels of the hydroxy metabolite (DS-3701) by the maceration technique. The study showed the two techniques to give comparable results for field- incurred residues. A similar conclusion had been reached for bananas by the 1979 Meeting. Both procedures were validated by fortifying just prior to extraction, but the study does not provide information on the efficiency of extraction of field-incurred residues by either technique. However, since the maceration extraction conditions are similar to those used in earlier evaluations (FAO/WHO 1975), the efficiency for the surface extraction technique is as high as that considered adequate previously. Residues of chlorothalonil, HCB, PCBN and DS-3701 are shown to decrease with time from application. The Meeting reviewed the definition of the residue for chlorothalonil. The results of supervised trials conducted in different parts of the world between 1965 and 1983, which had been reviewed by the Meeting, showed that levels of the major metabolite, DAC 3701, were lower than the 0.1 mg/kg limit of determination in most of the crops for which it was analysed. DAC 3701 accounted for approximately 10 percent of the total residue in lima beans (in pod); 5 percent in cantaloupes, oranges and grapes; 2 percent in peaches and onions; 1 percent in celery and 0.1 percent in peanuts. The Meeting noted that the current 0.5 mg/kg limit for lime beans is based on data for beans without pods and concluded that this should be reflected in the commodity description. RECOMMENDATIONS Additional data reviewed by the Meeting do not warrant a revision of current limits or support the estimation of new limits. The Meeting concludes that the current 5 mg/kg temporary MRL for grapes should remain temporary, irrespective of the status of the ADI, pending the receipt of additional information on good agricultural practice and data on residue trials, preferably from countries providing information on good agricultural practices. The Meeting recommends that the definition of the residue be revised to include chlorothalonil only and concludes that no changes in the numerical values of the maximum residue limits are necessary as a result. The current 0.5 mg/kg limit for lima beans should be expressed as applying to lima beans (without pod) to reflect the data on which it was based. FURTHER WORK OR INFORMATION Required (by 1985) 1. Additional and more specific information on good agricultural practice in the use of chlorothalonil on grapes, as well as residue data from field trials in countries where uses on grapes are known to be good agricultural practice. 2. Information, if it exists, on good agricultural practice for those countries in which residue trials for data provided to the 1983 Meeting were conducted. Desirable 1. Analyses of chlorothalonil-treated animal feed items (for example, bean and peanut vines), processed and unprocessed, for residues of PCBN (from the 1981 Meeting). 2. Information on possible PCBN residues in tissues and milk of dairy cattle fed a diet containing chlorothalonil (from the 1981 Meeting). REFERENCES-RESIDUES Diamond Shamrock. (2, 4, 5, 6-tetrachloroisophthalonitrile), 1981 Compilation of Residue Data and Registration/Recommendation Information on Use of Chlorothalonil on Grapes. Submitted to FAO by Diamond Shamrock Corporation. Netherlands. Information submitted to FAO by the government of The 1983 Netherlands, September 1983, MVD, Ref.: CL1983/22-Fr. SDS. The effects of cooking 2,4,5,6-tetrachloroisophthalonitrile 1983a (chlorothalonil, DS-2787) with vegetables. Document No. 372-3EF-83-0004-001 submitted to FAO by SDS Biotech Corporation. SDS. Validation of analytical procedures for determining residues of 1983b 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil, DS-2787), 4-hydroxy 2,5,6-trichloroisophthalonitrile (DS-3701), hexachlorobenzene (HCB) and pentachlorobenzonitrile (PCBN) on oranges. Document No. 506-3CR-81-0182-001 submitted to FAO by SDS Biotech Corporation. Sweden. Chlorothalonil residues in imported and domestic commodities - 1983 1981 to 1983. Data submitted to FAO from The Government of Sweden.
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: 1985 evaluations Part II Toxicology) 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)