AMITROLE JMPR 1974 IDENTITY Chemical name 3-amino-1,2,4-triazole; 3-amino-S-triazole. Synonyms Aminotriazole, ATA, AT, 3-AT, Weedazol(R) Structural formula Other information on identity and properties Amitrole is a white, crystalline solid with a molecular weight of 84 and a melting point of 150-153°C. It is soluble in water to the extent of 28 g/100 g and in ethanol and methanol to an extent of 26 g/100 g. It is sparingly soluble in ethyl acetate and insoluble in ether, acetone and most other organic solvents. Amitrole forms neutral aqueous solutions but acts as a weak base. When exposed to UV light, amitrole breaks down to form CO2, urea and cyanamide. Potts (1961) has reviewed the chemistry of s-triazoles in considerable detail. Amitrole behaves chemically as a typical aromatic amine. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOCHEMICAL ASPECTS Biotransformation The metabolic fate of amitrole in animals has not yet been fully elucidated. It is rapidly excreted from the body. Following intraperitoneal administration, at least 90% of the injected dose was observed in the urine unchanged within 24 hours (Bagdon et al., 1965). When amitrole (5-14C) was fed to rats (Fang et al., 1964), 70-96% of the radioactivity was excreted in the urine during the first 24 hours as amitrole and two unidentified metabolites. There were traces of 14C in the expired air and the faeces contained a small but variable amount of activity. After absorption, amitrole was distributed throughout most of the tissues. The maximum radioactivity in all tissues was generally reached within one hour and started to decrease 3-4 hours after dosing. Elimination of amitrole from all tissues was rapid. The liver contained a metabolite but no free amitrole. The rate of elimination of this metabolite from liver and kidney was much slower than that of the parent compound. Further studies on rats (Fang et al., 1966) have shown that amitrole is not metabolically acetylated, and that the average half-time for amitrole clearance in various tissues was 4.2 hours. These studies were extended to include the metabolism of two metabolites isolated from bean plants. One was readily excreated in the urine, mainly unchanged but partly as two new metabolites. The other bean metabolite was excreted much more slowly, apparently unchanged. Biotransformations in plants and soil are discussed in the section "Fate of residues". Effects on enzymes and other biochemical parameters Acute administration of amitrole to rats results in depression of catalase and peroxidase enzyme activity as well as the activity of several other enzymes. Liver peroxidase was found to recover within 24 hours, while liver and kidney catalase depression was slower to recover. Catalase returned to normal after seven days (Heim et al., 1956). Insect-derived catalase was also shown to regenerate its activity slowly following exposure to aminotriazole (Samio et al., 1972). Groups of rats (15 males per group) were administered radioactive iodine alone or in combination with 0.15 or 0.78 mg amitrole administered by intraperitoneal injection. Uptake of radioactivity by the thyroid gland was significantly depressed at 0.78 mg/kg. The lower dose reduced iodine uptake but the reduction was not statistically significant. Amitrole significantly decreased fecal radioactivity, while the urinary radioactivity was not significantly affected (Fregly, 1968). Amitrole inhibits peroxidase activity of rat thyroid and salivary gland (Alexander, 1959a and b); liver and kidney catalase activity of rats (Tephly et al., 1961; Heim et al., 1956); catalase activity of human red blood cell (Margoliash and Novogrodsky, 1958; Magos et al., 1974; Kudsk, 1969) catalase activity from other sources such as plant tissue and commercial crystallized enzymes; the synthesis of tryptophan peroxidase of rats (Auerbach et al., 1959); delta-aminolevulinic acid dehydrase activity of mouse and rat liver (Baron and Tephley, 1969; Tschudy and Collins, 1957); the inductive effect of phenobarbital on hepatic microsomal cytochrome P-450 (Baron and Tephly, 1969); the incorporation of iron (59FeCl3) into rat hepatic microsomes (Tephly et al., 1971); microsomal heme synthesis (Tephly et al., 1971); and hydroxylation of exogenous substrates by the liver (Raisfeld et al., 1970). Amitrole caused no proliferation of the microsomal endoplasmic reticulum (Raisfeld et al., 1970). Several further studies have shown the effect of amitrole on certain aspects of the drug metabolizing enzyme system in rat liver (Feytmans and Leighton, 1973; Lotikar et al., 1973; Stenger and Johnson, 1972; Levine, 1973; Matsushima and Weisburgh, 1972; Langhans and Shimassek, 1974). There is no clear picture of the effect of amitrole on microsomal metabolizing enzyme systems in mammals although it does serve to inhibit or reduce several specific biochemical reactions. Kudsk (1969) observed that peroxide generating systems, such as seen with methylene blue, accelerated the uptake of mercury in vitro by human red blood cell preparations. Amitrole and methylene blue which promoted the catalase-peroxide-complex I, had no effect on mercury uptake. Magos (1974) suggested that the ability of red blood cells to take up mercury from air saturated with mercury vapor was reduced when cells were treated for three hours with amitrole and methylene blue. In hemolysates mercury uptake was stimulated by amitrole. In vivo administration of amitrole to rats resulted in decreased lung concentration and increased liver concentrations of mercury. Tryptophan synthesis in plants was shown to be inhibited by amitrole (Smith and Chang, 1973). Amitrole stimulated respiration in Azotobacter and concurrently inhibited growth possibility through an uncoupling of oxidative phosphorylation (Kretschmar and Günther, 1970). In algae, amino acid synthesis was inhibited and glucosamine synthesis was stimulated (Schroeder, 1970). In vitro, amitrole does not inhibit cholinesterase activity of brain, submaxillary gland, serum, or ilium. Three hours after intraperitoneal administration of 4 mg/kg to male rats cholinesterase activity of brain, submaxillary gland, and serum was normal (Bagdon et al., 1965). TOXICOLOGICAL STUDIES Special studies on carcinogenicity Mouse A testing procedure using groups of hybrid strains of mice (18 mice of each sex and each of two strains) evaluated amitrole for carcinogenicity. The mice were administered amitrole orally from day 7 to day 28 of age at doses of 1000 mg/kg and thereafter in the diet at a dose of 2192 ppm. None of the mice survived the 18 month test interval. Hepatomas were evident in most animals, 67/72, and carcinoma of the thyroid was reported in 67/71 of the animals examined (Innes et al., 1969). A further study was designated as subcutaneous although no details were given of the experimental design (Innes, 1966). The majority of animals survived the 18 month test interval. There was no evidence of heptoma or carcinoma of the thyroid. Groups of mice (50 of each sex) were either treated with amitrole by a single subcutaneous administration of 10 mg per mouse or by a weekly dermal application of 0.1 mg/mouse. There was no abnormal behaviour observed. When the animals were sacrificed and examined, no signs of cancer were evident. No mention was made of examination of the thyroid gland in this study (Hodge et al., 1966). Rat Amitrole was found to have an effect on the induction of liver carcinogenesis by 4-dimethylaminoazobenzene. In an attempt to determine the relationship between liver catalase activity and carcinogenesis, Hoshino (1960) administered amitrole intraperitoneally to albino rats at a dose of 1000 mg/kg every other day for 150 days while feeding 4-dimethylaminoazobenzene at a dose that was known to induce liver carcinoma. There was a reduction in the occurrence of liver tumours in those animals administered amitrole (4/14) when compared to the animals administered the carcinogen alone (12/16). Napalkov (1962; 1969) administered amitrole to rats by subcutaneous injection twice weekly (later reduced to a weekly injection) at a dose of 125 mg/animal; in the water at a dose of 20-25 mg/day; in the diet at a dose of 250-500 mg/day; or by subcutaneous implantation combined with subcutaneous administration twice weekly at a dose of 125 mg/rat. Thyroid hyperplasia was observed after seven to eight months. Liver tumours were observed initially in those animals receiving amitrole in the diet and soon thereafter in those animals receiving it by injection or via the drinking water. Sarcomas developed where amitrole was implanted or injected. Concurrent daily injections of 2.5 µg/100 g bw thyroxine and oral administration of 300 mg/day/rat amitrole in food for the length of the experiment resulted in the development of only one thyroid adenoma but 9 liver tumors in 12 males. Amitrole alone induced thyroid tumors in 7/22 and liver tumors in 12/23 male rats of the same age. No liver tumors but 2 thyroid cystic adenomas were observed in 51 control animals. A comprehensive review of the carcinogenic risk of amitrole was made by the International Agency for Research on Cancer commenting on the animal and human data (IARC, 1974). Special studies on mutagenicity Mohandas and Grant (1972) observed that amitrole significantly increased the frequency of chromosomal aberrations in root tips of higher plants. Mutagenic tests using Salmonella strains and yeast cells as test indicators have been negative. A host mediated assay has been reported to be negative although details of the test are not yet available (Weir, 1974). A cytogenetic study, utilizing examination of bone marrow cells arrested in C-metaphase from rats treated with amitrole at levels of 0, 2.5, 25 and 250 mg/kg daily for 5 days, was negative (Fabrizio, 1973). Special studies on reproduction Rat Groups of rats (10 male and 10 female Sherman strain rats per group) were fed amitrole in the diet at levels of 0, 25, 100, 500 and 1000 ppm for approximately two months and mated. In the first generation at 500 and 1000 ppm the number of pups born and pup survival was reduced. It was noted that almost all of the pups died within one week after weaning and these dietary levels were terminated. Dietary levels of 0, 25, and 100 ppm were fed through two generations (two litters from the first generation and one litter from the second generation). At 100 ppm in the diet there was no effect on reproduction and all pups survived. At 100 ppm all animals were found to have thyroid hyperplasia while the incidence of thyroid hyperplasia was sporadic at 25 ppm (Gaines et al., 1973). Oral administration of amitrole at doses of 400 and 1000 mg/kg to rats on days 8 through 13 of gestation resulted in no signs of embryo toxicity or of teratogenic effects. Groups of rats (5 males and 5 females per group) were administered amitrole orally at 100 mg/kg or at 100 ppm in the drinking water for three months prior to mating and in the females up to day 15 of gestation. There was no indication of any effect on reproduction in either male or female rats (Hapke, 1967). The effect of oral administration of amitrole on reproduction was studied in three groups of eight pregnant Sherman strain rats administered dosage levels of 0, 20 and 100 mg/kg bw/day from day 7 through 15 of gestation. There was no effect of amitrole on reproduction and no abnormalities were observed in the offspring through weaning (Gaines et al., 1973). Special studies on teratogenicity Amitrole injected into the yolk of chicken eggs at doses of 20-40 mg/egg, produced a dose dependent malformation of the beak and abnormalities of the tibia shaft. This latter abnormality occurred less frequently. At doses of 2 mg/egg, no effects ware noted. It was observed that when amitrole was dissolved in DMSO it was more active than when it was administered in water (Landauer et al., 1971). Acute toxicity TABLE 1. Acute toxicity of amitrole LD50 Species Route (mg/kg bw) References Rat oral >25 000 Bagdon et al., 1965 dermal > 2 500 Gaines et al., 1973 ip > 4 000 Bagdon et al., 1965 Mouse oral >14 700 Fogleman, 1954 iv > 1 600 Bagnon et al., 1965 Rabbit dermal >10 000 Elsea, 1954 oral > 2 150 Fogleman, 1954 Dog iv > 1 800 Fogleman, 1954 Cat iv > 1 750 Bagdon et al., 1965 Signs of poisoning include: depression, dyspnea, diarrhoea, ataxia, altered respiration, coma and death. The G.I. tract was severely irritated following acute doses. Acute doses administered to dogs (50-1000 mg/kg) intravenously produced an immediate fall in blood pressure followed by increased respiration. Following a dose of 1000 mg/kg, the pressor response to adrenalin was blocked suggestive of an adrenalytic action with respect to blood pressure (Fogleman, 1954). TABLE 2. Acute toxicity of glucose adduct LD50 Species Sex Route (mg/kg bw) References Rat M oral >10 000 Bagdon et al., 1965 Mouse M oral >10 000 Bagdon et al., 1965 M ip >10 000 Bagdon et al., 1965 M iv > 1 600 Bagdon et al,, 1965 Amitrole applied to unabraded skin as a paste at doses of 1-10 g/kg for 24 hours caused a mild dermal irritation at all dose levels. The effect, a mild erythemia, was reduced within 24 hours. No other effects were noted. Gross and microscopic examination of tissues and organs was normal (Elsea, 1954). Amitrole (3 mg) was applied to the conjuntival sac of rabbits. Although mild irritation lasting 24-48 hours was observed, no permanent damage was noted (Elsea, 1954). Short term studies Rat Groups of rats (5 males and 15 females per group) were fed amitrole in the diet at levels of 0, 0.01%, 0.1%, and 1.0% for 63 days. There was no mortality over the course of this study. Food consumption and growth were depressed at 0.1% and above in both males and females. Histological examination from selected tissues at the conclusion of the study (thyroid was not examined) revealed vacuolation of liver cells in those animals fed 0.1% and above. The vacuoles were identified as fat globules indicative of fatty metamorphosis associated with liver cell damage. No histological effects were noted at 0.01% (Fogleman, 1954). Groups of weanling rats were administered amitrole for up to 56 days by intraperitoneal injection three times/week for eight weeks at 1000 mg/kg. The administration of a recrystallized amitrole resulted in no growth depression in two separate groups. The administration of a "pure" material, not recrystallized, resulted in growth depression (Heim et al., 1956). Groups of rats (10 males and 10 females per group) were administered amitrole at 1000 mg/kg by intraperitoneal administration on alternate days for 42 days. Amitrole had to effect on bw gain or food consumption but caused a 3-4 fold increase in thyroid weight (Bagdon et al., 1965). Groups of rats were administered amitrole daily, 5 days per week, for four weeks at levels of 0, 100, 200 and 400 mg/kg. Growth rate was reduced, relative thyroid weight increased, and iodine content of the thyroid was reduced (Hapke, 1967). Administration of dietary levels of 60 and 120 ppm for two weeks resulted in an enlargement of the thyroid gland of rats and a pronounced lowering of iodine uptake. Over this two week interval there were no significant changes at levels of 15 and 30 ppm (Jukes and Shaffer, 1960). Administration of a high dose (0.04%) of amitrole in the drinking water (approximately 60 mg/kg/day) has been shown to produce goitres in rats within three days (Strum and Karnovsky, 1971; Tsuda et al., 1973). Groups of rats (10 male rats per group) were fed dietary concentrations of amitrole for 32 days. One group was fed 500 ppm for 32 consecutive days, another group received 1000 ppm on alternate days for the duration of the study and a group was fed the basal diet. Behavior and mortality were not affected. Food intake and growth at 500 ppm were reduced. In the group receiving 1000 ppm on alternate days food intake and growth data did not differ significantly from the controls. At autopsy, gross examination of animals receiving 500 ppm showed the thyroid gland to be hyperaemic and enlarged. The thyroid of animals fed 1000 ppm appeared to be slightly hyperaemic but were otherwise comparable to the controls. Mean thyroid weight/body weight ratio for the 0, 1000 ppm, and 500 ppm groups was 58, 77, and 303 respectively (Shaffer et al., 1958). A group of 30 male weanling rats was fed amitrole in the diet at 1000 ppm for two weeks. A comparable group received a similar dietary level of propylthiouracil for two weeks. At the end of the feeding interval several animals were sacrificed and the thyroid glands weighed. The remaining animals were placed on control diets and sacrificed at either the third or fourth week of feeding. Amitrole, after two weeks produced a significant increase in the mean thyroid weight (comparable to that found with propylthiouracil). Daring the week following removal from the diet, the weight of the thyroid gland diminished and by the end of the second week on a control diet regression to normal size was almost complete (Bagdon et al., 1965). Groups of rats (20 rats per group) were fed dietary l levels of 0 and 316 ppm for 100 days to examine the goitrogenic potential of amitrole. Over the course of this experiment amitrole had a significant effect on growth. Exophthalmia was not observed as in the long term rat studies. Food intake was not drastically reduced. Gross examination of the thyroid showed a laterally enlarged thyroid with enlarged blood vessels. Microscopic examination showed hyperplasia of the thyroid. There was no evidence of abnormalities observed in either the liver or the kidney (Sanderson and Row, 1962). Groups of rats (10 males per group) were administered amitrole in the drinking water at concentrations of 0, 50, 250 and 1250 ppm for 106 days. The administration of amitrole resulted in a dose-dependent depression of growth with a corresponding reduction of food and water intake. Appearance, behaviour, and mortality were not affected by amitrole. Gross and microscopic examination of tissues and organs showed a marked increase in thyroid size at all dose levels. In rats where reduced growth was noted, the kidneys, adrenals, liver and spleen were proportionately smaller. Reproductive organs were not affected. Microscopic examination showed general enlargement of the thyroid at 50 ppm, with moderate stimulation of the thyroid epithelium (no evidence of hyperplasia). At 250 ppm, thyroid hyperplasia was evident (Bagdon et al., 1965). Groups of rats (10 male rate per group) were fed dietary levels of amitrole for 11-13 weeks at dosages of 0, 0.25, 0.50, 2, 10 and 50 ppm. At 0.5 ppm in the diet, no effect was observed in any of seven separate measurements of thyroid function although iodine uptake was slightly reduced and serum PBI concentrations were slightly increased. Significant effects were noted at 2 ppm in the diet, especially with regard to reduced PBI and reduced iodine uptake by thyroid. Gross and microscopic examination of the thyroid confirmed the effects noted at 2 ppm (Fregly, 1968). Dog Groups of beagle dogs (3 males and 3 females were controls; 2 males and 4 females - 0.25 mg/kg; 3 males and 3 females - 1.25 mg/kg; 2 males and 4 females - 2.50 mg/kg; and 2 males and 2 females - 12.5 mg/kg) were administered amitrole orally by gelatin capsule six days a week for 52 weeks. One male and one female per group were sacrificed at 26 weeks. There was no mortality over the testing interval. Growth, appearance, and behaviour were normal in all test animals. Results of biochemical, haematological, and urological examinations were normal. Gross and microscopic examination of tissues and organs showed no evidence of abnormality associated with amitrole. There was no apparent evidence of cytotoxic effects associated with the administration of 12.5 mg amitrole kg bw/day for one year (Weir, 1958; Dardin, 1958). Long term studies Dermal Rat Two groups of rats (25 males and 25 females per group) were administered amitrole dermally at a level of 2.4 mg/kg weekly for 23 months. The application was allowed to contact the skin for thirty minutes, after which the skin was rinsed and dried. Amitrole was found to be non-irritating to the skin. There was no apparent effect of amitrole on growth, behaviour, tumour formation or when tissues and organs were observed on gross and microscopic examination. Statistical studies with the liver and thyroid organ weight and organ-to-body weight ratio did not reveal any differences in this experiment between treated animals and controls (Rausina et al., 1972). Inhalation Rat Groups of rats (25 males and 25 females per group) were exposed to amitrole by inhalation for a one hour period every week for two years. The animals were exposed to an aerosol of 0.2% (w/v) aqueous solution by a head-only exposure to minimize oral ingestion. The average analytical concentration of aerosol in the exposure chamber was 2 mg/l air. There were no significant differences between the treated and the control groups when examined with respect to mortality, behaviour, growth, gross and microscopic examination of thyroid and liver, or the incidence of tumor formation. No abnormal effects were noted in this inhalation exposure (Grapenthien et al., 1972). Feeding Rat Groups of rats (35 males and 35 females per group) were fed amitrole in the diet for two years at levels of 0, 10, 50, 100 and 500 ppm. Animals were sacrificed periodically over the course of the experiment. The appearance of animals (especially females) fed 100 ppm in the diet was altered. The incidence of protruding eyes (exophthalmia) was considerably higher in the higher dose groups than in the controls. Growth was reduced at 500 ppm. There was no apparent effect of amitrole in the diet on survival, growth, or mortality in the animals fed 50 ppm. Gross examination of animals revealed a consistent finding of thyroid enlargement. At 13 weeks, thyroid enlargement in males and females was observed at 100 ppm. Microscopic examination suggested hypofunction and a non-functional hyperplasia evident at 50 ppm and above. The animals fed 500 ppm were removed from the diet at 19 weeks and examined after two weeks on control diets. No evidence of enlargement or hyperplasia was noted. At 26 weeks, thyroid enlargement was evident at 50 ppm. Liver and kidney weight in both sexes was decreased at 100 ppm. No effects were noted at 10 ppm. At 52 weeks, growth of males was depressed at 100 ppm. At 68 weeks, growth of males was again depressed at 100 ppm accompanied by enlargement of the thyroid, pituitary, and liver. A cystic adenomatous structure was observed at 100 ppm in the thyroid. At 50 ppm, thyroid hyperplasia and hypofunction were noted. At 10 ppm, thyroid hypofunction was observed in one male animal of the three males and three females examined. At 104 weeks, body weights were normal in all groups. At 100 ppm, liver and kidney weights were normal while the thyroid was enlarged and adenomas ranging from benign cysts to foetal malignant carcinoma were observed. At 50 ppm, the thyroid was not enlarged. Adenomas were observed in 3/16 rats. At 10 ppm, one rat showed an adenomatous nodule with cellular hyperplasia (other sections suggested slight enlargement while most control sections were normal). Thyroid enlargement was observed only at 100 ppm. A no-effect level was not observed in this study. Ten ppm was a minimal effect level (Keller, 1959; Dardin, 1959). OBSERVATIONS IN MAN A 39 year old woman ingested 20 mg/kg. This dose caused no signs of intoxication and within a few hours of ingestion the compound passed rapidly through the body and began to appear in concentrations up to 100 mg/100 ml in the urine (Geldmacher-von Mallinckrodt, 1970). Amitrole has been produced industrially since 1955 with no evidence of ill effects other than mild contact dermatitis to the occupationally exposed workers (Smagghe, 1974; Clyne, 1970). It was suggested that from 1955-60, sixteen employees were exposed for five to six months per year and from 1967 to 1970, nine employees were exposed for approximately ten months per year with no ill effects. No thyroid or liver tumors were observed "in excess of the general population". Oral administration of 100 mg amitrole to humans for treatment of overactive thyroids inhibited the 131I-intake of the thyroid for 24 hours in normal persons and in hyperthyreotics. A dose of 10 mg had only a very slight effect (Astwood, 1960). A preliminary study of Swedish railway workers exposed to amitrole showed two lung cancer cases. Although the number of subjects was small, a combination of amitrole with smoking (or other interacting substances) might have been responsible for the excess lung cancer (Axelson et al., 1964). COMMENTS Amitrole is rapidly absorbed and eliminated primarily in urine. It has an effect on a variety of biochemical systems including catalase, peroxidase and certain enzymes associated with oxidative metabolism. Amitrole is goitrogenic on continuous long-term exposure; probably as a result of continuous inhibitions of peroxidase activity. At high levels of exposure, antithyroid effects of amitrole have been seen within three days. In adult female rats fed amitrole in the diet, no effects on reproduction were noted below 500 ppm although goitre was observed. At 500 ppm, reduced fecundity and a reduced lactation index was observed but no malformation of pups was observed. Oral administration to rats from days 7 to 15 of pregnancy resulted in no effect on reproduction and no abnormal offspring. Amitrole, in the presence of dimethyl sulfoxide, injected into chicken eggs produced beak abnormalities and tibial malformations. Results of mutagenicity tests using currently defined protocols were negative. In two long-term studies, hepatomas have been produced in mice and rats administered amitrole at exceptionally high levels. However, a long-term feeding study at high dietary levels in rats did not result in hepatomas. In a one-year dog study, no hepatic, goitrogenic or other effects were noted at a dietary level of 12.5 mg/kg bw. The no-effect level was based on a short-term study where normal PBI values, a sensitive biochemical parameter of thyroid function, were noted at 0.5 ppm. In addition, since no goitrogenic effect on discontinuous exposure was observed, a conditional ADI was allocated. The Meeting was reassured that in the use of amitrole, man has only a remote, if any, chance of achieving the conditions where continuous exposure is maintained. The Meeting emphasized that the ADI was allocated with the condition that the uses of amitrole be restricted to those where food residues would be unlikely to occur and further to recommend that the use of materials in combination in the same formulation be restricted, especially where effects on specific target organs are expressed by both materials. TOXICOLOGICAL EVALUATION Level causing no toxicological effect Rat: 0.5 ppm in the diet, equivalent to 0.025 mg/kg bw. Dog: 12.5 mg/kg bw. ESTIMATE OF CONDITIONAL ACCEPTABLE DAILY INTAKE FOR MAN 0.00003 mg/kg bw. RESIDUES IN FOOD AND THEIR EVALUATION USE PATTERN Amitrole is a broad spectrum herbicide effective against a wide range of grasses and broad-leafed weeds when applied as a foliar spray. It was introduced as a herbicide in 1954. The major herbicidal uses are on industrial land, roadsides, rights of way, railways, forests, irrigation channels and other ditches, either used alone or in admixture with other herbicides or as a combination with ammonium thiocyanate. There are numerous important uses on crop land and these are summarized in Table 2. Amitrole is not selective and therefore all applications in the vicinity of crop plants must be made in such a way that growing parts of the plant are avoided. Because of its quick action but relatively poor residual effect amitrole is widely combined with triazine, substituted urea and uracil herbicides to widen their spectrum of activity and to enhance the knock down effect. Amitrole is translocated within many plants and is thereby effective against rhizomatous and stoloniferous grasses and bulbous plants. Once translocated to the root systems of such plants amitrole appears to remain effective until the next growing season or at least its effect is observed in the regrowth, possibly owing to the destruction of certain essential growth factors. In order to evaluate properly the possibility of residues of amitrole in raw agricultural commodities and foods it is important to understand the mode of use. The following is an outline of the application to crop land. Apples and pears In the spring, before fruit starts to form or after harvest, amitrole is applied to the floor of the orchard to control broadleaf weeds and grasses. The chemical treatment is a replacement for mechanical cultivations designed to maintain the area at the base of the trees free from all unwanted vegetation. Label directions state that application should be made to weeds and that spray should be kept off the trunk or foliage of trees. Actually, at the time applied, there may be blossoms on the tree but very little foliage. Because of this, very little transpiration is taking place and there is a minimum of fluid transport within the tree. Thus, any amitrole that does come into direct contact with the trunk or the roots would not be expected to move around in the tree in detectable amounts. Extensive data were available to the Meeting to judge the possible effect of accidental misapplication or treatment in mid-summer when fruit is on the tree and foliage is at a maximum. Grapes In many grape-growing areas where there is a winter rainfall, winter annual weeds are a serious cultural problem. These weeds sprout profusely with the first rains of the winter wet season and grow strongly during winter and early spring while adequate moisture is available. Several residual herbicides are effective for controlling these weeds provided they are applied before the weeds emerge. However, if application is made too late or if the amount of moisture is inadequate these materials are often ineffective and must be supplemented by a foliage-absorbed herbicide. During this period, the vines are completely dormant with no leaves or berries. The spray is directed to the weed foliage at the base of the grapevine with instructions to keep all chemical off the grape plant itself. When grapes are dormant, there is no transpiration of fluids within the grapevine. Therefore, no moisture is going into the vine and thus there is no vehicle for the amitrole. By the time the grape breaks dormancy and transpiration and water uptake are significant, amitrole apparently is degraded and not available for uptake. The residual herbicides suffer the disadvantage of being ineffective against some species. If these are left uncontrolled they soon colonize the whole area and interfere with cultural practices. Also there are numerous instances where the farmer has a perennial weed problem in his cropped fields. This can either be an entire field or a large patch within a field or merely scattered plants. The use of amitrole alone or in combination with other herbicides as a spot treatment is recognized as an important agricultural practice essential to the maintenance of the productivity of perennial cultures such as vineyards. TABLE 3. Pattern of use of amitrol based herbicides Rate Use kg/ha When applied Remarks Chemical fallow 1 Autumn/winter Apply after weeds emerge. Cropland 4-8 After harvest Do not plant crops or cutting. or graze for 8 months. Grapes 2 When vines are Directed spray. dormant. Orchards (apple 2 Before fruit Do not spray and pear) forms/after foliage. picking. Corn 2 10-14 days Spray weeds 10-14 pre-planting. days before ploughing. Irrigation drains 2-8 When weeds Do not allow and ditches 15 cm high. grazing. Orchards/vineyards 2-8 When weeds are Spot treatment. (perennial weeds) actively growing. Non-crop areas 1-8 When weeds are Boom sprays- actively growing. spot sprays. Aerial Not to be made application where spray or drift might contaminate crops or potable water. Maize Selective herbicides have revolutionised the growing of maize and many other crops but there still remains a serious problem of perennial weeds such as thistles, Agropyron repens and convolvulus. In the spring amitrole is applied to growing weeds. After roughly 10-14 days, during which the chemical is allowed to penetrate the weed foliage and translocate to the actively growing meristematic tissue, the weedy field is ploughed in the normal fashion. That is, the weed foliage is buried roughly 15.25 cm in the ground and soil which was well under the surface is brought to the top. Emphasis is placed on burying all treated weeds. Chemical fallow In areas where a fallow period is maintained because conservation of all available moisture is essential to produce a satisfactory crop, amitrole has been used alone or mixed with 2,4-D to control annual weeds that sprout during the fallow period. If allowed to grow, these weeds remove much of the moisture reserves in the soil. Depending upon winter conditions, it may be desirable to spray in the autumn or wait until weed growth starts in the spring. This initial treatment is followed by one or more mechanical cultivations to maintain the area fallow until the next crop is planted. It may be necessary to repeat the herbicide application if fresh weeds emerge. Practically speaking, there is no need to apply it less than 30 days before planting cereal crops although the residue data show that application could be as late as several days before planting without producing detectable residues. There is no control whatsoever of weeds that germinate after application. Irrigation channels and ditch banks Amitrole has been used for many years in some countries where difficulty is found in maintaining irrigation channels and ditch banks free from encroaching vegetation, particularly against those weeds which grow prolifically in such situations and where a herbicide which is rapidly translocated but rapidly disappears from soil or water is required. In some countries there are restrictions on use near potable water or catchments. Pre-harvest treatments There are no approved uses for direct application to crop plants. Lack of selectivity precludes the possibility of such treatments. Other uses There are no known domestic or industrial uses which could subject the general public to exposure to amitrole in any form. RESIDUES RESULTING FROM SUPERVISED TRIALS A considerable number of studies have been carried out to determine whether, following the use of amitrole for the control of weeds, residues could possibly occur in raw agricultural commodities or food. Virtually without exception these studies indicate that the parent compound is not found in any food commodity even when grossly excessive rates are used or when the compound is applied at times and under conditions not recommended as good agricultural practice. Apples and pears Otten (1970) reported before the Amitrole Advisory Committee of the U.S. Environmental Protection Agency the results of many studies on apple and pear trees. The approved treatment is 2-4 kg/ha under trees in the spring before fruit forms or after harvest. In one series of trials amitrole was applied for five successive years at the rate of 4 kg/ha per year, or a total of 20 kg/ha during the 5 year period. This is 10 times the normal use rate over the 5 year period. The bottom 50 cm of the trunk was wet if necessary. No amitrole residues were detected in the fruit. Otten reported other tests in which amitrole was applied for 8 successive years using 4 or 20 kg/ha each year, a total of 160 kg of amitrole per hectare during the period. Apple samples were taken from trees with or without white shoots at the base (indicating amitrole in the leaves) and analysed separately. No amitrole was detected in any samples. The limit of determination of the method used is 0.01-0.02 mg/kg. In both series of tests amitrole was applied during the summer when the fruit was on the tree. Only when spray was applied directly to the fruit was a residue detected. Schubert (1965) carried out trials continuously over the period 1957-1964 in numerous orchards which received treatments of amitrole over ground cover, fruit and leaf. Even in those trials where amitrole was applied beneath trees right through the summer, mature fruit were free from amitrole at harvest. In the early years apparent amitrole residues were reported at levels in the range 0.05-0.09 ppm. Subsequently Storherr and Burke (1961) developed improved methods to overcome the extremely high absorbance backgrounds of most crops. Even where chlorotic shoots were present on the trunk of trees, fruit at harvest was found to have no residues (limit of determination 0.02 mg/kg). Substantial amitrole residues were found in mature apples where foliage and/or fruit were directly treated with amitrole in mid-summer. The residues from direct application to foliage were similar to quantities found after dipping the fruit. When both leaves and fruit were sprayed the residues were approximately doubled. The residues resulting from these direct applications during the growing season were in marked contrast to the results of a ground cover application that avoided spray contact with foliage or fruit, where no amitrole residues were found at harvest. Maier-Bode and Bechtel (1968) reported trials with apple trees carried out in Germany. When amitrole was applied at 2.5-4.2 times the officially recognized dosage (6 kg/ha), no amitrole was detected in the apples (residues of less than 0.01 ppm). Not until 50 or 100 kg/ha of amitrole was used, i.e. 8.3-16.7 times the recognized dosage, were small residues of amitrole, between 0.02 and 0.09 mg/kg found in the apples. In practice such high doses are never used; the foliage of the trees in these plots was distinctly chlorotic. Maier-Bode and Bechtel (1968) also found that when grossly excessive amounts of amitrole were applied to the soil, the amitrole residues in apples were much higher when there was no weed cover beneath the trees than when the herbicide was applied to densely weed-covered plots. This was presumably because the lack of weed cover to absorb and metabolize the amitrole allowed it to reach the root zone of the trees. Amchem (1965) reports show that apples, grown in orchards where the floor of the orchard was cleared of poison ivy and general broadleaf and grass weeds according to label instructions, show no residues at harvest. These studies extended over 8 locations and numerous varieties of apples. The methods of analysis used showed 96± 15% recovery at the 0.1 mg/kg level with a limit of determination of 0.01 mg/kg. Only when partially grown apples hanging on the trees were sprayed to run-off with an amitrole spray mixture, could residues of amitrole be found in the mature apples. Moore (1968, 1969, 1970) reports three series of trials at various locations in Australia in which amitrole was applied beneath apple trees at varying rates between 2 and 4 kg/ha, at petal fall or in mid-summer. To obtain the most severe conditions possible, weed-free plots beneath trees were also sprayed in mid-summer. To ensure the most complete uptake of herbicide by the tree an area of 5 metres × 5 metres was treated instead of the usual 3 metres × 3 metres. The author concludes that if any residues were present they were below the analytical limit of determination of 0.01 ppm. There was no build-up of residues in the second year of treatment. Bayer (1973) reports studies carried out in Germany which showed that approved uses of mixed herbicides containing amitrole applied for the control of weeds in apple orchards did not produce detectable residues of amitrole in mature apples (limit of determination 0.05 ppm). The Netherlands authorities have submitted results of studies carried out to determine amitrole residues in some fruits following approved uses in that country. Pears and apples harvested in 1961 and 1962 following treatment of orchards for weed control 4-18 months previously showed no indication of residues when analysed by the method of Storherr and Burke (1961) having a limit of determination of 0.025 mg/kg (Wit and Van der Kamp, 1963). Cherries Maier-Bode and Bechtel (1968) report a number of experiments which show that when amitrole is applied for weed control beneath cherry trees 100 days before harvest no residues can be detected in mature fruit by methods capable of determining 0.01 ppm. Bayer reports experiments where amitrole was applied for weed control in sour cherry orchards at the rate of 4 kg/ha. No residues could be detected by methods sensitive to 0.05 ppm. Citrus An extensive study of the accumulation and depletion of amitrole residues in citrus fruit and foliage was published by Day and Hendrixson (1959) from California. Amitrole was usually applied to the soil at rates of 2 and 4 kg/ha; in two instances a logarithmic series of applications were made from 2-64 kg/ha. Treatments were replicated 4 times in each of 13 different orchards in various parts of the citrus growing area. Treatments were applied during the winter months, usually on dry soil surfaces, and orchards were irrigated during the period of observation. The analytical method used was effective down to 0.01 mg/kg. Several varieties of oranges and lemons were included and samples were taken each month for the four months following application. In spite of obvious symptoms of damage to the lower skirt of the citrus trees most samples from the 2 and 4 kg/ha experiments showed no detectable residues although a few were found to have residues up to 0.05 mg/kg. In the logarithmic plots the level of residues increased with increasing concentration of amitrole applied. Those plots receiving 64 kg/ha showed 0.15 mg/kg in the whole oranges 4 months after application. Less was found after shorter intervals. The authors concluded that citrus fruit from trees receiving either 2 or 4 kg/ha contained residues only rarely. The residue level found in citrus fruit does not appear to be affected by fruit variety, soil type, climate, geographical area, cultural operations or rate of application to the soil (except at grossly exaggerated rates). Albinism appears only on the lower skirt area indicating foliar contact rather than root uptake as the source of entry. Amitrole is not re-distributed beyond the immediate area of contact when the herbicide is applied at recommended rates. Field observations indicate general systemic distribution at high rates of application. Coffee beans Information was available from only one trial carried out in Hawaii. Hylin (1962) reports results of analysis of coffee beans from trees which had been treated twice within 6 weeks with amitrole, applied at the rate of 2 and 4 kg/ha to substantially bare ground. A total of 40 trees were involved in the trial. Great difficulty was apparently encountered in developing a suitable analytical procedure to deal with the coffee beans. Apparent amitrole residues were reported to occur in the green beans at levels ranging from 0.02 - 0.48 mg/kg. In view of the analytical difficulties reported and the experience of other workers, however, caution should be exercised in interpreting the significance of these results. Cranberries Following the disclosure by Fleming (1959) that the practical use of amitrole for the control of weeds in cranberry bogs led to significant residues of amitrole in cranberries, the U.S. Food and Drug Administration and the U.S. Department of Agriculture undertook field and laboratory studies extending over 19 months (Onley et al., 1963). Following the application of 4 and 8 kg of amitrole per ha of cranberry bog the level of amitrole was determined in samples of cranberry vines, soil, roots and fruits (when available). Results indicated that though the residues in the soil declined steadily and disappeared at the end of 12 months there was a distinct concentrating effect in the roots and a pronounced effect in bushes where the residues declined more slowly than in the soil. Cranberry fruit harvested 3 and 12 months after application showed residues ranging up to 0.4 mg/kg. The authors were unable to conclude whether the residue represented the parent compound or metabolites. Grapes Studies using radio-labelled amitrole (Leonard and Weaver 1961) showed that when amitrole was applied to the leaves, stem, clusters or shoots of the grapevine a modest upward translocation occurred for about 3 days. After this period the amitrole appears to be either complexed or broken down. Amitrole was not recoverable, as such, from the clusters except within 3 days after treatment. Leonard and Lider (1961) studied the translocation of amitrole and a number of other herbicides in the grapevine. Grape rootings were allowed to absorb solutions of herbicide for 3 days before being planted in pots. The distribution and fate of the herbicide was studied by radio-autographs. There was very little indication of translocation throughout the plant. Fruit from vines grown in amitrole-treated soil under greenhouse conditions showed no positive evidence of amitrole. Trials with grapes in Germany involved the application of a maximum of 4 kg/ha of amitrole weeds in vineyards, during both the dormant and vegetative stages (Maier-Bode and Bechtel 1968). No trace of amitrole was found in grapes when using a method capable of detecting 0.01. Similar results are reported by Bayer (1973) from trials involving the application of 4 kg of amitrole per ha of vineyard. Maize Otten (1970) reports results obtained by both chemical analysis and radio-labelling which show that no amitrole was detected in either immature maize plants or at normal harvest time when amitrole was applied to weeds 10 days before ploughing for the planting of maize seed. When exaggerated rates were applied or maize was planted 1 or 2 days after spraying, amitrole was detected in the seedling plant but had disappeared well before the crop would be used for silage or mature grain. In a statement before the Amitrole Committee, Amchem (1970) summarized investigations carried out by a number of official research workers investigating the possibility of amitrole residues finding their way into corn plants and grain. Ercegovich (1957) analysed corn from plots treated with amitrole at 0, 1, 2, 4 and 8 kg/ha and planted 1, 5, 9 and 13 days later. Harvests were made at 3, 4, 6, 10 and 16 weeks after planting. No residues of amitrole were found in any samples. Boyd applied radio-labelled amitrole directly to young corn plants and followed the rapid decrease in residues over a 47-day period. Initial residues of 4-10 mg/kg decreased to final levels of 0.1 - 0.2 mg/kg. Nuts Studies carried out in California involved the application of amitrole to the orchard floor at rates of 1, 4 and 8 kg/ha. Application was made during the winter months. Mature nuts collected the following season were analysed for residues of amitrole. None of the many samples examined was found to contain amitrole residues above the limit of determination (0.02 mg/kg) (Hill 1962-63). Peaches and plums The Meeting had available numerous reports on residue trials carried out in peach and plum orchards where amitrole was used for weed control. None of the many samples analysed by methods capable of determining as little as 0.02 mg/kg showed any indication of amitrole residues (Hill 1962/63; Maier-Bode and Bechtel 1968; Bayer 1973). Soybeans The Meeting examined summaries of many separate trials carried out in different areas of the USA and analysed by three separate laboratories to determine the level and fate of amitrole in soybean plants, pods and mature beans following different patterns of using amitrole for weed control. In all cases samples were from treatments at rates of 1´ - 2 times the maximum recommended rate. In no instances were the residues, even in immature plants, found to be above the limit of determination, 0.06 mg/kg (Amchem, 1969). Montgomery and Freed (1963), wishing to know whether the proposed Pre-plant use of amitrole could possible result in residues in soybeans, studied various methods of extraction and analysis. They reported that by each procedure samples from fields treated with up to 4 times the recommended rate of amitrole gave exactly the same results as samples from untreated control fields. They applied a statistical analysis to the extensive data and concluded that there was no reason to suspect that any residues could occur following pre-plant application. Further evidence of freedom from residues was provided by failure to demonstrate any difference between the absorption spectra of paired samples from control and treated plots when using a double-beam spectrophotometer to compare them. Sugar cane Amitrole has been found to be useful for the control of certain perennial grass and broad leaf weeds common in sugar cane fields and on irrigation ditch banks. Application of directed sprays in sugar cane produced a moderate amount of chlorosis which persisted for 3-4 weeks in the leaves without appearing to affect yields or subsequent growth. Hilton et al. (1963) carried out a residue study designed to determine the residual amitrole in sugar cane from several successive applications made over the period of the crop cycle (2 years). Hilton and Uyehara (1966) reporting on these studies noted that when sugar cane was double treated with amitrole at 5, 10 or 20 kg/ha, 12 and 20 weeks after planting, the residues of amitrole diminished to less than 0.002 mg/kg by harvest time. When 10 times as much cane was taken for analysis and treated in a manner to approximate the early raw sugar processing, residues, if present, were less than 0.01 mg/kg even from plots receiving the grossly exaggerated rate of 20 kg/ha. Wheat and oats Amitrole proved to be an effective means of controlling couch grass (Agropyron repens) in cereal crops. Most proposed treatments involved the application of amitrole to the grass 10-14 days before ploughing in preparation for seeding with cereals. In Sweden the procedure was to apply amitrole at the 3 leaf stage at a rate of 0.5 to 1 kg/ha. Svensson (1971) investigated the effect of such treatments on oats and the fate of residues in the oat plants and grain. He reports that when the treatment was carried out strictly in accordance with approved directions no detectable residues could be found in the grain. However when the rate was increased or application withheld for 2 weeks until the 5-leaf stage of the crop, the grain contained about 0.05 mg/kg. If the rate of application is increased to 2 kg/ha the residue can exceed 0.1 ppm. Determinations were made on straw from a number of trials but analytical difficulties rendered the results somewhat unreliable. However it appears that application of amitrole at any stage after the 3-leaf stage increases the likelihood that residues will be found in the straw at harvest. Much higher residues were found in the grain of oats treated with amitrole at the heading and milk stages. 0.5 kg/ha applied at the milk stage gave rise to a residue in the grain of 2.9 mg/kg. Amchem (1970) provided a summary of a number of studies by official workers in the USA who investigated the incidence, level and fate of residues of amitrole in wheat grain and straw from crops grown on soil treated according to label directions 14-28 days before ploughing for planting. In some of these studies the application rate was increased to 4 times the recommended rate and the time interval was reduced to 1 day before planting. In no instance was any amitrole residue found in cereal grains or straw at harvest time. Soil studies on the same test plots showed detectable residues of amitrole on the day of treatment but no detectable residues the day after treatment even at the doubly exaggerated rate. The limits of detection were 0.02 ppm in grain and straw. Recoveries were 86-96% at levels of 0.2-0.4 ppm. On the basis of these data it was concluded that the use of up to 4 kg/ha of amitrole, one or more weeks before planting wheat or other cereals, should cause no residues in the crop. FATE OF RESIDUES General Kröller (1966) and Menzie (1969) have reviewed the metabolism of amitrole in animals, plants, micro-organisms, etc. There is an extensive literature on the mode of action of amitrole and on its metabolism in plants. Many of these investigations were carried out with the object of determining uptake, translocation, site of action, reason for selectivity, and possible means of enhancing the activity. Among the most useful publications are those of Boyd (1964/65), Carter (1969), Herrett and Linck (1961a), Jukes (1961), Moser (1968), and Rogers (1957a,b). Many of these studies reveal that amitrole does not persist for extended periods in plant tissues though there is considerable disagreement as to whether it becomes conjugated, converted into a more active material or destroyed and its fragments taken up into the plant. There appears to be no clear-cut mode of action, but rather multiple pathways by which it interferes with plant metabolism. Carter (1969) reviewed the available literature on the effects of amitrole on amino acid and protein metabolism, purine metabolism, flavin synthesis, enzyme activity, chlorophyll synthesis and plastid development. It is obvious that there is no single site of action but the relative importance of the various effects has apparently not been unravelled. In animals The fate of amitrole in rats is discussed in the section "Biotransformation". There do not appear to be any grounds for assuming that livestock grazing on plant materials growing on land that had been treated with amitrole for the control of weeds would absorb or retain significant amounts of amitrole or its metabolites. In plants The s-triazole nucleus is highly stable (Potts 1961); hence it is not surprising that few workers have reported evidence of ring cleavage under physiological conditions. Yost and Williams (1958) reported disappearance of radio-labelled amitrole from corn plants in approximately 6 weeks with a half-life of about 8 days. Disappearance was also observed in soybeans but at a much slower rate. Miller and Hall (1961) could not detect amitrole in cotton 4 days after treatment. However, large quantities of metabolic products were present. Fang et al. (1967) report the half-life of amitrole in several plants as 18 to 28 hours. Little of the applied material was recovered as CO2 from beet, corn or beans. Freed et al. (1961) reported evolution of radio-active, CO2 from treated oats and barley, indicating ring cleavage. Montgomery and Freed (1963) observed some evolution of 14CO2 from treated soybeans, with the remaining radioactivity apparently bound to components of plant tissues. Resistant oats released CO2 more readily than sensitive barley. Massini (1963) found no loss of CO2 from beans or tomatoes. Muzik (1965) observed chlorosis in scions grafted onto tomato plants 103 days after treatment with amitrole, indicating long persistence of the toxic moiety. Studies of amitrole degradation in plants have been complicated by the fact that the material is available commercially only with the 5-carbon labelled. Apparently, the 5-carbon is quickly lost (as CO2 or formate) when ring rupture occurs and the remaining fragments are thus unlabelled. However, if significant amounts of CO2 or 14C-formate were produced from 14C-amitrole in higher plants, one would expect to find some incorporation of 14C into normal metabolites. This is not the case. The vast majority of the literature indicates that the extractable 14C from plants treated with amitrole-5-14C remains in the intact ring as free amitrole or conjugates. Considerable amounts of amitrole are attached to protein (Brown and Carter 1968; Castelfranco and Brown 1963) or somehow bound in an insoluble form (Racusen 1958). There is evidence of activation to a free radical form which can react with amino acids (Carter and Naylor 1960, 1961a, b; Herrett and Bagley 1964; Herrett and Linck 1961a; Miller and Hall 1961). Conclusive evidence of rapid and extensive ring cleavage by higher plants has not been reported. Most literature on metabolic alteration of amitrole in plants deals with the formation and properties of conjugates between amitrole and endogenous plant constituents. These "degradation" products contain the intact triazole nucleus which often may be regenerated by chemical treatment. Rogers (1957a,b) reported a derivative of amitrole in several plants which was "chromatographically identical" with an amine-glucoside derivative of amitrole. The glucose derivative forms quite readily in vitro (Fredrick and Gentile 1960) and its occurrence in plant extracts is probably an artifact (Naylor 1964), since numerous attempts by other workers to detect the compound in plant extracts failed (Miller and Hall 1961; Massini 1963; Carter and Naylor 1959). However, Gentile and Fredrick (1959) and Frederick and Gentile (1960, 1969, 1962 and 1965) have published a series of studies on the properties and metabolism of the glucose derivative. These same authors suggest that the triose derivative represents the true structure of the amitrole derivatives reported by other workers. Massini (1963) and Carter and Naylor (1960, 1961a) reported studies of amitrole metabolism in which Massini identified one of the major metabolites as 3-(3-amino-1,2,4-triazole-1-yl)-2-amino propionic acid (3-ATAL), also referred to as 3-(3-amino-1,2,4-triazol-1-yl)-d-alanine. The formation of 3-ATAL apparently represents a detoxication, since the derivative does not appear to be nearly as toxic as amitrole, or as mobile. Furthermore ammonium thiocyanate, which synergizes the action of amitrole, inhibits the formation of 3-ATAL. Smith and Chang (1973) studied the metabolism of amitrole in Canada thistle (Cirsium arvense) and peas. They showed that excised leaves of thistles metabolized amitrole into three major products one of which was 3-ATAL. The other two were shown to be its metabolic products, one being the precursor of the other. An enzyme preparation from peas capable of synthesizing tryptophan was also able to metabolize amitrole. Tryptophan synthesis with the enzyme preparation was inhibited by amitrole and the authors deduced that amitrole metabolism may follow a similar pathway to tryptophan synthesis. A number of workers including Racusen (1958) reported another metabolite which was stable to 6N HCI for 5 hours at 100°C. Verification of the structure of this material and others awaited further studies. The most important outstanding question is whether the conjugates and/or metabolites represent biologically active derivatives of amitrole and if so whether these are available to react physiologically and biochemically with animals receiving such conjugates and metabolites in plant materials as part of their ration. However, use patterns at present approved avoid the possibility that significant residues are ingested by livestock or man. In soil Amitrole disappears rapidly from soils as shown by Sund (1956), Bondarenko (1958), Ashton (1962), Riepma (1962), and Ercegovich and Frear (1964). Disappearance has been attributed to absorption (Sund, 1956, Ercegovich and Frear, 1964) and microbial degradation, although attempts to isolate organisms capable of degrading amitrole have not been successful. Kretschmar (1970) showed that amitrole was not degraded by Azotobacter. Kaufmann (1965), Kaufman and co-workers (1968) and Plimmer et al. (1967), have proposed that most of the amitrole degradation occurring in soils proceeds by non-biological reactions. They showed that approximately 69% of the radio-label from amitrole was released as CO2 in 20 days by non-sterilized soil. Although autoclaved soil released only 2% in a comparable period, soil treated with potassium azide or ethylene oxide released 46 and 35% respectively, and reinoculation of autoclaved soil did not restore the capacity to metabolize amitrole. These authors propose that amitrole is degraded in soil by an oxidative mechanism involving an attack on the triazole nucleus. Microbial attack is not discounted however. Soil moisture, temperature and pH markedly affected amitrole degradation (Riepma 1962), indicating possible microbial involvement. The studies by Ercegovich and Frear (1964) show that amitrole degradation obeys first-order kinetics, suggesting a chemical reaction. No-one has reported an investigation of the possible involvement of extra-cellular enzymes. Whatever the mechanisms by which the triazole ring is opened, there appears to be little doubt that ring opening does occur rapidly in soils and the resulting products (urea, cyanamide and nitrogen) should be readily metabolized by soil micro-organisms. Norris (1970) showed that amitrole was rapidly lost from the forest floor but that degradation was not completely biological, since there was considerable loss in steam-sterilized material. He found that ammonium thiocyanate applied with amitrole in the proportions found in the commercial herbicide had no effect on the degradation of amitrole. Sund (1956) showed that amitrole becomes tightly adsorbed to soil particles. He concluded that it takes part in the soil's base exchange system, but also it has the tendency to complex metals. Studies of amitrole toxicity to tomato seedlings were correlated with chemical analysis and it was found that the biological response of plants is proportional to the amount recoverable in any soil type. MacRae and Alexander (1965) are in agreement with the results of Ashton (1963) that microflora have an important function in the degradation of amitrole. They confirmed the rapid biodegradation in soil by plant bioassay. Ercegovich and Frear obtained evidence of complex formation between soil clay and amitrole by means of X-ray diffraction measurements. Ashton (1963) noted that 13 compounds were formed in addition to CO2 when amitrole was added to unsterile soil. One or more metabolic compounds formed from amitrole are tenaciously bound to the soil and appear to be resistant to degradation. Day et al. (1960) studied the effect of soil type, temperature, moisture and sterilization by steam on the fate of amitrole in 55 types of Californian citrus soil. Rates of decomposition in steam-sterilized soils were much lower than in unsterilized soil, apparently indicating that the decomposition is primarily due to the action of soil microorganisms (but compare the results of Kaufman, Plimmer and co-workers quoted above). Rates of decomposition of amitrole were highly variable among the soils studied, apparently because of differences in populations or levels of activity of the soil micro-organisms concerned. A short residual life for amitrole was more frequently found in the more highly evolved soils having finer textures and more highly developed colloidal properties. Freed and Furtick (1961) showed by simultaneous bioassay and chemical analyses capable of detecting less than 0.05 mg/kg of amitrole that it would be highly improbable that any residue of amitrole would remain in the soil even a short while after application. It has generally been assumed that failure to recover amitrole from soils following its use as a herbicide was due to elimination by such processes as leaching, volatalization, or biological or chemical destruction. Work by Groves and Chough (1971) demonstrated that new solvent mixtures gave much better recoveries of amitrole from soil than water extraction. Concentrated ammonium hydroxide/glycol mixture (5/20) gave distinctly higher recoveries than water as indicated by the following table. TABLE 4. Extraction (%) of amitrole from soils with water and ammonium hydroxide/glycol (A/G) mixture (5x20) Time after Non-sterile soil Sterile soil application (days) Water A/G Water A/G % % % % 0 48.1 1 36.7 97.6 49.6 97.3 12 21.3 55.9 36.1 77.9 17 3.2 15.2 36.9 67.7 These results point to a need for caution in interpreting some of the earlier results. In water Since amitrole is widely used for the control of ditch bank weeds many studies have been carried out to determine the fate of any amitrole reaching the water. Many of these studies (Segal 1960; Marston et al. 1968; Frank 1969; Dunstar 1969; and Demint et al. 1970) have largely involved the study of the rate of dissipation of amitrole in irrigation water or flowing streams. Since only a small proportion of the herbicide applied in such operations ever reaches the water, dissipation and dilution is a recognized means of reducing any risk to crops and municipal users. Where application has been made by aerial spraying, measurable amounts were found in samples of water near the downstream edge of the sprayed area for up to 5 days after spraying (Marston et al. 1968). Such studies are however of little value in determining whether residues are lost by physical, chemical or biological means. Studies were reported by Thoman (1963) from Florida where ponds with static water levels were treated at the rate of 3 kg/ha of water surface, giving a concentration of 0.45 mg/l in the water immediately after treatment. Samples collected every 7 days thereafter were analysed. The residue level declined slowly but 46 days elapsed before it reached the limit of determination (0.02 mg/l). Samples taken thereafter until the 74th day gave results comparable to pre-treatment levels. Nicholson (1963) reported experiments carried out in a fish hatchery in South Carolina where ponds of 1 acre surface area were treated with amitrole to yield a concentration of 1 mg/l in 80,000 cu.ft of pond water. Samples were taken each 7 days for 5 weeks and thereafter at slightly greater intervals until the 201st day when the pond became flooded. The concentration following treatment (1.16 mg/l) gradually declined through 0.49 mg/l on the 68th day to 0.07 mg/l on day 201. No information is given on the quality of the water in the ponds but it is obvious that under these conditions amitrole remains relatively stable for long periods. In processing and cooking No information was available on the effect of processing and cooking on residues in plant materials. In view of the fact that none of the recommended use patterns give rise to detectable residues in raw agricultural commodities such studies are unlikely to have been carried out. RESIDUES IN FOOD IN COMMERCE OR AT CONSUMPTION Food moving in commerce No data were available to the Meeting to indicate the level and incidence of amitrole residues in food moving in commerce. Food at the time of consumption Results of several total diet studies in which an attempt was made to find amitrole residues in many separate food groups were available to the Meeting. Duggan et al. (1966) reported that amitrole residues were not found in any food composites in the total diet study carried out in the USA in 1964/65; the method used had a limit of determination of 0.05 mg/kg. The same authors (1967), having repeated the total diet study, reported that amitrole residues were not found at or above the prescribed sensitivity limit. Corneliussen (1969, 1970), reporting the results of the 4th and 5th total diet studies carried out in the USA, states "amitrole has never been found in any total diet composites". The limit of determination was again 0.05 mg/kg. METHODS OF RESIDUE ANALYSIS One of the first methods for the determination of amitrole residues was that of Sund (1956). This was a colorimetric method applicable to soils, depending upon the formation of a green colour with sodium nitroprusside reagent. It is sensitive to 1 mg/l of amitrole in aqueous solutions and Sund reported that it conformed to Beer's law. Bioassay with tomato seedlings correlated with chemical analysis. American Cyanamid (1958) published a colorimetric method for the estimation of residues of amitrole in cranberry fruit. The extract is purified by means of a cation exchange resin, treated with nitrous acid and coupled with N-1-naphthyl-ethylenediamine dihydrochloride to produce a pink-coloured solution. The intensity of the colour is proportional to the amitrole concentration and is measured spectrophotometrically at 512 nm. The limit of determination is 0.03 mg/kg. Herrett and Linck (1961b) published what they describe as a simple reproducible method for the separation and quantitative determination of amitrole in biological systems. The method consists of diazotization followed by coupling with 1-amino-8-naphthol-3,6-disulfonic acid (H-acid). The authors describe the method as being sensitive in the range of 0.1-3.3 mg/kg. Storherr and Burke (1961) in describing an improved method for the determination of amitrole in crops refer to previous methods published by the Food and Drug Administration which were applicable to only a limited number of food crops. With their modifications the method involves extraction of vegetable crops with ethanol, adsorption on cation-exchange resin and desorption with aqueous ammonia. The ammoniacal concentrate is subjected to a clean-up procedure using acetonitrile and filter aid followed by acid digestion and clean-up with activated carbon. The resulting solution is diazotized and coupled with H-acid. The pink colour is measured at 455 nm. It is necessary to carry out a blank determination. The apparent amitrole found in control crops ranged from 0.003 to 0.02 mg/kg. The limit of determination is considered to be 0.025 ppm using a 40 g sample. Storherr and Onley (1962) later published a procedure for the clean-up and separation of amitrole and its metabolites from vegetable crops. This method utilizes a chromatrographic column packed with dry cellulose for the removal of interfering substances from plant extracts. It is reported to recover amitrole from some conjugates. Hilton et al. (1963) modified the method of Storherr and Burke, (1961) for use with sugar cane and sugar juice and were able to lower the limit of determination to 0.002 mg/kg. Their modifications involved an increase in the size of sample taken and a reduction in the amount of activated carbon used in the acid digestion and cleanup steps. Segal (1960) and Hilton and Uyehara (1966) described a number of minor modifications to the method of Storherr and Burke which lowered the limit of determination of amitrole to 0.001 mg/kg in water and sugar cane respectively. Meissner (1971), on behalf of the Amitrole Advisory Committee to the US Environmental Protection Agency, commenting on the claim that the method of analysis developed by Storherr and Burke (1961) determines not only amitrole but all known metabolites drew attention to the lack of detailed information available on the metabolites of amitrole in plants or on the influence of environmental and chemical factors on plant metabolism. Groves and Chough (1971) report an improved solvent mixture (concentrated ammonium hydroxide and ethylene glycol) to extract amitrole from soil. Further details are given above (see "Fate of residues in soil"). NATIONAL TOLERANCES The information available to the Meeting indicated that most countries with pesticide residue tolerance legislation either registered amitrole on a "no-residue" basis or provided a zero tolerance. It is assumed that zero would in effect mean "at or about the limit of determination". In the Netherlands there is a general tolerance of 0.02 mg/kg in fruit and vegetables. In Australia there is a maximum residue limit for amitrole in water of 0.01 mg/l. This is at or about the limit of determination. In the Federal Republic of Germany, use of amitrole where potable water might be contaminated is prohibited. APPRAISAL Amitrole is a non-selective, foliage-absorbed herbicide widely used since 1954 for the control of unwanted vegetation on industrial land, roads, railways, rights of way, ditch banks and similar situations. It is also used for the destruction of weeds beneath trees and vines in permanent horticultural crops, for spot treatment of perennial weeds and for their destruction prior to the planting of cereal crops. There are no known uses where application is made to crop plants. Extensive information indicates that amitrole is rapidly lost from soil by a combination of chemical, biological and microbiological attack. There is no indication that amitrole is taken up by the roots of plants if currently approved practices are followed. Approved uses involve the application of amitrole to weeds between the crop when fruit trees, vines and similar horticultural crops are dormant or the use of spot sprays and directed sprays which avoid contamination of crop plants. In these cases there is no uptake of residues by crop plants. Studies with radio-labelled amitrole confirm that it is not taken up from soil following simulated normal practices. Extensive residue trials have been carried out under many conditions in many countries and all indicate that there is no residue in fruit, vegetables or grain following the recommended use of amitrole as a herbicide. Residues have been found experimentally only when crop plants have been treated with excessive quantities of amitrole applied directly over the crop when the fruiting parts are already well formed. There appears to be no single mode of action and the metabolic pathways in plants appear most complex. There is evidence that when amitrole is applied to the leaves of plants, most of the material absorbed is metabolized. Some is complexed with various plant materials, but the Meeting considered that the nature and biological significance of conjugation products would only be of importance when considering residues resulting from direct application over food-crop plants. Where amitrole was administered to laboratory animals it was shown that similar conjugates with sugars and proteins were formed in the animal body. There was some doubt whether the methods of analysis for residues of the parent compound would adequately recover and determine all conjugated materials or other biologically active metabolites. The method of Storherr and Onley (1962) is the most sensitive and specific method available, and is reported by several authors to recover amitrole from some conjugates. Under the circumstances the Meeting felt confident in recommending a maximum residue limit at or about the limit of determination by the beet available method. The Meeting however proposed certain precautions to avoid contamination of food crops. RECOMMENDATIONS There is no reason to believe that significant residues occur in any raw agricultural commodities when amitrole is used for the control of weeds according to approved directions. To reduce the possibility of contaminating food crops with residues of amitrole, use-patterns should avoid the direct treatment of food crops and should be limited to directed sprays, spot sprays and, in the case of pre-planting and stubble treatments, application to weeds at least 10 days before ploughing (see Table 2), the interval to be conditioned by the temperature of the soil. For regulatory purposes and as a means of determining whether amitrole herbicides have been misused or incorrectly applied the Meeting recommends a maximum residue limit at or about the limit of determination by the best available analytical method. CONDITIONAL TOLERANCE mg/kg Raw agricultural commodities of plant origin 0.02* * at or about the limit of determination. FURTHER WORK OR INFORMATION DESIRABLE 1. Long term feeding studies in a sufficient number of rats and mice with low levels of amitrole of known composition and purity. 2. 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See Also: Toxicological Abbreviations Amitrole (EHC 158, 1994) Amitrole (HSG 85, 1994) Amitrole (ICSC) Amitrole (Pesticide residues in food: 1977 evaluations) Amitrole (Pesticide residues in food: 1993 evaluations Part II Toxicology) Amitrole (Pesticide residues in food: 1997 evaluations Part II Toxicological & Environmental) Amitrole (IARC Summary & Evaluation, Supplement7, 1987) Amitrole (IARC Summary & Evaluation, Volume 7, 1974) Amitrole (IARC Summary & Evaluation, Volume 41, 1986) Amitrole (IARC Summary & Evaluation, Volume 79, 2001)