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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY



    ENVIRONMENTAL HEALTH CRITERIA 158





    AMITROLE







    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    Published under the joint sponsorship of
    the United Nations Environment Programme,
    the International Labour Organisation,
    and the World Health Organization

    First draft prepared by Dr P.J. Abbott,
    Department of Health, Housing and
    Community Services, Canberra, Australia

    World Health Orgnization
    Geneva, 1994


         The International Programme on Chemical Safety (IPCS) is a
    joint venture of the United Nations Environment Programme, the
    International Labour Organisation, and the World Health
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    disseminate evaluations of the effects of chemicals on human health
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    the development of epidemiological, experimental laboratory, and
    risk-assessment methods that could produce internationally
    comparable results, and the development of manpower in the field of
    toxicology. Other activities carried out by the IPCS include the
    development of know-how for coping with chemical accidents,
    coordination of laboratory testing and epidemiological studies, and
    promotion of research on the mechanisms of the biological action of
    chemicals.

    WHO Library Cataloguing in Publication Data

    Amitrole.

        (Environmental health criteria ; 158)

        1.Amitrole - standards  2.Environmental exposure
        3.Herbicides        I.Series

        ISBN 92 4 157158 6        (NLM Classification: WA 240)
        ISSN 0250-863X

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    letters.





    CONTENTS

    1. SUMMARY

         1.1. Identity, physical and chemical properties, and analytical
              methods
         1.2. Sources of human and environmental exposure
         1.3. Environmental transport, distribution and transformation
         1.4. Environmental levels and human exposure
         1.5. Kinetics and metabolism in laboratory animals and humans
         1.6. Effects on experimental animals and  in vitro systems
         1.7. Effects on humans
         1.8. Effects on other organisms in the laboratory and field

    2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES AND ANALYTICAL
         METHODS

         2.1. Identity
         2.2. Physical and chemical properties
         2.3. Conversion factors
         2.4. Analytical methods
              2.4.1. Plants
              2.4.2. Soil
              2.4.3. Water
              2.4.4. Formulations
              2.4.5. Air
              2.4.6. Urine

    3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

         3.1. Natural occurrence
         3.2. Anthropogenic sources
              3.2.1. Production levels and processes
              3.2.2. Uses

    4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

         4.1. Transport and distribution between media
              4.1.1. Air
              4.1.2. Water
              4.1.3. Soil
                        4.1.3.1   Adsorption
              4.1.4. Vegetation and wildlife
              4.1.5. Entry into food chain
         4.2. Biotransformation
              4.2.1. Biodegradation and abiotic degradation
                        4.2.1.1   Plants
                        4.2.1.2   Soils
              4.2.3. Bioaccumulation
         4.3. Ultimate fate following use

    5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

         5.1. Environmental levels
              5.1.1. Air
              5.1.2. Water
              5.1.3. Soil
         5.2. General population exposure
              5.2.1. Environmental sources
              5.2.2. Food
         5.3. Occupational exposure during manufacture, formulation
              or use

    6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS

         6.1. Absorption, distribution and excretion
              6.1.1. Mouse
              6.1.2. Rat
              6.1.3. Human
         6.2. Metabolic transformation

    7. EFFECTS ON EXPERIMENTAL ANIMALS  IN VITRO TEST SYSTEMS

         7.1. Single exposure
              7.1.1. Oral
              7.1.2. Other routes
         7.2. Short-term exposure
              7.2.1. Oral
                        7.2.1.1   Dietary
                        7.2.1.2   Drinking-water
                        7.2.1.3   Intubation
              7.2.2. Inhalational
              7.2.3. Intraperitoneal
         7.3. Long-term exposure
              7.3.1. Oral
                        7.3.1.1   Mouse
                        7.3.1.2   Rat
                        7.3.1.3   Other species
              7.3.2. Other routes
         7.4. Skin and eye irritation; skin sensitisation
         7.5. Reproduction, embryotoxicity and teratogenicity
              7.5.1. Reproduction
              7.5.2. Embryotoxicity and teratology
         7.6. Mutagenicity and related end-points
              7.6.1. DNA damage and repair
              7.6.2. Mutation
              7.6.3. Chromosome damage
              7.6.4. Cell transformation
              7.6.5. Other end-points

         7.7. Carcinogenicity
              7.7.1. Mouse
              7.7.2. Rats
              7.7.3. Other species
              7.7.4. Carcinogenicity of amitrole in combination
                        with other agents
         7.8. Other special studies
              7.8.1. Cataractogenic activity in rabbits
              7.8.2. Biochemical effects
         7.9. Mechanisms of toxicity - mode of action

    8. EFFECTS ON HUMANS

         8.1. General population exposure
         8.2. Occupational exposure

    9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

         9.1. Laboratory experiments
              9.1.1. Microorganisms
              9.1.2. Aquatic organisms
                        9.1.2.1   Plants
                        9.1.2.2   Invertebrates
                        9.1.2.3   Vertebrates
              9.1.3. Terrestrial organisms
                        9.1.3.1   Plants
                        9.1.3.2   Invertebrates
                        9.1.3.3   Birds
         9.2. Field observations
              9.2.1. Terrestrial organisms
                        9.2.1.1   Plants
                        9.2.1.2   Invertebrates

    10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE
         ENVIRONMENT

         10.1. Evaluation of human health risks
         10.2. Evaluation of effects on the environment

    11. CONCLUSIONS AND RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH
         AND THE ENVIRONMENT

         11.1. Conclusions
         11.2. Recommendations for protection of human health and the
              environment

    12. FURTHER RESEARCH

    13. PREVIOUS EVALUATIONS BY NATIONAL AND INTERNATIONAL BODIES

    REFERENCES

    RESUME

    RESUMEN
    
    WHO TASK GROUP FOR ENVIRONMENTAL HEALTH

    CRITERIA FOR AMITROLE

     Members

    Dr   P.J. Abbott, Chemicals Safety Unit, Department of Health,
         Housing and Community Services, Canberra, Australia
          (Rapporteur)

    Professor J.F. Borzelleca, School of Basic Health Sciences,  
         Department of Pharmacology, Richmond, Virginia, USAa

    Professor V. Burgat-Sacaze, Ecole Nationale Vtrinaire, Toulouse,
         France

    Dr   E.M. den Tonkellar, Toxicology Advisory Centre, National 
         Institute of Public Health and Environmental Protection, 
         Bilthoven, The Netherlands

    Dr   S. Dobson, Institute of Terrestrial Ecology, Monks Wood
         Station, Abbots Ripton, Huntingdon, Cambridgeshire, United
         Kingdom

    Dr   R. Fuchs, Department of Toxicology, Institute for Medical 
         Research and Occupational Health, University of Zagreb, Zagreb,
         Croatia

    Dr   D. Kanungo, Division of Medical Toxicology, Central
         Insecticides Laboratory, Department of Agriculture and
         Cooperation, Directorate of Plant Protection, Quarantine and
         Storage, Faridabad, Haryana, India

    Professor M. Kessabi-Mimoun, Institut Agronomique et Vtrinaire
         Hassan II, Rabat, Morocco

    Professor M. Lotti, Universit di Padova, Istituto di Medicina  del
         Lavoro, Padua, Italy  (Chairman)

    Professor A. Rico, Ecole Nationale Vtrinaire, Toulouse, France
          (Vice-Chairman)

     Observers

    Mr   C. Chelle, CFPI, Gennevilliers, France (GIFAP Representative)

    Dr   L. Diesing, Bayer AG, Institute of Toxicology and Agriculture,
         Wuppertal, Germany (GIFAP Representative)

             
    a Invited but unable to attend

    Dr   B. Krauskopf, Bayer AG, Leverkusen-Bayerwerk, Germany (GIFAP
         Representative)

    Dr   Rouaud, Agrochemicals Division, CFPI, Gennevilliers, France
         (GIFAP Representative)

     Secretariat

    Dr   D. McGregor, Unit of Carcinogen Identification and Evaluation,
         International Agency for Research on Cancer (IARC), Lyon,
         France

    Dr   R. Plestina, International Programme on Chemical Safety, World
         Health Organization, Geneva, Switzerland  (Secretary)

    NOTE TO READERS OF THE CRITERIA MONOGRAPHS

         Every effort has been made to present information in the
    criteria monographs as accurately as possible without unduly
    delaying their publication. In the interest of all users of the
    Environmental Health Criteria monographs, readers are kindly
    requested to communicate any errors that may have occurred to the
    Director of the International Programme on Chemical Safety, World
    Health Organization, Geneva, Switzerland, in order that they may be
    included in corrigenda.


                                  *   *   *


         A detailed data profile and a legal file can be obtained from
    the International Register of Potentially Toxic Chemicals, Case
    postale 356, 1219 Chtelaine, Geneva, Switzerland (Telephone No.
    9799111).


                                  *   *   *


         This publication was made possible by grant number 5 U01
    ES02617-14 from the National Institute of Environmental Health
    Sciences, National Institutes of Health, USA.

    ENVIRONMENTAL HEALTH CRITERIA FOR AMITROLE

         A WHO Task Group on Environmental Health Criteria for Amitrole
    met at the Ecole Nationale Vtrinaire, Toulouse, France, from 18 to
    22 May 1993, the meeting being sponsored by the Direction gnrale
    de la Sant, Ministre des Affaires sociales, de la Sant et de la
    Ville, Paris. Professor A. Rico welcomed the participants on behalf
    of the host institute. Dr R. Plestina, IPCS, opened the meeting and
    welcomed the participants on behalf of Dr M. Mercier, Director of
    the IPCS, and the three IPCS cooperating organizations
    (UNEP/ILO/WHO).

         The first draft was prepared by Dr P.J. Abbott, Department of
    Health, Housing and Community Services, Canberra, Australia.
    Extensive scientific comments were received following circulation of
    the first draft to the IPCS contact points for Environmental Health
    Criteria monographs and these comments were incorporated into the
    second draft by the Secretariat. The Group reviewed and revised the
    draft document and made an evaluation of the risks for human health
    from exposure to amitrole.

         Professor M. Lotti deserves special thanks for skilfully
    chairing the meeting and for assistance to the Secretariat in
    finalizing the monograph. Special thanks are also due to Professor
    A. Rico for his technical support and exceptional hospitality.
    Thanks are also due to Mrs A. Rico and the staff of the Ecole
    Nationale Vtrinaire responsible for administrative aspects of the
    meeting.

         The fact that Bayer AG and Union Carbide made available to IPCS
    and the Task Group proprietary toxicological information on their
    products is gratefully acknowledged. This allowed the Task Group to
    make its evaluation on a more complete data base.

         Dr R. Plestina and Dr P.G. Jenkins, both members of the IPCS
    Central Unit, were responsible for the overall scientific content
    and technical editing, respectively, of this monograph. The efforts
    of all who helped in the preparation and finalization of the
    monograph are gratefully acknowledged.

    ABBREVIATIONS

    3-ATAL    3-(3-amino- s-triazole-1-yl)-2-aminopropionic acid

    ACGIH     American Conference of Governmental and Industrial
              Hygienists

    ADI       acceptable daily intake

    DAB       4-dimethylaminobenzene

    DES       diethylstilbestrol

    DHPN       N-bis(2-hydroxypropyl) nitrosamine

    DIT       diiodotyrosine

    EC        emulsifiable concentrate

    GSH-Px    glutathione peroxidase

    HPLC      high performance liquid chromatography

    IC50      median immobilization concentration

    MIT       monoiodotyrosine

    MTD       maximum tolerated dose

    NBU        N-nitrosobutylurea

    NOAEL     no-observed-adverse-effect-level

    NOEC      no-observed-effect concentration

    NOEL      no-observed-effect level

    OECD      Organisation for Economic Co-operation and Development

    PBI       protein-bound iodine

    PHS       prostaglandin-H-synthetase

    T3        L-triiodothyronine

    T4        L-thyroxine

    TC        thin layer chromatography

    TLV       threshold limit value

    TSH       thyreostimulating hormone

    TWA       time-weighted average

    WP        wettable powder

    1.  SUMMARY

    1.1  Identity, physical and chemical properties, and analytical
         methods

         Amitrole (3-amino-1,2,4-triazole) is a colourless, crystalline
    powder. It is thermally stable and has a melting point of
    156-159 C. It is readily soluble in water and ethanol and only
    sparingly soluble in organic solvents such as hexane and toluene.
    Chemically, amitrole behaves as a typical aromatic amine as well as
    an  s-triazole. A wide range of analytical methods are available
    for detection and quantification of amitrole in plants, soil, water,
    air and urine.

    1.2  Sources of human and environmental exposure

         Amitrole does not occur naturally. It is manufactured by the
    condensation of formic acid with aminoguanidine bicarbonate in an
    inert solvent at 100-200 C. Amitrole is used as a herbicide with a
    wide spectrum of activity and appears to act by inhibiting the
    formation of chlorophyll. It is commonly used around orchard trees,
    on fallow land, along roadsides and railway lines, or for pond weed
    control.

    1.3  Environmental transport, distribution and
    transformation

         Owing to its low vapour pressure, amitrole does not enter the
    atmosphere. It is readily soluble in water with a photodegradation
    half-life in distilled water of more than one year.
    Photo-degradation does occur in the presence of the photosensitizer
    humic acid potassium salt, reducing the half-life to 7.5 h.

         Amitrole is adsorbed to soil particles and organic matter by
    proton association. The binding is reversible and not strong, even
    in favourable acid conditions. Measured  n-octanol/water partition
    coefficient values classify amitrole as "highly mobile" in soils of
    pH > 5 and "medium to highly mobile" at lower pH. There is
    considerable variation in leaching of the parent compound through
    experimental soil columns. Generally, movement is most readily seen
    in sand; increasing the organic matter content reduces mobility.

         Degradation in soils is usually fairly rapid but variable with
    soil type and temperature. Bacteria capable of degrading amitrole
    have been isolated. The herbicide can act as sole nitrogen source,
    but not also as sole carbon source, for the bacteria. Microbial
    degradation is probably the major route of amitrole breakdown;
    little or no breakdown has been recorded in studies with sterilized
    soil. However, abiotic mechanisms, including the action of free
    radicals, have also been proposed as a means of degradation.

    Laboratory studies have indicated degradation to CO2 with a
    half-life of between 2 and 30 days. A single field study suggests
    that the degradation may take longer at lower temperatures and
    different soil moisture levels; the half-life was about 100 days in
    a test clay.

         Although the parent compound leaches through some soils,
    degradation products are tightly bound to soil. Since amitrole is
    degraded rapidly in soil, the high leaching potential of the
    herbicide does not seem to be realized in practice. Occasional
    damage to trees reported during the early usage of amitrole has not
    been a regular feature of its use.

         When applied to vegetation, amitrole is absorbed through the
    foliage and can be translocated throughout the plant. It is also
    absorbed through roots and transported in the xylem to shoot tips
    within a few days.

         High water solubility, a very low octanol-water partition
    coefficient and non-persistence in animals means that there is no
    possibility for bioaccumulation of amitrole or transport through
    food chains.

    1.4  Environmental levels and human exposure

         Particulates containing amitrole may be released from
    production plants; atmospheric levels of 0 to 100 mg/m3 have been
    measured close to one plant.

         The use of amitrole in waterways and watersheds has led to
    transitory water concentrations of up to 150 g/litre.
    Concentrations fall rapidly to non-detectable (<2 g/litre) levels
    in running water within 2 h. Application to ponds gave an initial
    water concentration of 1.3 mg/litre falling to 80 g/litre after 27
    weeks. Close to a production plant, river concentrations ranged from
    0.5 to 2 mg/litre.

         No residues of amitrole have been detected in food following
    recommended use. Spraying of ground cover around fruit trees did not
    lead to residues in apples. Wild growing fruit in the vicinity of
    control areas can develop residues.

         There have been no reports of amitrole in drinking-water.

    1.5  Kinetics and metabolism in laboratory animals and humans

         Following oral administration, amitrole is readily absorbed
    from the gastrointestinal tract of mammals. It is rapidly excreted
    from the body, mainly as the parent compound. The main route of
    excretion in humans and laboratory animals is via the urine, and the

    majority of excretion takes place during the first 24 h. Metabolic
    transformation in mammals produces two minor metabolites detectable
    in the urine of experimental animals. When an amitrole aerosol is
    inhaled, a similar rapid excretion via the urine takes place.

    1.6  Effects on experimental animals and  in vitro test
    systems

         Amitrole had low acute toxicity when tested in several species
    and by various routes of administration (LD50 values were always
    higher than 2500 mg/kg body weight). It was found to affect the
    thyroid after single, short-term and long-term exposures. Amitrole
    is goitrogenic; it causes thyroid hypertrophy and hyperplasia,
    depletion of colloid and increased vascularity. In long-term
    experiments these changes precede the development of thyroid
    neoplasia in rats.

         The carcinogenic effect of amitrole on the thyroid is thought
    to be related to the continuous stimulation of the gland by
    increased thyroid stimulating hormone (TSH) levels, which are caused
    by the interference of amitrole with thyroid hormone synthesis.

         Equivocal results have been reported in some studies on the
    genotoxic potential of amitrole. In carcinogenicity testing in rats,
    amitrole did not induce tumours in organs other than the thyroid.
    However, high doses of amitrole caused liver tumours in mice.

         Several criteria have been used to assess the early effects of
    amitrole on the thyroid. The lowest no-observed-adverse-effect level
    (NAOEL) derived from these studies was 2 mg/kg in the diet of rats
    and was assessed on the basis of thyroid hyperplasia.

    1.7  Effects on humans

         A single case of contact dermatitis due to amitrole has been
    reported. Amitrole did not cause toxic effects when ingested at a
    dose of 20 mg/kg. In a controlled experiment, 100 mg was found to
    inhibit iodine uptake by the thyroid at 24 h. Weed control operators
    exposed dermally to approximately 340 mg amitrole per day for 10
    days exhibited no changes in thyroid function.

    1.8  Effects on other organisms in the laboratory and field

         Several studies on the growth of cyanobacteria (blue-green
    algae) have shown no effect of amitrole at concentrations at or
    below 4 mg/litre. No consistent adverse effects on nitrogen fixation
    have been reported. Bacteria from soil were unaffected by
    concentrations of 20 mg/litre medium in the case of nitrogen-fixing
     Rhizobium and 150 mg/kg in the case of cellulolytic bacteria.

    There were no effects on nitrification or soil respiration at 100 mg
    a.i./kg dry soil, 5 times the maximum recommended application rate.
    Reduced nodulation in sub-clover was reported at concentrations of
    up to 20 mg/litre.

         Various unicellular algae have been tested for
    growth-inhibiting effects. At 0.2 - 0.5 mg amitrole/litre, the
    growth inhibition of  Selenastrum was the most sensitive reported
    effect.

         Most aquatic invertebrates show high tolerance to technical
    amitrole: LC50 values were > 10 mg/litre for all organisms other
    than the water flea  Daphnia magna, where the acute 48-h EC50
    (immobilization) was 1.5 mg/litre. Fish and amphibian larvae are
    also tolerant to amitrole with LC50 values above 40 mg/litre.
    Longer-term studies indicated that young rainbow trout survive an
    amitrole concentration of 25 mg/litre for 21 days.

         Two earthworm species  (Eisenia foetida and  Allolobophora
     caliginosa) were unaffected by amitrole (SP50) at 1000 mg/kg
    soil and Amitrole-T at 100 mg/kg soil, respectively. Carabid beetles
    were unaffected after direct spraying with amitrole at rates
    equivalent to 30 kg/ha. Effects on nematodes only occurred at high
    concentrations of amitrole (the LC50 was 184 mg/kg).

         Amitrole was reported to be non-hazardous to bees in field
    trials. Amitrole has low toxicity to birds, all reported dietary
    LC50 values being above 5000 mg/kg per diet. Acute oral dosing
    killed no mallard ducks at 2000 mg/kg body weight.

    2.  IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL
        METHODS

    2.1  Identity

    Common name:                  Amitrole

    CHEMICAL STRUCTURE

    Chemical formula:             C2H4N4

    Relative molecular mass:      84.08

    CAS chemical names:           1 H-1,2,4-triazol-3-amine (9C1)
                                  3-amino- s-triazole (8CI)

    IUPAC names:                  1 H-1,2,4-triazol-3-ylamine
                                  3-amino-1 H-1,2,4-triazole
                                  3-amino- s-triazole

    CAS registry number:          61-82-5

    RTECS registry number:        XZ3850000

    Common synonyms:              aminotriazole; 2-aminotriazole;
                                  3-aminotriazole; 3-amino-1,2,4-
                                  triazole; 2-amino-1,3,4-triazole;
                                  3-amino-1H-1,2,4-triazole; AT;
                                  3AT; ATA; 3,A-T; ATZ; AT-90;
                                  triazolamine; 1,2,4-triazol-3-amine;
                                  5-amino-1H-1,2,4-triazole.

    Common trade names:           Amerol; Aminotriazole Weedkiller
                                  90; Aminotriazol Spritzpulver;
                                  Amitril; Amitril T.L.; Amitrol;
                                  Amitrol 90; Amitrol Plus;
                                  Amitrol-T; Amizine; Amizol;
                                  Amizol DP; Amizol F; AT Liquid;
                                  Azaplant; Azolan; Azole; Azaplant
                                  Kombi; Campaprim A1544; Cytrol;

                                  Cytrole; Destraclol; Diurol. 5030;
                                  Domatol; Domatol 88; Elmasil;
                                  Emisol; Emisol 50; Emosol F; ENT
                                  25445; Exit; Fenamine; Fenavar;
                                  Fyrbar; Kleer-Lot; Lancer;
                                  Nu-Zinole-AA; Orga 414; Preceed;
                                  Radoxone TL; Ramizol; Sapherb;
                                  Solution Concentree T271; Ustinex;
                                  Vorox; Vorox AA; Vorox AS;
                                  Weedar ADS; Weedar AT; Weedazin;
                                  Weedazin Arginit; Weedazol;
                                  Weedazol GP2; Weedazol Super;
                                  Weedex Granulat; Weedoclor; X-All 
                                  Liquid.

         Technical grade amitrole contains a minimum of 95% active
    ingredient and is formulated as a solution of 250 g/litre in water,
    usually with an equimolar concentration of ammonium thiocyanate, or
    as a 400 g/kg wettable powder, usually in combination with other
    herbicides.

         The major impurities are 3-(N-formylamino)-1,2,4-triazole,
    4 H-1,2,4-triazole-3,4-diamine, and
    4 H-1,2,4-triazole-3,5-diamine.

    2.2  Physical and chemical properties

         Some of the physical and chemical properties of amitrole are
    shown in Table 1.

         Amitrole is readily soluble in water, methanol, ethanol and
    chloroform, sparingly soluble in ethyl acetate, and insoluble in
    hydrocarbons, acetone and ether. It forms salts with most acids or
    bases and is a powerful chelating agent. It is corrosive to
    aluminium, copper and iron. Chemically, amitrole behaves as an
     s-triazole and also as an aromatic amine, and hence will diazotize
    and couple several dyes.

    2.3  Conversion factors:

              1 mg/kg = 3.43 mg/m3    1 mg/m3 = 0.29 mg/kg

    
    Table 1. Some physical and chemical properties of amitrole
                                                                        

    Physical state                          crystalline

    Colour                                  colourless

    Taste                                   bitter

    Odour                                   none

    Thermal stability                       stable at 20 Ca

    Hydrolytic stability (pH 4-9; 90 C)    stableb

    Melting point                           157-159 Cc

    Water solubility (25 C)                280 g/litrec

    Water solubility (53 C)                500 g/litred

    Ethanol solubility (75 C)              260 g/litred

    Solubility in n-hexane (20 C)          < 0.1 g/litred

    Solubility in dichloromethane (20 C)   0.1-1 g/litred

    Solubility in 2-propane                 20-50 g/litred

    Solubility in toluene (20 C)           <0.1 g/litred

    Vapour pressure (20 C)                 55 nPac

    Octanol/water partition coefficient     (21 C)

    (log Pow)                               -0.969e
                                                                        

    a    Klusacek & Krasemann (1986)
    b    Krohn (1982)
    c    Worthing & Hance (1991)
    d    Personal communication from Bayer AG to the IPCS (1993)
    e    Hazleton Laboratories, USA Report HLA-6001-187
    
    2.4  Analytical methods

    2.4.1  Plants

         Early methods for the detection of amitrole by paper
    chromatography or for its quantitative determination by
    spectrophotometry involved extraction by ethanol or water,
    diazotization of the 3-amino group and, finally, coupling with
    either phenol in 20% HCl (Aldrich & McLane, 1957),
     N-(1-naphthyl)ethylenediamine dihydrochloride (Storherr & Burke,
    1961),  H-acid (8-amino-1-naphthol-3,6-disulfonic acid, monosodium
    salt) (Racusen, 1958; Herrett & Linck, 1961; Agrawal & Margoliash,
    1970) or chromotropic acid (Green & Feinstein, 1957). This technique
    has been used for residue analysis in plants (Aldrich & McLane,
    1957; Herrett & Linck, 1961), and vegetable crops (Storherr & Burke,
    1961). The detection limit was found by Aldrich & McLane (1957) to
    be approximately 0.1 g/spot. The method outlined by Storherr &
    Burke (1961) is sensitive to 0.025 mg/kg. Recovery was described by
    Herrett & Linck (1961) to be close to 100%. Storherr & Onley (1962)
    found that dry-packed cellulose column chromatography was preferable
    to paper chromatography for separation of amitrole from some crops.

         Several gas chromatographic methods have been developed to
    determine amitrole residues in plants (Jarczyk, 1982a, 1985; Jarczyk
    & Mllhoff, 1988). The principle of all these methods is similar.
    After extraction with an ethanol-water mixture, acetylation with
    acetic anhydride (conversion of amitrole to the monoacetyl
    derivative) and a clean-up step by gel chromatography, the residue
    is dissolved in acetone or ethanol and determined by a gas
    chromatograph equipped with a nitrogen-phosphorus detector.

         Weber (1988) developed a method for the determination of
    amitrole in plant material by high performance liquid chromatography
    (HPLC). Amitrole was extracted with an acetone-water mixture and the
    water phase was extracted with dichloromethane to remove lipophilic
    compounds. After a further clean-up step with column chromatography
    on a cation exchange resin and on aluminium oxide, the residues were
    determined by HPLC with ion pairing reagent and electrochemical
    detection. In plants the detection limit was 0.01 mg/kg and the
    recovery was between 91 and 99% in the range 0.01-1.0 mg/kg.

         The Codex Committee on Pesticide Residues has recommended the
    methods of Lokke (1980) and Van der Poll et al. (1988).

         The method of Lokke (1980) uses ion-pair HPLC, which in
    potatoes or fodder beets had a limit of detection between 0.005 and
    0.01 mg/kg.

         The method of Van der Poll et al. (1988) is capable of
    determining amitrole in plant tissues and sandy soils by capillary
    gas chromatography with an alkali flame ionization detector. Samples
    are extracted with ethanol, absorbed on resin and desorbed with
    ammonia. After acetylation with acetic anhydride and clean-up with a
    SEP-PAK silica cartridge, the residue is determined by gas
    chromatography (GC). The limit of detection is 0.02 mg/kg and
    average recoveries are 76-81% in the range from 0.05 to 0.2 mg/kg.

    2.4.2  Soil

         An early method for the determination of residues in soil was
    developed by Sund (1956) which involved extraction with water
    followed by colour reaction with nitroprusside in alkaline
    solutions.

         Groves & Chough (1971) developed an improved procedure for the
    extraction of amitrole from soil using concentrated ammonium
    hydroxide and glycol (1:4). Pribyl et al. (1978) investigated the
    extraction of amitrole from soils and its identification and
    quantitation by photometry and thin layer chromatography (TLC). The
    limit of detection was 0.05 mg/kg. They proposed analysis by TLC
    after reaction with 5-dimethylaminonaphthalene-1-sulfonyl chloride
    (dansylation), in preference to HPLC. Lokke (1980) suggested that
    HPLC separation could be used if preceded by clean-up on a polyamide
    column. Both the GC method (Jarczyk, 1985; Jarczyk & Mllhoff, 1988)
    and the HPLC method (Weber, 1988) described in section 2.4.1 for
    plants are also suitable for the determination of amitrole residues
    in soil.

    2.4.3  Water

         Marston et al. (1968) and Demint et al. (1970) have used cation
    ion-exchange column chromatography to extract amitrole from
    contaminated creek and canal waters. This is followed by
    diazotization and coupling as described by Storherr & Burke (1961).
    Alary et al. (1984) have modified these methods to achieve a
    spectrophotometric determination of amitrole in waste water in the
    vicinity of production plants in the presence of interfering amino
    compounds.

         A more recent capillary gas-liquid chromatographic method for
    determining amitrole in ground water and drinking-water, using an
    alkali flame ionisation detector, has been described, the reported
    limit of detection being 0.1 g/litre (Van der Poll et al., 1988).

         Legrand et al. (1991) formed a nitroso derivative of amitrole
    concentrated from surface and ground waters prior to HPLC analysis.
    The nitroso derivative showed an absorption maximum in the near UV
    spectrum. Aqueous solutions of amitrole in the range of
    0.25-0.50 g/litre were measurable, and the recoveries were 70  8%
    (n = 11). The limit of determination was 0.1 g/litre.

         Pachinger et al. (1992) developed an HPLC analytical method
    with amperometric detection for the determination of amitrole
    without derivatization in drinking-water and ground water. Detection
    limits were 1 mg/litre for directly injected samples and
    0.1 g/litre following an evaporation step to concentrate the
    samples. Recoveries were close to 100%.

         Both the GC method (Jarczyk, 1985; Jarczyk & Mllhoff, 1988)
    and the HPLC method (Weber, 1988) described in section 2.4.1 for
    plants are also suitable for the determination of amitrole residues
    in water.

         An immunochemical approach to the detection of amitrole has
    been recently described by Jung et al. (1991). Development of this
    rapid and sensitive method is likely to lead to a very effective
    method for detecting amitrole in waterways.

    2.4.4  Formulations

         Ashworth et al. (1980) described a potentiometric precipitation
    titration method using silver nitrate and silver/silver chloride or
    silver/mercurous sulfate electrode. This method can be used for the
    determination of amitrole in its formulations or in the presence of
    triazines, substituted urea herbicides or plant growth regulators
    such as bromacil and ammonium thiocyanate. Another method for the
    determination of amitrole in its formulations has been described by
    Gentry et al. (1984). This involves dissolving or extracting the
    sample with dimethylformamide, acidifying by adding 0.5 N HCl and
    back-titrating the excess acid with 0.5 N sodium hydroxide.

         Jacques (1984) has described a simple GC method for the
    detection and quantification of amitrole in technical and formulated
    products.

         A TLC method for the routine identification of amitrole in
    pesticide mixtures has been developed by Ebing (1972).

    2.4.5  Air

         Alary et al. (1984) have described a method for the analysis of
    air samples collected on glass-fibre filters followed by
    diazotization and coupling to produce a colour reaction. The
    detection limit was not reported.

    2.4.6  Urine

         In order to assay amitrole in urine samples, Geldmacher-von
    Mallinckrodt & Schmidt (1970) separated the amitrole by paper
    chromatography using phenol saturated with water, or a mixture of
     n-butanol:water (15:1) and propionic acid:water (7:6), and
    identified amitrole by spraying with a solution of
     p-dimethyl-aminobenzaldehyde in acetic acid or hydrochloric acid.
    In a more recent paper by Archer (1984), a proposed method for
    biological monitoring of urine samples used HPLC separation with a
    visible light detector following diazotization and coupling. The
    detection limit was 200 g/litre.

    3.  SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

    3.1  Natural occurrence

         Amitrole does not occur naturally.

    3.2  Anthropogenic sources

    3.2.1  Production levels and processes

         The synthesis of amitrole was first reported by Thiele &
    Manchot in 1898 and involved the reaction of aminoguanidine with
    formic acid (Carter, 1975). The current industrial production
    process, described by Allen & Bell (1946), involves the same
    reaction, in which an aminoguanidine salt is heated to 100-120 C
    with formic acid in an inert solvent (Carter, 1975; Sittig, 1985).

         Amitrole is currently manufactured or formulated in several
    countries. Its use has declined, particularly in the USA. However,
    in spite of some recent replacements, amitrole remains a widely used
    herbicide.

    3.2.2  Uses

         Amitrole is primarily used as a post-emergent non-selective
    herbicide and has a very wide spectrum of activity against annual
    and perennial broad leaf and grass type weeds. Its primary mode of
    action is unknown but a prominent feature is its inhibition of the
    formation of chlorophyll, and weeds initially change colour to
    white, brown or red, and subsequently die (Carter, 1975). This
    herbicidal activity is enhanced by the addition of ammonium
    thiocyanate as a synergist. Amitrole can be used alone as a
    concentrated solution in water or as a wettable powder in
    combination with other herbicides. It is primarily used as a
    herbicide and as a brush killer. It is also used as a non-selective
    pre-emergent herbicide on fallow land before planting kale, maize,
    oilseed rape, potatoes and wheat, and in other non-crop situations
    (Worthing & Hance, 1991). It is also used along roadsides and
    railway lines to control weeds. Approved uses of amitrole on soil
    are either for non-crop land prior to sowing, or for inter-row weed
    control in tree and vine crops, where contact with food plants is
    avoided. Amitrole is also used for the control of pond weeds and is
    an especially effective herbicide in the control of water hyacinth
     (Eichhornia crassipes).

         Amitrole has also been used as a cotton defoliant in some
    countries (Hassall, 1969).

    4.  ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

    4.1  Transport and distribution between media

    4.1.1  Air

         The very low vapour pressure of amitrole (Table 1) means that
    it will not enter the atmosphere.

    4.1.2  Water

         Amitrole is readily soluble in water (280 g/litre at 25 C) and
    has a half-life of more than one year at 22 C and pH 4-9 (Worthing
    & Hance, 1991). Although no direct photolysis occurred in doubly
    distilled water, the photodegradation rate increased in the presence
    of humic acid, potassium salt (100 mg/litre), a natural
    "photosensitizer", resulting in a half life of 7.5 h (Jensen-Korte
    et al., 1987).

    4.1.3  Soil

    4.1.3.1  Adsorption

         Amitrole is adsorbed to soil particles and organic matter by
    proton association. The adsorbed aminotriazolium cation will enter
    into cationic exchange reactions (Nearpass, 1969). Binding is
    strongly pH dependent, and the cation is adsorbed to a greater
    extent in acid conditions. The aminotriazolium cation is bound more
    strongly than sodium but is displaced by calcium ions. The binding
    is reversible and not strong, even in favourable acid conditions.
    The binding capacity of soils at pH 5 or more is limited (Nearpass,
    1970). Anderson & Hellpointner (1989) determined the Koc values
    for amitrole in four soils i.e. silty clay, sandy loam, sand and
    silt, to be 112, 30, 20 and 52, respectively. Adsorption increased
    at lower pHs; adjustment of pH to a constant 4.5 resulted in Koc
    values ranging from 77 to 356. The authors classified amitrole as
    highly mobile in the soils at their equilibrium pH values of 5.6 to
    7.4 and medium to highly mobile with the pH adjusted from 4.2 to
    4.5.

         There is considerable variation in the literature, both old and
    recent, in reported adsorption and leachability of amitrole. Sund
    (1956) described adsorption to soil as strong. He demonstrated that
    amitrole could be efficiently removed from aqueous solution with a
    resin cation exchanger and argued that soil would also bind the
    compound efficiently. A correlation between the base exchange
    capacity of soil and binding of amitrole was postulated. This agrees
    with the theoretical and experimental work of Nearpass (1969, 1970),
    although the latter author does not support the strength of
    adsorption proposed by Sund (1956). Day et al. (1961) investigated

    leaching of amitrole through 400-g, 4-cm diameter columns of three
    different soils from a citrus growing area of California following
    occasional reports of damage to trees after application of high
    rates of amitrole for the control of perennial weeds. Amitrole moved
    readily with the leaching water for all soil types (two sandy loams
    and one silt loam) and most readily through quartz sand. Zandvoort
    et al. (1981) supported this conclusion, suggesting that the high
    water solubility of amitrole could result in leaching from sandy
    soils. Weller (1987) investigated leaching of amitrole through
    27-cm, 5-cm diameter columns of two soils, a sandy "standard soil
    2.1" and a second soil with substantially higher organic content.
    Immediately after incorporation of the 14C-amitrole to give 2 mg
    on the surface area of the column, leaching with deionized water
    began, 393 ml being pumped onto the soil column over 2 days. The
    leachate was collected in two fractions: 175-191 ml and 185-200 ml.
    Duplicate experiments showed 24 and 31% of the initial radioactivity
    in the leachate (entirely in fraction II) in the sandy soil, with
    11%, 16% and 46%, respectively, remaining in the upper, middle and
    lower third of the soil column. The second soil leached markedly
    less of the added radioactivity (1.4 and 1.8% for the duplicate
    columns); this also appeared in fraction II. The radioactivity in
    the leachate was unchanged amitrole.

         Since amitrole is degraded rapidly in soil (section 4.2.1), the
    high potential of amitrole to leach through sandy soils does not
    seem to be realized in practice. The occasional damage to trees
    reported in the study by Day et al. (1961) has not been a regular
    feature of the use of amitrole. Degradation products of amitrole do
    not leach significantly through soil (section 4.2.1).

    4.1.4  Vegetation and wildlife

         When applied directly to vegetation as a herbicide, amitrole is
    absorbed through the foliage and can be translocated throughout the
    plant. Translocation occurs in the photosynthetic stream and is
    dependent on light. When applied to soil, amitrole can be adsorbed
    through the roots and transported in the xylem, within a few days,
    to the tips of the shoots (Carter, 1975).

    4.1.5  Entry into food chain

         Amitrole is not to be used on food crops and therefore food
    residues should not occur. Grazing animals could consume amitrole as
    surface residues on vegetation after application or as residues
    within the plant. Amitrole is not persistent in animals and would
    not be expected to pass through the food chain.

    4.2  Biotransformation

    4.2.1  Biodegradation and abiotic degradation

    4.2.1.1  Plants

         Racusen (1958) reported the first comprehensive studies of
    amitrole metabolism in plants. Two major metabolites were isolated,
    neither of which were as phytotoxic as amitrole. These results were
    supported by studies by Carter & Naylor (1960). One metabolite was
    identified as the product of the reaction of amitrole with serine,
    namely, 3-(3-amino- s-triazole-1-yl)-2-aminopropionic acid
    (3-ATAL). Formation of 3-ATAL is considered to represent
    detoxification since ammonium thiocyanate, which synergizes the
    action of amitrole, inhibits the formation of 3-ATAL (Smith et al.
    1969). Other products of amitrole metabolism in plants have not been
    identified. Fang et al. (1967) found that metabolism of amitrole in
    leaves was exponential, with half-lives in sugar beet, corn and bean
    leaves being 18.7, 28.0 and 23.2 h, respectively. A review of the
    degradation of amitrole in plants has been presented by Carter
    (1975).

         The soluble metabolites of [3,5-14C]-amitrole in apples were
    examined by Schneider et al. (1992) following soil application.
    Significant proportions of the radioactivity were found as bound
    residues, but 69-90% were extractable with acetonitrile. In addition
    to 3-ATAL, 3-(1,2,4-triazole-1-yl)-2-aminopropionic acid
    (3-aminotriazolylalanine) was also identified, in both the free form
    and as conjugates. This was the major metabolite in apple cell
    cultures treated with amitrole (Stock et al., 1991).

    4.2.1.2  Soils

         There is general agreement that degradation of amitrole in soil
    is usually fairly rapid and variable with soil type and temperature.
    However, there is no clear consensus on the relative roles of biotic
    and abiotic processes in the breakdown of the compound.

         Day et al. (1961) measured amitrole colorimetrically in 55
    different soils of 5 main types from California and estimated the
    depletion after 2 weeks of incubation. The results were very
    variable, 26 soils having no measurable amitrole after 2 weeks, 6
    soils showing traces and the remaining 23 soils having higher
    quantities, in some cases comparable to initial levels. Four soils
    had more than half of the original amitrole after the 2-week
    incubation. It was not possible to correlate depletion of amitrole
    to soil type. The authors classified the soils according to general
    type and ranked them in terms of "heaviness"; the four soils
    retaining most amitrole ranked 7, 23, 30 and 54 in the list. There
    was a geographical correlation with reported incidents of non-target

    effects of the herbicide. Some specific characteristic of a variety
    of soils from a single location had led to movement of the herbicide
    and its retention longer than in apparently comparable soils
    elsewhere. Decomposition rates in steam-sterilized soils were much
    lower than in unsterilized soils, which led the authors to conclude
    that breakdown was principally due to microorganisms. Decomposition
    was optimal at temperatures between 20 and 30 C and at medium to
    high soil moisture content. Breakdown was not well correlated with
    soil classification, texture, base-exchanged capacity or adsorption
    capacity for amitrole. Differences in microbial populations were
    cited as the most likely explanation for the variation.

         Kaufman et al. (1968) also found that sterilization of soil
    reduced the breakdown of amitrole. Within 20 days, 69% of the
    radioactivity of 14C-labelled amitrole was released as 14CO2
    in unsterilized soil. Soil treated with sodium azide or ethylene
    oxide released 46% and 35%, respectively, whilst autoclaved soil
    released only 25%. Reinoculation of soil with microorganisms
    isolated from the original soil failed to restore the capacity to
    degrade amitrole. Amending the soil with other organic compounds
    reduced amitrole degradation. The authors concluded that degradation
    of amitrole was largely a chemical process and that microbial action
    was indirect. Free radicals (such as HO.) were proposed as agents
    for oxidation of the amitrole nucleus. Plimmer et al. (1967) studied
    the degradation of amitrole by free-radical generating systems. They
    demonstrated that riboflavin (and light) or an ascorbate-copper
    reagent (Fenton's reagent) promotes oxidation of amitrole, resulting
    in ring cleavage, loss of CO2 and production of urea, cyanamide
    and possibly molecular nitrogen. Riepma (1962) observed a lag-phase
    which he considered typical of microbial breakdown. Carter (1975)
    concluded that "whatever the mechanism, triazole ring opening occurs
    rapidly in soils and the resulting products ... should be readily
    metabolized by soil microorganisms".

         Campacci et al. (1977) reported the isolation of bacteria
    capable of degrading amitrole, strengthening the argument for
    microbial involvement. Only one of three media tested succeeded in
    growing organisms that could degrade amitrole. Of 36 isolates from
    this culture, 10 were found to be capable of degrading amitrole.
    Nine of these were gram-positive rods  (Bacillus spp. and
     Corynebacterium spp.) and one was identified as a  Pseudomonas
    sp. The growth of these bacteria was roughly proportional to
    amitrole concentration up to 256 mg/litre. The organisms could
    degrade amitrole as sole nitrogen source but not also as sole carbon
    source; the medium was enriched with sucrose. This explained
    previous failures to isolate organisms capable of degrading the
    herbicide.

         Various studies have quantified the degradation of amitrole in
    soil. Scholz (1988) observed the release of 48% of applied
    radioactivity (14C-amitrole) as 14CO2 after 5 days. In

    degradation studies in the laboratory, half-lives of between 2.4 and
    9.6 days were observed in different soils. DT90 (the time required
    for degradation of 90% of the amitrole) values were in the range of
    13 to 22 days (LUFA, 1977; Jarczyk, 1982b,c,d). Hawkins et al.
    (1982b) measured 70-80% degradation to CO2 in standard soil and
    40-50% in English clay soil within 28 days. There was no release of
    14CO2 from autoclaved soil. Hawkins et al. (1982a) measured
    decomposition in the same English clay in the field. Here 53% of the
    applied radioactivity remained after 112 days. The slower rate of
    breakdown in the field was ascribed to the temperature and soil
    moisture content. Schneider et al. (1992) suggested that amitrole
    can be deaminated in soil to give triazole.

         A study by Weller (1987) examined the leaching of "aged"
    residues of amitrole. Soils, with 14C-amitrole incorporated as
    described in section 4.1.3.1, were incubated for 30 and 92 days, in
    duplicate experiments, and then used in leaching tests as for the
    initial soils. For both the "standard soil 2.1" and the second soil,
    between 50 and 73% of initial radioactivity was lost as 14CO2
    during incubation. Of the remaining radioactive material, negligible
    amounts leached through the soil column in tests after 30 and 92
    days. Almost all of the activity remained in the upper third of the
    soil column. After 30 days 4% or less of the activity was unchanged
    amitrole. The breakdown products of amitrole (not characterized
    except for traces of urea) were tightly bound to the soil and were
    not leachable or easily extractable.

    4.2.3  Bioaccumulation

         Flow-through studies on fish using 14C-amitrole indicated
    that the bioaccumulation of amitrole in bluegill sunfish  (Lepomis
     macrochirus) and in channel catfish  (Ictalurus punctatus),
    exposed to 1 mg/litre, was only slight after 21 days of exposure
    (approximately 1.7-3.0 times the amitrole concentration in the
    water). When the fish were returned to untreated water, the amitrole
    concentration in their organs fell rapidly (Iwan et al., 1978).

         Bioaccumulation of amitrole by aquatic organisms would not be
    expected because of its high water solubility and very low
    octanol-water partition coefficient (Table 1).

    4.3  Ultimate fate following use

         MacCarthy & Djebbar (1986) described a method using chemically
    modified peat to decontaminate eluant from chemical production
    plants before it enters major waterways. When converted to a
    granular product suitable for column chromatography, the peat can
    act as an efficient ion-exchange material for the removal of
    amitrole and other basic chemicals.

         Amitrole is resistant to hydrolysis and the action of oxidizing
    agents. Burning the compound with polyethylene is reported to result
    in > 99% decomposition (Sittig, 1985).

    5.  ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

    5.1  Environmental levels

    5.1.1  Air

         Amitrole-containing particles are released from the stack of
    production plants during dry crushing and, to a lesser extent,
    bagging operations. Atmospheric levels in the range of 0 to
    100 g/m3 were measured in the vicinity of such a plant (Alary
    et al., 1984). Severe chlorosis and defoliation was noted following
    atmospheric fallout in the vicinity of the plant.

    5.1.2  Water

         Grzenda et al. (1966) studied the persistence of amitrole and
    three other herbicides in pond water following an aquatic weed
    control programme. The initial level on day one of 1.34 mg/kg
    decreased gradually to 1.03 mg/kg on day 11, 0.73 mg/kg on day 25,
    0.49 mg/kg at 9.5 weeks and 0.08 mg/kg at 27 weeks.

         In a study by Marston et al. (1968) in which 100 acres of a
    watershed in Oregon was sprayed, the levels of amitrole in water
    samples were measured on the downstream edge of the sprayed area. A
    maximum concentration of 155 g/litre was found 30 min after
    application began, but this decreased to 26 g/litre by the end of
    the application. No amitrole could be detected 6 days after
    spraying. The detection limit was 2 g/litre.

         Demint et al. (1970) measured the amitrole concentration in two
    flowing water canals following treatment of a single ditchbank of
    each canal with amitrole at the normal treatment rate. Samples taken
    at stations up to 7.2 km downstream indicated rapid decreases in
    amitrole levels following passage of the initial amitrole-bearing
    water, the levels having declined to 1 g/litre within 2 h. In a
    preliminary environmental survey conducted in 1984 in Japan,
    amitrole was not detected (detection limit 4 g/litre) in any of 24
    water samples nor was it detected in any of the 24 bottom sediments,
    the detection limit being 5-20 g/kg (Environment Agency Japan,
    1987).

         Alary et al. (1984) measured the level of amitrole in water
    samples collected in a river downstream from the discharge of an
    aeration pond in the vicinity of a production plant. The levels were
    in the range of 0.5 to 2 mg/litre while the concentration in the
    water of the aeration pond was in the range of 50 to 200 mg/litre.
    Legrand et al. (1991) tried to detect 38 compounds including
    amitrole in different areas of France (13 sampling points) with a
    detection limit of 0.1 g/litre, but no amitrole was found.

    5.1.3  Soil

         As discussed in chapter 4, amitrole, when applied to soil, is
    readily degraded or adsorbed to the soil particles.

    5.2  General population exposure

    5.2.1  Environmental sources

         No exposure would be expected from environmental sources.

    5.2.2  Food

         Amitrole is not to be used on food crops, and food residues
    should therefore not occur. Using a limit of determination of
    0.05 mg/kg, amitrole was not detectable in a wide range of food
    crops (Duggan et al., 1966, 1967; Corneliussen, 1969, 1970). This
    was confirmed by several studies (Bayer AG, 1993a,b).

         Experimental studies in West Virginia, USA, indicated that
    residues of amitrole on whole apples could not be detected 3 months
    after ground cover application, but could be detected when either
    fruit or foliage or both were directly treated with amitrole
    (Schubert, 1964). The analyses were conducted using the method of
    Storherr & Burke (1961) with a detection level of 0.025 mg/kg.

         Similarly, residue trials conducted in Tasmania and New South
    Wales on apples did not reveal amitrole at a detection limit of
    0.01 mg/kg following ground cover application (Moore, 1968, 1969,
    1970). A slight modification of the method of Storherr & Burke
    (1961) was used.

         In one study, residues of amitrole were found in blackberries
    growing very near a railway line that was sprayed by amitrole in the
    normal way by the railway authorities. Thirteen days after spraying
    at a dose 3,5 kg a.i./ha, blackberries were picked close to the
    railway at two different locations. The mean residues found at the
    two locations were 0.67 (0.2-1.4) mg/kg and 2.0 (0.1-3.8) mg/kg. The
    places where the blackberries were picked was prohibited to the
    general public. This study shows that spraying of amitrole on
    blackberries results in considerable residues (Dornseiffen &
    Verwaal, 1988).

    5.3  Occupational exposure during manufacture, formulation or use

         The potential for toxicity via the dermal or inhalational
    routes appears to be low. A threshold limit value (TLV) of
    0.2 mg/m3, as an 8-h time-weighted average (TWA), has been set for
    amitrole by the American Conference of Governmental & Industrial
    Hygienists (ACGIH, 1991-1992). Amitrole is a mild skin and eye
    irritant.

    6.  KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS

    6.1  Absorption, distribution and excretion

    6.1.1  Mouse

         The distribution of [5-14C]-radiolabelled amitrole has been
    examined in non-pregnant C57BL strain female mice (Tjalve, 1975) and
    in the fetuses of pregnant NMRI strain mice (Tjalve, 1974). In each
    case, the mice received amitrole (5 Ci) either intravenously or
    orally, and the distribution of radioactivity was determined by
    whole-body autoradiography of the adult or fetus at intervals of
    5 min to 5 days after administration. In the non-pregnant animals,
    further analysis of the distribution of radioactivity was performed
    by microautoradiography of the spleen and thymus, and by subcellular
    fractionation of the liver, spleen and thymus. The highest
    radioactivity was found in tissues with rapid cell turnover, e.g.,
    bone marrow, spleen, thymus and gastrointestinal mucosa. Only a
    moderate level of radioactivity was found in the thyroid. The level
    of radioactivity in the tissues was the same whether the treatment
    was intravenous or oral. In all cases, there was a significant
    decrease over the 5-day period. Microautoradiography indicated
    amitrole was located mostly in the cytoplasm. 14C-labelled
    amitrole crossed the placental barrier and could be detected in
    fetal tissues 4 and 8 h after administration to the dams by
    intravenous injection or gavage.

         Following intravenous administration of 14C-amitrole
    (3.4 mg/kg body weight), adult ICR mice were killed at given
    intervals (5 min, 30 min, 8 h and 24 h) and submitted to whole-body
    autoradiography and microautoradiography. The liver had the highest
    accumulation of radioactivity and two distribution patterns were
    observed: a homogenous distribution up to 8 h following injection,
    and a subsequent heterogenous one. Liver sections were extracted
    with trichloroacetic acid and methanol, but considerable amounts of
    radioactivity remained non-extractable. A microauto-radiography of
    the liver 8 h after 14C-amitrole injection revealed that the
    radioactivity was localized in the centrolobular areas (Fujii
    et al., 1984).

    6.1.2  Rat

         Kinetic studies on amitrole in rats were performed by Fang
    et al. (1964). Groups of Wistar rats were administered 1 mg
    14C-amitrole by gavage and the distribution of radioactivity was
    analysed at various time intervals between 30 min and 6 days. High
    levels of radioactivity (70-95% of the administered radioactivity)
    were found in the urine during the first 24 h, but only low levels
    in the faeces, indicating rapid and almost complete absorption from
    the gastrointestinal tract followed by rapid excretion. Tissue

    levels were very low after 3 days, and significant amounts were
    found only in the liver. In a second experiment (Fang et al., 1966),
    14C-amitrole was administered at various dose levels (1-200 mg/kg
    body weight). The average total radioactivity found in urine and
    faeces (as a percentage of the administered dose) was comparable for
    all the doses applied. The difference in average half-time for
    clearing of radioactivity from various organs was considered to be
    insignificant between dosages of 1 and 200 mg/kg. The fate of two
    unidentified plant metabolites of amitrole, i.e. 14C-metabolite 1
    and 14C-metabolite 3 (isolated from bean plants), was also
    examined by Fang et al. (1966). Radioactivity from metabolite-1 was
    excreted rapidly in the urine in the first 48 h and identified as
    unchanged metabolite-1. Elimination of metabolite-3 was mainly in
    the faeces. In a study by Grunow et al. (1975), 14C-amitrole was
    administered to rats by gavage at a dose level of 50 mg/kg, and the
    urine and faeces were examined over 3 days. The major route of
    excretion of radioactivity was the urine, the majority of the
    radioactivity being excreted in the first 24 h.

         Two groups of five male and five female Sprague-Dawley rats
    weighing 200-250 g were exposed (either nose only or whole body) to
    atmospheres of 5-14C-amitrole (radiochemical purity > 97%) in
    water aerosols at concentrations in air of 49.2 g/litre
    (2.6 Ci/litre) or 25.8 g/litre (1.4 Ci/litre), respectively, for
    1 h, and then observed for 120 h (MacDonald & Pullinger, 1976). The
    particle size distribution of the aerosols was not reported. The
    calculated elimination half-life of radioactivity was approximately
    21 h for both exposures; approximately 75% of the radioactivity was
    eliminated in the urine within 12 h.

    6.1.3  Human

         Urinary excretion of unchanged amitrole has been reported in a
    woman who ingested approximately 20 mg/kg of the herbicide
    (Geldmacher-von Mallinckrodt & Schmidt, 1970).

    6.2  Metabolic transformation

         The limited data available indicates that little metabolic
    transformation of amitrole occurs in mammalian species. In the
    mouse, tissue residues were identified by TLC as mainly unchanged
    amitrole (84% of the detected radioactivity) when measured 8 h after
    exposure (Tjalve, 1975). Similarly, paper chromatographic analysis
    of rat liver residues following oral administration revealed
    unchanged amitrole plus one unidentified metabolite (Fang et al.,
    1964). In the urine of rats, the majority of the radioactivity was
    also unchanged amitrole; one unidentified metabolite was isolated
    which represented approximately 20% of the total radioactivity. The
    liver was the site of the unidentified metabolite-1 formation and
    the rate of elimination of this metabolite from liver and kidney was
    much slower (Fang et al., 1964).

         In a more extensive analysis of urinary metabolites in the rat
    by Grunow et al. (1975), the major part of the radioactivity
    identified by paper chromatography corresponded to unchanged
    amitrole. Two urinary metabolites were identified as
    3-amino-5-mercapto-1,2,4-triazole and
    3-amino-1,2,4-triazolyl-(5)-mercapturic acid, which together
    amounted to approximately 6% of the administered dose.

         In a metabolic study (Turner & Gilbert, 1976), which was
    supplementary to the inhalation exposure experiment and is described
    in section 6.1.2 (MacDonald & Pullinger, 1976), it was found that
    approximately 60% of the urinary radioactivity chromatographed on
    silica gel 60 TLC in methanol: 880 ammonia (100: 1.5, s/s) as
    amitrole, 15-20% remained at the origin and 5-8% migrated faster
    than amitrole. Treatment with -glucuronidase had no effect upon
    this TLC distribution.

    7.  EFFECTS ON EXPERIMENTAL ANIMALS AND  IN VITRO TEST SYSTEMS

    7.1  Single exposure

    7.1.1  Oral

         The acute oral toxicity data for amitrole when administered as
    an aqueous suspension are presented in Table 2.

    
    Table 2. Acute oral toxicity of amitrole
                                                                             

    Species             LD50 (mg/kg              Reference
                        body weight)a
                                                                             

    Rat                 > 4080 (m and f)         Gaines et al. (1973)

                        > 4200                   Seidenberg & Gee
                        24 600 (m)               Bagdon et al.(1956)
                        > 10 000                 Hecht (1954)
                        > 2500                   Kimmerle (1968)
                        > 5000 (m)               Thyssen (1974)
                        > 5000 (m)               Heimann (1982)
    Mouse               11 000                   Hapke (1967)
                        14 700 (m)               Fogleman (1954)
    Cat                 > 5000 (m and f)         Bagdon et al. (1956)
                                                                             

    a    m = males; f = females
    
         In cats and dogs the general signs of toxicity were dyspnoea,
    ataxia, and diarrhoea with vomiting. Coma and death appeared to be
    associated with profound respiratory depression. Gastro-intestinal
    irritation and haemorrhage were the only treatment-related findings.

         The toxicity of a mixture of amitrole and ammonium thiocyanate
    (1:1), referred to as Amitrol-T, appeared to be slightly higher than
    that of amitrole itself, but was still very low. LD50 values
    obtained following oral administration in rats were 3500 mg/kg and
    10.5 ml/kg of the commercial product (DeProspo & Fogleman, 1973;
    Field, 1979).

         The possibility that amitrole might form a Schiff's base with
    glucose was investigated by Shaffer et al. (1956). An
    amitrole-glucose adduct was prepared and administered orally to rats
    and mice (10 mg/kg), intraperitoneally to mice (10 mg/kg), and
    intravenously to mice (1.6 mg/kg). There were no deaths or signs of
    toxicity following treatment.

    7.1.2  Other routes

         The acute toxicity of amitrole by other routes of
    administration is very low, as shown in Table 3.

    
    Table 3.  Acute, dermal, intraperitoneal and intravenous toxicity
              of amitrole
                                                                                            

    Species          Route                LD50 (mg/kg bw)        Reference
                                                                                            

    Rat              dermal               > 2500 (m and f)       Gaines et al. (1973)
                     intraperitoneal      > 4000 (m)             Shaffer et al. (1956)
    Mouse            intravenous          > 1600 (m)             Shaffer et al. (1956)
                     intraperitoneal      > 10 000               Shaffer et al. (1956)
                     intraperitoneal      5470 (m)               Nomiyama et al. (1965)
                     subcutaneous         5540 (m)               Nomiyama et al. (1965)
    Rabbit           dermal               > 10 000               Elsea (1954)
    Dog              intravenous          > 1800 (m)             Fogleman (1954)
    Cat              intravenous          > 1750 (m and f)       Shaffer et al. (1956)
                                                                                            

    a    m = males; f = females
    
         Amitrole applied in water formulations to the unabraded skin of
    rabbits for 24 h caused a very mild and reversible erythema (Elsea,
    1954). Intraperitoneal administration in mice and rats and
    intravenous administration in either mice, dogs or cats produced no
    signs of toxicity (Fogleman, 1954).

         No toxicity was observed in rats after inhalation of amitrole
    following either head-only (approximately 50 g/litre) or whole-body
    (approximately 25 g/litre) exposure for a period of one hour
    (MacDonald & Pullinger, 1976).

    7.2  Short-term exposure

    7.2.1  Oral

    7.2.1.1  Dietary

         When groups of Carworth Farm male and female rats (five per
    group) were administered amitrole in the diet at dose levels of 0,
    100, 1000 or 10 000 mg/kg for 63 days, reduced body weight gain for
    both males and females was observed at the two highest dose levels,
    this being accompanied by reduced food consumption. There were no
    deaths or clinical signs of toxicity. Histo-pathological examination
    of the liver, kidney, portions of the small intestine, spleen, and
    testes revealed increased vacuolization of the liver cells around
    the central vein and steatosis at the two highest dose levels
    (Fogleman, 1954).

         Mayberry (1968) studied the effects on the thyroid of a dietary
    level of 1000 mg amitrole/kg in rats during 83 days and compared
    this to the effects of other anti-thyroid chemicals,
    propylthiouracil (1000 mg/kg) and potassium chlorate (1000 mg/kg).
    At various time intervals, starting with 3 days, the relative
    thyroid weight and total iodine content of the thyroid were
    measured. An increase in thyroid weight and a decrease of total
    iodine were observed within the amitrole group; this was already
    observable after 3 days and becoming more pronounced during the
    course of the experiment. The effects of propylthiouracil were
    comparable, but, in the case of potassium chlorate, the weight
    increase was less pronounced and the iodine content was lower than
    with amitrole. In another experiment, uptake and release of
    radioactive iodine was measured after a single 131I injection to a
    control group of rats and to a group simultaneously receiving 10 mg
    amitrole subcutaneously. Animals were killed after 1, 2, 3, 4, 5 or
    6 days. The t for 131I in the thyroid was 4.9 and 1.3 days for
    control and amitrole-treated animals, respectively. Separation by
    paper chromatography of 131I-containing thyroid fractions showed
    that levels of monoiodotyrosine (MIT) were increased, diiodotyrosine
    (DIT) were constant and T3 and T4 were markedly reduced. The
    author concluded that amitrole not only interferes with
    organification of iodine but also inhibits the coupling of
    iodotyrosines to form iodothyronines (Mayberry, 1968).

         The effect of amitrole on thyroid hormones was studied by
    giving groups of male Sprague-Dawley rats (20 per dose level)
    amitrole (94.6% pure) in the diet at dose levels of 0, 30, 100 or
    300 mg/kg during 28 days, followed by a recovery period of 28 days
    (Babish, 1977). The assessment of thyroid function was performed by
    measuring T3 and T4 in blood by a radioimmuno-assay. On days 3, 7,
    14, 21 and 28 of the treatment period and on days 19, 21 and 28 of
    the post-treatment period, blood samples were collected from two

    animals which were then killed for autopsy. There were no adverse
    effects on the general health of the rats during the treatment or
    post-treatment period. Consumption of 100 or 300 mg amitrole/kg diet
    significantly depressed body weights during the 28-day treatment
    period. The mean weekly body weights in the rats given 100 mg/kg
    returned to control values by the third week of the post-treatment
    period, while the mean weights of animals in the highest-dose group
    did not return to control values during the post-treatment period.
    The depression of body weights correlated with decreased food
    consumption.

         Serum T3 levels were significantly depressed by day 7 at
    300 mg/kg (about 50%) and by day 14 at 100 mg/kg/diet (about 40%).
    An inexplicable return to control values was seen 21 days into the
    treatment period, followed by continued depression of T3 values on
    day 28 of the treatment period. The depression of T3 appeared to
    be dose related after 4 weeks of treatment. All treatments exhibited
    essentially normal T3 levels by day 19 of the post-treatment
    period. T4 levels followed exactly the same pattern as those of
    T3. However, the T3/T4 ratio (which fluctuated between 12 and
    18 in control rats) increased as the dose increased, being highest
    at 300 mg/kg after 14 days of treatment.

         From this study it may be concluded that amitrole, at levels of
    100 mg/kg diet or more, rapidly suppressed T3 and T4 hormone
    levels and maintained the depressed levels during the treatment
    period. Both T3 and T4 levels returned to control values within
    three weeks following withdrawal of amitrole from the diet. The
    no-observed-adverse-effect level (NOAEL) in this study was
    30 mg/kg/diet (Babish, 1977).

         Fregly (1968) investigated the dose-response relationship
    between amitrole administered in the diet and a variety of clinical
    parameters in order to establish the minimal dose with an effect on
    thyroid activity. Groups of male Spruce Farm strain rats (10 per
    dose level) were administered amitrole in the diet at dose levels of
    0, 2, 10 and 50 mg/kg diet for 13 weeks. Body weight gain, food
    consumption, haematocrit, haemoglobin concentration and rate of
    oxygen consumption were unaffected by the treatment. Mean body
    temperature was slightly increased but only at 50 mg/kg. During week
    12, uptake of radioactive iodine was measured at various times
    between 22-53 h after intraperitoneal injection of 131I. A
    slightly lower uptake was found at the highest dose level. At the
    end of the study, radioactivity in the thyroid gland, excised 24 h
    after intraperitoneal injection, was slightly decreased in all
    groups. At the end of the study, the protein-bound iodine (PBI) in
    blood, measured as an indicator for the concentration of thyroid
    hormones, was decreased at all dose levels. The values were
    51 g/litre (control) and 37, 38 and 33 g/litre for the 0, 2, 10
    and 50 mg/kg groups, respectively. In a second experiment the PBI
    levels were not affected by treatment with amitrole at 0.25 and

    0.5 mg/kg diet. Values were 32 g/litre in controls and 39 and
    45 g/litre, respectively, in treated groups. It should be noted,
    however, that PBI control values measured in the second experiment
    were much lower than those measured in the first experiment. This
    implies that there was no biologically significant effect on PBI
    since all values were within the same range. The thyroid weight was
    increased significantly only in the 50-mg/kg group. The number of
    blood vessels/thyroid section, which is a very sensitive measure of
    histopathological changes in the thyroid, was increased at 10 and
    50 mg/kg. It can be concluded that 2 mg/kg diet was the NOAEL in
    this study.

         Several short-term studies were carried out by Den Tonkelaar &
    Kroes (1974) in order to establish a no-observed-effect level on
    thyroid function tests. In all experiments the uptake of 131I by
    the thyroid was measured in an  in vivo test, 6, 24 and 48 h after
    the intraperitoneal administration of 0.6 c 131I per animal. In
    addition, thyroid weight and PBI were measured and the thyroid was
    studied histopathologically.

         In the first experiment, four groups of eight female Wistar
    rats received, respectively, 0, 2, 20 and 200 mg amitrole/kg in the
    diet for 6 weeks. After 5 days and 6 weeks the uptake of 131I was
    measured. On both occasions a significantly increased uptake was
    found in the 200-mg/kg group 6 h after injection, which decreased
    fairly rapidly after 24 and 48 h. At that time the radioactivity was
    lower than that of the controls. The thyroid weight was increased in
    the 200-mg/kg group and histopathologically goitre was found only in
    this group.

         In the second experiment, eight female animals per group
    received, respectively, 0, 20, 50 and 200 mg/kg diet for 6 weeks,
    and similar effects were found in the 200-mg/kg group to those
    observed in the first experiment. In addition, a significant
    decrease in PBI was observed at the end of the experiment compared
    with the control value. At 50 mg/kg, a statistically increased
    uptake was found 6 h after injection of 131I. However, in this
    case the radioactivity in the thyroid remained higher than that in
    the controls after 24 and 48 h. Histopathologically only a very
    slight activation was found. However, the 200-mg/kg group showed
    strong activation and goitre.

         In the third experiment 0, 20, 50 and 200 mg/kg diet were given
    to 10 female animals per group during 13 weeks. The uptake of 131I
    by the thyroid was significantly increased at 200 and 50 mg/kg after
    6 and 12 weeks. The difference between the groups was that at
    50 mg/kg the radioactivity in the thyroid remained high after 24 and
    48 h, whereas with 200 mg/kg a very high uptake was found 6 h after
    injection of 131I but this was followed by a rapid decrease, with
    still lower values than the controls after 48 h. At 200 mg/kg the

    PBI was decreased and the thyroid/body weight ratio increased by a
    factor of 6. At 50 mg/kg only a slightly increased relative thyroid
    weight was found. Histologically, a strong activation and goitre
    were found at 200 mg/kg, and a slight activation at 50 mg/kg. In
    this experiment, a tendency to a higher uptake of 131I was found
    in the 20-mg/kg group.

         The above-mentioned experiments were carried out with a
    relatively low iodine content in the diet (about 0.2-0.3 mg/kg
    diet). In the fourth experiment, a diet containing 2 mg iodine/kg
    was used. In this experiment, eight female rats per group received,
    respectively, 0, 20, 50, 200 and 500 mg amitrole/kg in the diet for
    6 weeks to see whether iodine could protect against the antithyroid
    action of amitrole. At 500 mg/kg, a small increase in iodine uptake
    was found 5 h after 131I injection, but thereafter there was a
    very rapid decrease. At 200 mg/kg, the uptake was much higher and
    the same type of decrease was found as in the other experiments,
    whereas at 50 mg/kg a significantly increased thyroid radioactivity
    was found at all times. PBI was decreased at 200 and 500 mg/kg only.
    Histopathologically, goitre and strongly activated thyroids were
    found at the two highest dose levels. Some activation was found in
    the 50-mg/kg group and a very slight activation was also found in
    the 20-mg/kg group. It can be concluded that measurement of 131I
    uptake at different time points is a sensitive method for the
    effects of amitrole on the thyroid. At 20 mg/kg only slight effects
    were found on uptake and thyroid histopathology. The NOAEL was
    2 mg/kg diet, equivalent to 0.1 mg/kg body weight.

    7.2.1.2  Drinking-water

         When groups of male albino rats (10 per dose level) were
    administered amitrole in the drinking-water at dose levels of 0, 50,
    250 or 1250 mg/litre for 106 days, there was a dose-related decrease
    in body weight gain in all treated groups with a corresponding
    reduction in food and water intake. There were no clinical signs of
    toxicity. At the end of the study, histopathological examination was
    performed on the thyroid, hypophysis, liver, kidney, spleen,
    stomach, small intestine, large intestine, bladder, testis, adrenal
    and lung. The major gross pathological finding was an increase in
    size and vascularity of the thyroid. At the high dose level, colloid
    was absent in large and medium size thyroid follicles. High-dose
    animals also displayed liver steatosis (Bagdon et al., 1956).

         The time-course for development of goitre in rats was examined
    by Strum & Karnovsky (1971). Sprague-Dawley rats were administered
    amitrole in the drinking-water (2.5 mg/ml), and the thyroid of each
    animal was examined by light microscopy at various periods from 3
    days to 6 months. Each rat drank approximately 30 ml water per day.
    After 3 days, the thyroid size was unchanged although cellular
    changes were apparent. By one week, the thyroid was twice its normal

    size with marked structural changes. These changes continued to
    progress with prolonged administration of amitrole. Goitre formation
    was accompanied by increased vascularity and decreased colloid
    content in the follicular cells. Electron microscopy revealed
    pronounced dilation of the endoplasmic reticulum of thyroid cells.
    Thyroid peroxidase activity progressively decreased with
    administration of amitrole.

         The effects of amitrole on thyroid histology were examined in
    seven groups of five female Wistar rats (weighing about 200 g),
    which were given amitrole in their drinking-water (2.5 mg/ml) and
    killed after 1, 2, 3, 10, 30, 50 or 100 days (Tsuda et al., 1973).
    Water consumption was not reported. After 1 and 2 days of exposure
    the only change noted was a slight enlargement of some endoplasmic
    cisternae of the follicular cells. After 3 days the thyroid gland
    was slightly enlarged, follicular colloid was slightly reduced and
    in some follicular cells the cisternae were clearly dilated and
    stained more lightly for peroxidase activity than did normal cells.
    By 10 days the glands had doubled in size, the follicular epithelium
    consisted of low, columnar cells, and colloid had been severely
    depleted. Nuclei had become located basally and slightly elongated
    microvilli projected into the lumen. Peroxidase activity was no
    longer detected in the endoplasmic reticulum cisternae, but in
    portions of perinuclear cisternae. These changes had progressed in
    the 30-day samples, so that the glands were now several times their
    normal size. In addition, fibrous thickening of both stroma and
    capsule was prominent and cisternal peroxidase activity was absent.
    Administration for 50 days resulted in increased irregularity in
    follicular size, more prominent papillary growth of the follicular
    epithelium and greatly diminished peroxidase activity throughout the
    cells.

         Histopathological changes induced by amitrole in the liver of
    mice were investigated by Reitze & Seitz (1985). Groups of male
    albino mice were exposed to amitrole in the drinking-water at dose
    levels of 0.5%, 1.0% or 2% for 30 days (water consumption not
    reported). Light microscopy revealed dose-related hypertrophy of
    hepatocytes, increased pyknotic nucleoli, and increased vacuoles in
    the cytoplasm. Electron microscopy revealed also a dose-related
    proliferation of smooth endoplasmic reticulum.

    7.2.1.3  Intubation

         No data available.

    7.2.2  Inhalational

         Groups of Fischer-344 rats (15 of each sex per dose level) were
    exposed to an atmosphere containing amitrole (94.6% pure) at
    concentrations of 0, 0.1, 0.32, 0.99 or 4.05 mg/litre (nominal
    concentrations adjusted for non-nebulized material) for 5 h/day, 5

    times per week, for 4 weeks (particle size not provided). There were
    no adverse effects on behaviour, and no body weight changes were
    noted. T4 levels were significantly depressed by the 27th day at
    the two highest dose levels. T3 levels were significantly
    depressed by 14 days at all but the lowest dose level. Pathological
    changes were confined to the thyroid, and hyperplasia was noted at
    all but the lowest dose level (Cox & Re, 1978).

    7.2.3  Intraperitoneal

         Alexander (1959a) investigated the uptake of 131I by the
    thyroid gland in Sprague-Dawley rats following intraperitoneal
    injection of approximately 5 or 250 mg/kg body weight. At both dose
    levels thyroid 131I uptake was inhibited, whereas catalase
    activity was decreased by about 50% at the highest dose level only.

         When 328 White Leghorn 3-day old chicks were injected with
    amitrole (500 or 1000 mg/kg day), 5 days per week for 5 weeks,
    increases in the relative thyroid-to-body weight ratio was observed
    in all birds from day 10 onward. In addition, two groups of chickens
    were injected the same doses of amitrole but for 17 consecutive
    days. At the cessation of amitrole treatment, an increase in the
    relative thyroid-to-body weight ratio was observed until day 13;
    this was followed by a decrease and then a stabilization, which
    occurred between days 17 and 41. However, the ratio never attained
    the levels observed in control animals. Histopathological
    examination of the thyroid gland revealed epithelial hyperplasia,
    hyperaemia, obliteration of the follicular lumina and disappearance
    of the colloid. In birds that were injected with amitrole only for
    17 days, the thyroid histology returned to normal two weeks after
    treatment (Wishe et al., 1979).

    7.3  Long-term exposure

    7.3.1  Oral

    7.3.1.1  Mouse

         Reversible thyroid hyperplasia has been reported during
    long-term feeding studies with amitrole at levels of 1000 mg/kg diet
    in both C3H and C57BL mice (Feinstein et al., 1978a). The
    acatalasemic C3H mice survived longer on the amitrole diet than did
    their normal catalasemic counterparts (mean survival times in weeks,
    both sexes combined, were 35  10, n = 141, and 26  10, n = 146,
    respectively, P < 0.001). Similar differences were observed with
    C57BL/6 mice, although group sizes were much smaller (57  5, n =
    12, and 42  7, n = 10, respectively). All mice given the treated
    diet had a reduced body weight gain compared with mice given the
    normal diet. In those mice for which the amitrole diet was withdrawn
    at 12 weeks, the thyroid weight reduced in size gradually but the

    gland was still enlarged after 60 weeks. A larger proportion of the
    acatalasemic C3H mice developed liver tumours, as compared with
    normal catalasemic C3H mice. Out of 87 mice in the acatalsemic
    group, 21 developed liver tumours that were detected earlier
    (beginning at 35 weeks) compared with the normal catalase mice (6/85
    beginning at week 50) (Feinstein et al., 1978b).

         In a life-time study in NMRI mice (dose levels 0, 1, 10,
    100 mg/kg diet), the appearance, behaviour, food intakes, body
    weights and survival times of the treated mice did not differ from
    those of the controls. The frequency of pituitary hyperaemia was
    slightly elevated in the high-dose group; no treatment-related
    histological lesions were otherwise found. The frequency of types
    and distribution of tumours in the control and treated groups were
    similar. The thyroid weights were elevated in male high-dose group
    mice at all dose levels and were up to three times the weights in
    the control group. The percentage of iodine accumulation and the
    iodine level in the thyroid were elevated in the male mice of the
    100-mg/kg group. The sum of PBI in the male mice was elevated nine
    months after study initiation, but was depressed at later test
    dates. Comparable results were observed in the female high-dose
    group mice. However, the deviations from control group values were
    generally smaller than in the males, and were not significant in
    most cases (Weber & Patrick, 1978; Steinhoff & Boehme, 1979b).

    7.3.1.2  Rat

         The long-term effects of oral administration of amitrole in
    rats have been described in two detailed reports by Keller (1959)
    and Johnson et al. (1981). The details and results of these studies
    are given below.

         In the study by Keller (1959), groups of Carworth Farm Wistar
    rats (35 of each sex per dose level) were administered amitrole in
    the diet at dose levels of 0, 10, 50 or 100 mg/kg diet for two
    years. After 13 and 68 weeks, 5 and 3 animals of each sex and dose
    level, respectively, were killed for organ weight measurement and
    histopathological examination. A separate group received 500 mg/kg
    diet for 19 weeks, followed by the control diet for 7 weeks, and
    then were killed. In this group, body weight gain and food
    consumption were markedly reduced during the amitrole
    administration. Animals in all groups, including the controls,
    suffered from apparent respiratory infection and 67 of them died,
    but there was no relationship with the treatment. Body weight gain
    was reduced at 100 mg/kg in male animals during the first 13 weeks
    of the study. After 68 and 104 weeks of treatment, relative thyroid
    weight was increased at 100 mg/kg (not measured after 13 weeks).
    Histopathological examination after 13 weeks showed hyperplasia and
    hypertrophy of the thyroid at 500 mg/kg; these effects were found to

    be reversible. Histopathological changes in the thyroid were also
    seen at 100 mg/kg and in one animal at 50 mg/kg. At 68 weeks three
    animals given 50 mg/kg showed definitive evidence of hyperplasia,
    while all animals given 100 mg/kg displayed hyperplasia and
    hyperfunctioning of the thyroid. At 104 weeks tumours were found
    (see section 7.7.2). Based on thyroid hyperplasia, the NOAEL was
    10 mg/kg diet (equivalent to 0.5 mg/kg body weight).

         In a chronic toxicity study by Johnson et al. (1981), groups of
    Fischer-344 rats (75 of each sex per dose level) were administered
    amitrole. Group A were the controls. Group B rats were fed 5 mg
    amitrole/kg in their diet during weeks 1-39 and then 100 mg/kg
    during weeks 40-115 (for males) or 40-119 (for females). Rats in
    groups C, D and E received amitrole in their diet at pulsed
    intervals (alternate 4-week periods) of 1, 3 and 10 mg/kg,
    respectively, during weeks 1-39 and 20, 60 and 100 mg/kg,
    respectively, during weeks 40-115 (for males) or 40-199 (for
    females). There were no treatment-related clinical signs of toxicity
    or changes in body weight or food consumption. There were no
    consistent effects on serum T3 and T4 levels. Thyroid weight was
    increased in both males and females in groups B and E after 60 weeks
    and at termination. There were no treatment-related pathological
    changes up to 36 weeks (when only the lower dose levels were
    administered). Follicular epithelial hyperplasia in the thyroid was
    noted in groups B, D and E and to a much lesser extent in group C.
    An increased incidence in thyroid tumours was observed in male and
    female rats of groups B and E and in the male animals of group D.
    There was no significant difference in tumour incidence between
    groups B and E. It should be noted that this study was poorly
    reported.

         When amitrole was administered to groups of Wistar rats (75 of
    each sex) at concentrations in the feed of 0, 1, 10 or 100 mg/kg, no
    effect on body weight gain or food intake was observed but a slight
    decrease in survival time was found at 100 mg/kg. Thyroid weight was
    increased at 100 mg/kg as was uptake of 131I by the thyroid,
    measured 24 h after oral administration of 131I. For this
    measurement, five animals of each sex per group were killed at 3, 6,
    12 and 24 months. PBI, measured as the ratio between radioactivity
    in plasma protein and total plasma, was not affected. At the highest
    dose level, elevated incidences of haemorrhage and hyperaemia of the
    pituitary gland, as well as a very high rate of cystically dilated
    thyroid follicles, were seen. The tumour incidence is given in
    section 7.7.2. The NOAEL was 10 mg/kg diet, equivalent to 0.57
    (males) or 0.85 (females) mg/kg body weight (Weber & Patschke 1978;
    Steinhoff & Boehme, 1979a).

         Authors who have studied the time-course of the response of the
    thyroid to amitrole treatment (e.g., Strum & Karnovsky, 1971; Tsuda
    et al., 1973; Wynford-Thomas et al., 1983) have shown that, after a
    short lag phase of a few days, there is a rapid rise in TSH that is
    paralleled by thyroid hypertrophy and hyperplasia. These effects
    peak and plateau after 3-4 months and thereafter remain relatively
    stable despite further exposure. A number of studies have shown that
    the goitrogenic action of amitrole is reversible on cessation of
    exposure (Jukes & Shaffer, 1960).

    7.3.1.3  Other species

         Other species in which long-term amitrole treatment has been
    studied are the hamster and dog.

         In a carcinogenicity study on hamsters (Steinhoff & Boehme,
    1978; Steinhoff et al., 1983; see section 7.7), there were no
    pathological changes at dose levels of up to 100 mg/kg diet. In a
    one-year study in dogs, amitrole was given in capsules at dose
    levels of 0, 0.25, 1.25, 2.50 and 12.5 mg/kg body weight per day,
    6 days/week. There were no clinical signs of toxicity or
    pharmacological effects. Haematological, biochemical and urinalysis
    parameters were comparable to those of control dogs and were within
    normal limits. The dogs fed 12.5 mg/kg per day had a pale pancreas.
    Histopathological examination of all dogs did not reveal any
    treatment-related effects. The thyroid, in particular, was normal at
    all dose levels (Weir, 1958; Hodge et al., 1966).

    7.3.2  Other routes

         In a chronic 104-week study, 25 male and 25 female rats
    (Charles River strain) were exposed (head-nose only) to an amitrole
    aerosol (the purity of the amitrole used was not specified) for one
    hour each week. An aqueous 0.24% (w/v) solution of amitrole was used
    to generate the aerosol. The mean analytical concentration in the
    inhalation chamber was 2 mg aerosol per litre of air; based on dry
    amitrole, the level was 5 g/litre air. A control group was exposed
    to a water aerosol. No differences between the control group animals
    and those exposed to the test substance were found in the mortality,
    appearance, behaviour or body weight development. No
    treatment-related changes were observed at necropsy. No differences
    in the thyroid or liver weights, or in the incidence of tumours,
    existed between the two groups of animals (Grapenthien, 1972).

         In an inhalation study involving intermittent treatment, groups
    of 75 Fischer rats per dose and of each sex were exposed to aerosols
    at nominal amitrole levels of 0, 50, or 500 g/litre air (the purity
    of the amitrole used was not specified). The actual amitrole
    concentrations in the low-dose group varied between 15.8 and
    32.2 g/litre air on different days of exposure, and the levels

    measured in the high-dose group ranged between 97.9 and
    376.4 g/litre air. The animals were exposed for 5 h per day on 5
    days per week. The treatment phases during weeks 1-13, 40-52 and
    78-90 were interrupted by treatment-free intervals. Interim
    necropsies of five animals per dose group and of each sex were
    performed after 3, 9 and 18 months, and the study was concluded
    after 24 months. A total of 28 rats died in week 51 due to a defect
    in the air conditioning system, which led to an increase in the room
    temperature. Treatment of the high-dose group was thereupon
    concluded, and the surviving animals were necropsied.

         The food intake and body weight gain were decreased in the
    high-dose group, and the rate of mortality was elevated.

         Decreases in the T3 (significant) and T4 (non-significant)
    levels were only observed in the high-dose group, this being
    assessed in the 13th week of the study. However, values in the
    amitrole-treated animals were greater than, or equal to, those of
    control rats at all other test dates (weeks 39, 52, 78, 91 and 104).
    Epithelial hyperplasia of the thyroid follicles was observed in both
    dose groups at the end of the first treatment interval (week 13).
    This observation was no longer made after a treatment-free interval
    of 24 weeks, but the thyroid weights relative to those in the
    control animals were elevated in both dose groups. Follicular
    epithelial hyperplasia was again present in most of the animals of
    both treatment groups at the end of the second treatment phase (week
    51). This observation was still made after a treatment-free interval
    of 26 weeks, which indicates that complete reversion no longer
    occurred at this time at an amitrole level of 50 g/litre air.
    Neoplasms of the thyroid (adenomas and adenocarcinomas) were found
    in addition to hyperplasia at terminal necropsy (Becci, 1983).

         Twenty-five male and 25 female rats (Charles River strain) were
    dermally exposed to an 0.239% aqueous solution of amitrole (the
    purity of the amitrole used was not specified) once weekly for 30
    min over a period of 23 months (total of 100 exposures). The
    treatment volume was 1 ml/kg body weight, and about 20% of the body
    area was treated. The dermal exposure to amitrole thus amounted to
    2.39 mg/kg body weight per week. The treatment did not cause skin
    damage. No differences between the control group and animals exposed
    to the test substance were found with respect to mortality,
    appearance, behaviour or body weight development. No
    treatment-related alterations were determined at necropsy. No
    differences in the thyroid or liver weights, or in the incidence of
    tumours, were found between the two groups of animals (Rausina,
    1972).

    7.4  Skin and eye irritation; skin sensitisation

         The potential for dermal irritation by amitrole was examined in
    rabbits over a 24-h period following a single application of between
    10 and 100 mg/kg body weight (Elsea, 1954). Mild erythema was
    observed at the high-dose level only. By 48 h, the skin appeared
    normal. The potential for eye irritation by amitrole was examined in
    rabbits following application of 3 mg into the conjunctival sac of
    the left eye (Elsea, 1954). Observations were made at 1, 4, and 24 h
    and at daily intervals for 6 days. Mild irritation was observed at
    4 h in all animals, but the majority of animals had recovered by
    24 h.

         Amitrole was tested for possible dermal sensitization potential
    in guinea-pigs using the Magnusson-Kligman maximization test with
    Freund's adjuvant. The concentrations employed were 2.5% for
    intracutaneous induction, 25% for topical induction, and 12% for the
    first and second challenges. Evidence for moderate skin-sensitizing
    potential in amitrole was found after both challenges (Mihail,
    1984).

         No skin-sensitizing effect was observed in a Klecak open
    epicutaneous test involving treatment of three groups of animals
    with 3%, 10% or 30% amitrole formulations in the induction phase
    (Mihail, 1985).

    7.5  Reproduction, embryotoxicity and teratogenicity

    7.5.1  Reproduction

         In a preliminary one-generation reproduction study by Gaines
    et al. (1973), groups of 10 male and 10 female rats were fed
    amitrole in the diet at concentrations of 0, 500 or 1000 mg/kg for
    55 days before pair-mating. The offspring were weaned at 21 days.
    Complete autopsies were performed on the parents after a total
    exposure of 107-110 days. Ten weaning rats from each dose groups
    were killed. Mean body weight gain was reduced at all dose levels.
    The average number of pups per litter was significantly reduced at
    all dose levels, as were the number surviving to weaning. The body
    weight of pups at weaning was also reduced. Relative kidney, spleen
    and liver weights were also reduced in parents following treatment,
    while thyroid hyperplasia was noted in all treated animals.

         In a subsequent multi-generation study by Gaines et al. (1973),
    groups of 10 male and 10 female rats were fed amitrole at dietary
    levels of 0, 25 or 100 mg/kg for 61 and 173 days before pair-mating
    to produce the F1A and the F1B generations, respectively. The
    thymus and spleen were examined in weanling rats in the F1A
    generation. There was no treatment-related effect on body weight
    gain in the FO animals. Hyperplasia of the thyroid was observed in
    all animals at the highest dose level but not at 25 mg/kg.

    Reproduction parameters were normal at these dose levels. A slight
    decrease in body weight gain was noted at 25 and 100 mg/kg in pups
    of the F1A and F1B generation. Pathological examination revealed
    a slight but significant decrease in liver weight at 25 and
    100 mg/kg (male pups) and at 100 mg/kg (female pups). The thymus and
    spleen sizes were normal and no histopathological changes could be
    detected. F2A generation rats showed a decrease in the number of
    litters at 100 mg/kg but there were no other changes, such as
    survival to weaning and mean body weight at weaning.

    7.5.2  Embryotoxicity and teratology

         Teratology studies have been performed in rats, mice and
    chickens.

         In a study by Gaines et al. (1973), three groups of pregnant
    rats were administered amitrole by gavage at dose levels of 0, 20 or
    100 mg/kg body weight per day on days 7 to 15 of gestation, and the
    animals were allowed to litter and to wean. There was no evidence of
    gross abnormalities among the pups.

         In a further teratology study on rats by Machemer (1977b),
    groups of 20 presumed pregnant rats (strain FB30, Long-Evans) were
    administered amitrole by gavage at dose levels of 0, 100, 300 or
    1000 mg/kg body weight per day on days 6 to 15 of gestation, and
    fetuses were examined on day 20 of gestation. There were no deaths
    or signs of toxicity at any dose level. Body weight gain was not
    affected by treatment. There were no treatment-related effects on
    the resorption rate, fetal weight, number of live fetuses, placental
    weight or sex ratio. There was no treatment-related increase in
    gross, skeletal or visceral malformation.

         In a study by Tjalve (1974), pregnant mice were administered
    amitrole at 500, 1000, 2500 and 5000 mg/litre in the drinking-water
    on days 6-18 of pregnancy. There was a marked decrease (22-28%) in
    body weight gain in the dams and pronounced retardation in the
    development in their fetuses at dose levels > 1000 mg/litre. At
    the highest dose level used, maternal toxicity was associated with
    an increase in the rate of resorption. No teratogenic effects were
    observed at any dose level.

         Teratogenicity in chickens was investigated by injecting the
    yolk sac of eggs with amitrole at dose levels of between 0.5 and
    40 mg at 0, 24, 48 and 96 h of incubation (Landauer et al., 1971).
    Dose-dependent abnormalities of the beak were found to be present in
    chickens following the administration of 20-40 mg amitrole at 24 and
    48 h. When injected after 96 h of incubation, beak abnormalities
    could be found at dose levels of 10, 20 and 40 mg at a rate of 20,
    48, and 60%, respectively. No effects were seen at dose levels up to
    and including 2 mg/egg.

    7.6  Mutagenicity and related end-points

         A referenced summary of the test results with amitrole is given
    in Table 4. The important features of these data are described
    below.

    7.6.1  DNA damage and repair

         The possibility of DNA damage being induced by amitrole has
    been investigated frequently and in a number of different ways. In
    bacteria, the results have been negative, except in one experiment
    with the  rec assay, in which exogenous metabolic activation was
    provided by "liver" preparations from a mollusc and a fish. Among
    assays which could be evaluated, a DNA repair assay in yeast gave a
    positive result, as did a repair assay in mammalian cells.

    7.6.2  Mutation

         One study in a single laboratory with  Escherichia coli and
     Salmonella typhimurium strains gave significant responses (Venitt
    & Crofton-Sleigh, 1981). Amitrole did not induce joint mutations in
    histidine-requiring mutants of  S. typhimurium (Andersen et al.,
    (1972). An equivocal response was obtained in another bacterial
    mutation assay, but many other  in vitro assays gave negative
    results. A significant result was obtained in a mouse peritoneal
    host-mediated assay with  S. typhimurium (Simmon et al., 1979). No
    mutation induction has been observed in yeast or fungi. In
     Drosophila melanogaster, a significant response was obtained with
    a wing-spot test in a single study, but not with several sex-linked
    recessive assays. Mutations were not induced in mouse lymphoma
    cells, but  hprt locus and Na+/K+ ATPase locus mutations were
    induced in Syrian hamster embryo cells (Tsutsui et al., 1984). These
    latter results may hold particular significance in view of other
    properties of these cells described in sections 7.6.4 and 7.6.5.

    7.6.3  Chromosome damage

         In yeast, there is conflicting evidence for recombinogenic
    activity (intragenic and mitotic recombination), while numerical
    chromosomal aberrations were induced in three assays. Structural
    chromosomal damage was not induced by amitrole in cultured mammalian
    cells, but the frequency of sister-chromatid exchanges was increased
    in a single study. No effects of amitrole were observed in mice
    subjected to bone marrow micronucleus tests or male dominant lethal
    tests.

    7.6.4  Cell transformation

         Assays for anchorage-independent growth and cell transformation
    in several systems consistently gave positive results.



        Table 4.  Summary of mutagenicity and related end-point studies on amitrole
                                                                                                                                              

    Test                    Organism                                    Result                 LED or HIDe          Reference
                                                                        -S9h      +S9i
                                                                                                                                              

    Microorganisms

    Prophage induction       E. coli 58-161 enVA, lambda                 n.t.      -            10 000 g/ml         Thomson (1981)
                            prophage

    Prophage induction       E. coli GY5027, GY4015                      n.t.      -            2000 g/plate        Mamber et al. (1984)

     Rec assay              Bacillus subtilis H17 rec+, M45 rec-        -         n.t.         g/plate             Shirasu et al. (1976)

     Rec assay              Bacillus subtilis H17 rec+, M45 rec-        -         +a           1000 g/plate        Kada (1981)

     Rec assay              E. coli JC 2921, 9238, 8471, 5519,          -         -            500 g/ml            Ichinotsubo et al. (1981)
                            7623, 7689

     Rec assay              E. coli WP2, WP100                          n.t.      -            4000 g/ml           Mamber et al. (1983)

     Pol assay              E. coli pol A1, pol A+                      -         n.t.         g/plate             Bamford et al. (1976)

     Pol assay              E. coli WP3110, p3478                       -         -            333 g/plate         Rosenkranz et al. (1981)

    Differential killing     E. coli WP2, WP67, CM 871                   -         -            1000 g/ml           Tweats (1981)

    Reverse mutation         S. typhimurium TA1535, TA1537, TA1538       -         -            100 g/plate         Brusick (1975)

    Reverse mutation         S. typhimurium TA1535, TA1538,              -         -            1000 g/plate        Prince (1977)
                            TA98, TA100

    Reverse mutation         S. typhimurium TA1535, TA1536,              -         -            2000 g/plate        Carere et al. (1978)
                            TA1537, TA1538
                                                                                                                                              

    Table 4 (contd).
                                                                                                                                              

    Test                    Organism                                    Result                 LED or HIDe          Reference
                                                                        -S9h      +S9i
                                                                                                                                              

    Reverse mutation         S. typhimurium TA1535, TA1538               -         -            250 g/plate         Rosenkranz & Poirier
                                                                                                                    (1979)

    Forward mutation         S. typhimurium TM 677                       -         -            100 g/ml            Skopek et al. (1981)

    Reverse mutation         S. typhimurium TA1535, TA1537,              -         -            12 500 g/plate      Herbold (1980)
                            TA98, TA100

    Reverse mutation         S. typhimurium TA1535, TA1537,              -         -            2000 g/plate        Brooks & Dean (1981)
                            TA1538, TA98, TA100, TA92

    Reverse mutation         S. typhimurium TA1537, TA98, TA100          -         -            5000 g/plate        MacDonald (1981)

    Reverse mutation         S. typhimurium TA1535, TA1537, TA1538,      -         -            10 000 g/plate      Richold & Jones (1981)
                            TA98, TA100

    Reverse mutation         S. typhimurium TA1535,  TA1537, TA1538,     -         -            2000 g/plate        Rowland & Severn (1981)
                            TA98, TA100

    Reverse mutation         S. typhimurium TA1535, TA1537, TA1538,      n.t.      -            2500 g/plate        Trueman (1981)
                            TA98, TA100

    Reverse mutation         S. typhimurium TA98, TA100                  n.d.      +            f                    Venitt & Crofton-Sleigh
                                                                                                                    (1981)

    Reverse mutation         S. typhimurium TA98, TA100                                       500 g/ml            Hubbard et al. (1981)

    Reverse mutation         S. typhimurium TA1535, TA1537, TA98         -         -            1000 g/ml           Gatehouse (1981)

    Reverse mutation         S. typhimurium TA1535, TA1537, TA1538,      -         -            5000 g/plate        Moriya et al. (1983)
                            TA98, TA100
                                                                                                                                              

    Table 4 (contd).
                                                                                                                                              

    Test                    Organism                                    Result                 LED or HIDe          Reference
                                                                        -S9h      +S9i
                                                                                                                                              

    Reverse mutation         S. typhimurium TA1535, TA1537, TA1538,      -         -            333 g/plate         Dunkel et al. (1984)b
                            TA988, TA100

    Reverse mutation         E. coli WP2, WP2uvrA                        nd        +            f                    Venitt & Crofton-Sleigh
                                                                                                                    (1981)

    Reverse mutation         E. coli WP2uvrA                             -         -            500 g/plate         Gatehouse (1981)

    Reverse mutation         E. coli WP2uvrA                             -         -            5000 g/plate        Moriya et al. (1983)

    Reverse mutation         E. coli WP2uvrA                             -         -            333 g/plate         Dunkel et al. (1984)b

    Forward mutation         E. coli CHY832                              +         -            2500 g/ml           Hayes et al. (1984)

    Forward mutation         Streptomyces coelicolor A3(2)                        n.t.         g/plate             Carere et al. (1978)

    Host mediated            S. typhimurium TA1950 in NMRI mouse         -         n.t.         2900 mol/kg         Braun et al. (1977)
    reverse mutation

    Host mediated            S. typhimurium TA1530, TA1535, TA1538       +         n.t.         1585 mg/kg i.p.      Simmon et al. (1979)
    reverse mutation        in Swiss-Webster mouse

    DNA repair               Saccharomyces cerevisiae 197/2d             +         -            100 g/ml            Sharp & Parry (1981b)
                            rad 3, rad 18, rad 52, trp 2

    Reverse mutation