AMITROLE       JMPR 1974


    Chemical name

         3-amino-1,2,4-triazole; 3-amino-S-triazole.


         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.




         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

         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.,

         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

         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).


    Special studies on carcinogenicity


         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).


         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


         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,

         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

    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


    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


         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.,

         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).


         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



         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).



         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.,



         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,


         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

         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).


         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.


    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.


         0.00003 mg/kg bw.



         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

         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.


         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


    Use                   kg/ha   When applied        Remarks

    Chemical fallow       1       Autumn/winter       Apply after weeds

    Cropland              4-8     After harvest       Do not plant crops
                                  or cutting.         or graze for 8

    Grapes                2       When vines are      Directed spray.

    Orchards (apple       2       Before fruit        Do not spray
    and pear)                     forms/after         foliage.

    Corn                  2       10-14 days          Spray weeds 10-14
                                  pre-planting.       days before

    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.


         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.


         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

         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).


         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.


         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

         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.


         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.


         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


         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

         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.


         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).


         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.



         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

    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

         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

         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

         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.


    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.


         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

         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

         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").


         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


         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


         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.


    Raw agricultural commodities of plant origin                0.02*

    * at or about the limit of determination.



    1. Long term feeding studies in a sufficient number of rats and mice
    with low levels of amitrole of known composition and purity.

    2. Studies to elucidate the possible relationship between the effects
    of amitrole on the thyroid and on the liver.

    3. Studies to show that the analytical methods determine not only the
    parent compound but also biologically active metabolites.

    4. Studies to develop a specific method sensitive to 0.005 mg/kg.


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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)