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    PESTICIDE RESIDUES IN FOOD - 1980


    Sponsored jointly by FAO and WHO






    EVALUATIONS 1980





    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Expert Group on Pesticide Residues
    Rome, 6-15 October 1980




    IMAZALIL

    Explanation

    Imazalil was reviewed by the Joint Meeting in 1977 (FAO, 1978) and
    a temporary ADI for man was estimated to be 0-0.01 mg/kg body
    weight. Imazalil is a moderately toxic fungicide from the group of
    N-substituted imidazoles.  Studies in rats have suggested that
    imazalil is rapidly absorbed, distributed, metabolized, and
    excreted following oral administration, with the major quantities
    of metabolites occurring in urine and faeces within 3 days of
    administration.  Imazalil does not bioaccumulate.  Tissue residues
    are observed predominantly in those parts of the body associated
    with biodegradation and elimination.  Imazalil is not mutagenic as
    evidenced by a dominant lethal study in mice and is not teratogenic
    in rats.  Long-term studies in the rat and a 2-year study in the
    dog were believed to be inadequate because of unresolved pathology
    questions concerning rat kidney and dog liver.

    Based on these considerations, a temporary ADI was allocated and
    further work was requested with respect to additional long-term,
    short-term, acute toxicity, pharmacokinetic and metabolic studies.

    Additional data (and a further interpretation of the previously
    reviewed data) were submitted, reviewed, and are considered in this
    monograph addendum.

    Further metabolic studies with imazalil labelled with 14C and
    further information on the nature and level of metabolites and
    degradation products in plants were considered desirable.

    A study was set up in order to provide information on the metabolic
    fate of imazalil on growing banana plants.  The results of
    experiments have been submitted to the Joint Meeting for
    evaluation.  Additional information was obtained on use pattern and
    national tolerances.


    DATA CONSIDERED FOR DERIVATION OF ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, distribution, and excretion

    A single lactating goat was orally administered imazalil (3H,
    tritium-labelled) twice a day at a daily dose of 1.0 mg/kg body
    weight for 3.5 days.  Twenty-four hours after the final dose, the
    animal was sacrificed and tissue residues were determined. 
    Qualitative and quantitative evaluations for tissue and excreted
    residues were made.

    Within 24 hours of the last dose, 96.6% of the administered
    imazalil was excreted, predominantly in the urine (70%). 
    Approximately 0.14% of the administered radioactivity was secreted
    in milk.  (A slight translocation of the tritium isotope to water
    may have made this milk residue higher than the actual imazalil
    residue).  Tissue residues were extremely low, not exceeding 0.03
    mg/kg.  Over the course of the study, an attempt was made to
    determine whether translocation of the radioisotope tritium had
    occurred with water.  It was observed that very little tritiated
    water was recovered, confirming the stability of the radioisotope
    in the metabolism study.  Within 36-hours, a steady state appeared
    to be reached with respect to residues in milk and the plasma level
    of imazalil.  The steady-state concentrations in all cases were
    extremely low.  In urine, approximately 15% of the radioactivity
    was present as free base metabolites and 14% as conjugated basic
    metabolites.  About 30% of the urinary metabolites were acidic,
    polar products.  The metabolic sequence with respect to the goat
    was almost identical to that previously proposed for the rat.

    After the last dose, plasma radioactivity decreased rapidly with
    the half-life of approximately 19 hours.  Highest tissue levels
    were measured in the GI tract and in tissues associated with
    biodegradation and excretion.

    It was concluded that imazalil is rapidly absorbed and eliminated
    from the body (within 24 hours).  Approximately 70% of the residue
    was found in urine, 26% in the faeces, small fractions were
    observed in milk and as a residue in tissues (tissues predominantly
    related to the excretory system).  The steady-state condition was
    rapidly reached on multiple dosing and residue data did not suggest
    bioaccumulation (Meuldermans et al, 1979).

    Biotransformation

    Studies on the metabolic fate of imazalil (predominantly in
    excreta) showed that imazalil was extensively metabolised with only
    about 3% of the total administered dose excreted as unchanged
    imazalil in the faeces.  More than 10 acidic urinary metabolites
    were detected.  Five major basic metabolites were characterised. 
    Figure 1 demonstrates a qualitative evaluation of the metabolism of
    imazalil.

    Imazalil was metabolised extensively in the goat with only
    approximately 3% of the parent chemical recovered unchanged,
    predominantly in the faeces.  Five major basic metabolites were
    characterised resulting from oxidation and degradation of the
    imidazole ring, from epoxidation and subsequent hydration of the
    molecule; and oxidative O-dealkylation (Meuldermans et al, 1979).

    The same qualitative metabolic picture with imazalil and its
    congeners has been seen in various mammalian species.  In man and
    the rat, metabolism of a drug similar in chemical structure to
    imazalil (econazole) was reported to be similar.  Additionally, the

    same pathway was observed in monkey and dog as well as rats with 
    respect to other chemicals of this class of N-substituted imidazoles
    (Heykants, 1978).

    From all the data on imazalil and on the congeners of imazalil, it
    appears that the metabolic fate in all mammalian species is
    comparable.


    FIGURE 1

    TOXICOLOGICAL STUDIES

    Special studies on reproduction

    Groups of rats (10 male and 20 female rats/group) were fed imazalil
    in the diet at dosage levels of 0, 50, 200 or 800 mg/kg and
    subjected to a standard 3-generation, 2-litter per generation,
    reproduction study. Observations included: growth, food
    consumption, mortality, and the standard indices of reproduction
    (mating, fertility, gestation, viability and lactation).

    The reproductive performance of rats was unaffected by imazalil at
    any dose level.  There was no maternal mortality and the parental
    generations were unaffected by imazalil.  No foetal abnormalities
    nor embryonic effects were noted.  Litter size, weight and survival
    were normal.  There were no effects on the reproductive performance
    of rats continuously fed imazalil in the diet at levels up to and
    including 800 mg/kg (Marsboom, 1978).

    Acute toxicity

    Rat

                                                       
    Chemical Form    Sex               LD50
                               mg/kg          (CL)
                                                       

    Nitrate           M         343         (262-448)
                      F         288         (221-377)

    Sulphate          M         355         (272-464)
                      F         309         (237-404)

    Acetate           M         371         (284-485)
                      F         309         (237-404)

    Free Base         M         343         (262-448)
                      F         227         (174-297)
                                                       

    All data were generated with adult rats, administered imazalil
    orally as an aqueous suspension.  The acute signs of poisoning were
    the same for each chemical form of imazalil.  These included:
    ataxia, piloerection, hypotonia, hypothermia, exophthalmia,
    tremors, salivation, lacrimation, diuresis, diarrhoea, palpable
    ptosis, and loss of the righting reflex (Niemegeers, 1979).

    Long-term studies

    Rat

    Groups of rats (50 male and 50 female Wistar rats/group) were
    administered imazalil in the diet at dosage levels of 0, 25, 100 or
    400 mg/kg for 24 months.  The animals were 3-4 months of age at the
    initiation of the carcinogenicity study.

    Animals were examined daily for behaviourial changes and toxicity. 
    At the conclusion of the study, gross and microscopic examination
    was performed on all surviving animals.  During the course of the
    study, examinations were performed on animals that had died or were
    sacrificed in extremis.  Microscopic examinations were performed on
    the major tissues and organs and on all lesions that were noted
    during the course of gross examination.

    There was substantial mortality over the course of the study, and
    very few animals survived the full 24 months.  It was considered
    that this was due to the fact that the animals were 3-4 months old
    at the start of the study and that they were not maintained under
    SPF-conditions. Evaluation of the data, relating to the time on
    study at which animals died, did not suggest that imazalil
    contributed to the increased mortality or the accelerated death
    rate.  There were no dose-related events noted on gross pathology.

    There were no significant differences with respect to the overall
    tumour rate or the individual types of tumours reported in any of
    the does groups.  There is no evidence that imazalil was
    responsible for tumour formation in any organ or tissue examined in
    the study (Marsboom and Herin, 1979a).

    Mice

    Groups of mice (50 male and 50 female albino, Swiss mice/group)
    were administered imazalil in the drinking water for 18 months at
    dosage levels of 0, 6.25, 25, or 100 mg/kg.  It was approximated
    that the dietary levels reflect an intake of 0, 2.5, 10 or 40 mg/kg
    body weight based upon an assumption that each mouse drinks
    approximately 100 ml/week.

    All animals were examined daily for behaviour abnormalities and
    clinical toxicity.  At the termination of the study, gross and
    microscopic examinations were performed on all surviving animals. 
    A complete examination was performed on animals dying during the
    course of the study or that had to be sacrificed before
    termination.  Gross and microscopic examinations were made on
    selected tissues and organs. (Gross examinations did not include
    organ-weight data).  In addition, any tissue that was suspect of
    unusual lesion was examined histologically.

    There was no apparent dose-related effect on survival or on the
    time that mortality occurred.  At the conclusion of 18 months,

    there was a substantial number of animals (greater than 25% in all 
    cases) surviving.  There were no significant differences with 
    respect to survival in any of the test groups when compared to 
    control values. There were no dose-related effects on health, 
    behaviour, or appearance.  An examination of the data on the 
    occurrence of tumours in animals dying or sacrificed at the end 
    of the study showed that there were no substantial differences 
    between groups or with control values.  Imazalil in the drinking 
    water did not influence the occurrence of tumours.  It was 
    concluded that under the conditions of this assay, imazalil was 
    not a tumourigen or a carcinogen in mice (Marsboom and Herin, 
    1979b).

    RESIDUES IN FOOD

    USE PATTERN

                                                                  
    Crop/               Formulation and mode     Concentration
    disease                of application              ai
                                                                  

    Gherkin (outdoors)     20 EC, 0.025%          0.05 kg/ha
    powdery mildew

    Barley
    Pyrenophora spp.       liquid, 2.4%           48 mg/kg seed
    Fusarium

    Wheat, barley          seed dressing
    oats, rye              50 mg/kg
                                                                  

    RESIDUES RESULTING FROM SUPERVISED TRIALS

    The results of recent trials are summarised in Table 1.



        TABLE 1.  Residues resulting from supervised trials in the Netherlands

                                                                                                       
    Crop           Year of               Application                   Residues in mg/kg, at intervals
                    trial                                               (days) after (last) application
                             Number of    Rate per    Formulation
                             treatments   treatment                                                    
                                          kg ai/ha                         3         4          60
                                                                                                       

    wheat           1976         1          0.125        liquid                               <0.011
    wheat-straw     1976         1          0.125        200 g/l                              <0.11
    gherkin         1977         1          0.1          liquid          <0.011
    (glasshouse)                                         200 g/l
        "           1977         1          0.15         200 g/l                    0.04
                                                                                 (0.03-0.05)
                                                                                                       

    1 Limit of determination
    


    FATE OF RESIDUES

    Eleven growing banana plants with a minimum of six leaves were used in
    the study.  One of the banana plants (No. 1) was not treated and
    served for providing blank samples.  The others were sprayed uniformly
    from above with a distilled water solution of 3N-imazalil-sulphate
    labelled specially on the asymmetric carbon.  The plants were treated
    once, four, five or nine times consecutively at two-week intervals.

    The sampling of the individual plants was carried out as follows:

    One plant (No. 1) was sampled without any previous treatment, serving
    as a blank.  Plants Nos. 2, 3 and 4 were sampled two hours (the time
    necessary for drying), four days and fourteen days respectively after
    a single treatment, Plant No. 5 fourteen days after the fourth
    treatment, plants Nos. 6, 7 and 8 two hours, four days and fourteen
    days respectively after the fifth treatment and plants Nos. 9, 10 and
    11 two hours, four days and fourteen days respectively after the ninth
    treatment.

    Five different parts of the plants were investigated: mature leaves,
    new leaves emerged after the last treatment, psuedostem, rhizome and
    roots (petioles were considered part; of the psuedostem).

    The roots were out from the rhizome and the soil was washed off with
    water.

    The various parts of plants were minced and thereafter homogenised in
    water (1/5, w/v).

    The radioactivity levels of samples were measured in the aliquots of
    the homogenates immediately after homogenisation.

    The leaf homogenates of the treated plants and the spiked blank
    homogenate were subjected to repeated extraction.  The radioactivity
    of the various extracts was measured.  Some of the extracts were
    analysed with on-line radio HPLC after sample clean up on Sep-pak
    TMC18 cartridges.  A part of leaf and a part of cigar leaf of plant
    No. 11 were prepared for autoradiography.

    The radioactivity levels in the various plant samples indicated fairly
    uniform sprayings and the absence of tritiated water or other volatile
    radioactive compounds.  The leaves contained 95.2-100% of the total
    radioactivity recovered in the plants treated one to nine times.  The
    radioactivity was practically all concentrated at the upper surface of
    the leaf tested with autoradiography.  Transport of the radioactivity
    to the roots (0.32% maximally) or the rhizome (2.54% maximally) was
    minimal.  In the leaves emerged after the last treatment less than
    0.03% of the radioactivity recovered in the whole plant was detected.

    The metabolite pattern in the leaf homogenates was investigated after
    extraction.

    The scheme of extraction and the percentage ratio of the total
    radioactivity found in various extracts are shown in Figure 1.

    The differences between the various homogenates of the leaves of
    plants that were sprayed once, four, five or nine times were only
    minor.  Attempts to extract the radioactivity from the final residues
    by soxhlet with chloroform, ethyl acetate and acetone or by batch
    extraction with 19% trichloroacetic acid failed.

    Analyses of various plant extracts on radio-HPLC revealed that the
    main part of radio-activity originated from imazalil and its
    metabolite alpha-(2,4-dichloro-phenyl)-1H-imidazole-1-ethanol
    (R14821).  Unchanged imazalil constituted 33.8, 46.9, 37.1, and 22.3%
    and R 14821 23.4, 22.2, 27.9 and 37.8% of the radioactivity in the
    alkaline extracts of the leaves of plants Nos. 8, 9, 10 and 11
    respectively.  These results indicated that imazalil was degraded
    slowly as a function of time and that R 14821 was the main degradation
    product which was found in the pH11 extract too.  Although the
    radioactivity in the extracts of pH 11, pH 9, ph 5 and pH 2 amounted
    to 15.38% of the alkaline extract, only a few small radioactivity
    peaks could be detected, which may be formed at least partly
    artificially from imazalil and R 14821 as similar peak patterns
    appeared on the chromatograms of the extracts of blank homogenate
    spiked with these compounds.

    The remaining part of the radioactivity might be explained with the
    presence of a large number of minor metabolites.  R 14821 (45.4%) and
    imazalil (20.7%) were also major radioactive compounds in the soxhlet
    extract.

    The parent compound accounted for altogether about 20 and 15% of the
    total radioactivity while the proportion of R 14821 was 15% and 25% in
    the leaves of plants Nos. 8 and 11 respectively.

    A mixture of unidentified polar acid metabolites (7% of the total
    radioactivity) was found in both the pH 2 extract and the final
    aqueous layer.  From 7-17% of the radioactivity in the leaf
    homogenates could not be extracted.

    The rest of the radioactivity, 35 to 45%, was due to a very large
    number of minor metabolites (Meuldermans, W. et al, undated).


    EVALUATION

    COMMENTS AND APPRAISAL

    Imazalil was reviewed by the 1977 Joint Meeting and a temporary ADI
    was estimated to be 0-0.01 mg/kg bw/day.  Concern was expressed over
    the pathological changes observed in rat kidney and dog liver in
    two-year studies.  On the basis of a re-evaluation by an independent
    pathologist the meeting concluded there was no difference between the
    control and the treated animals.

    FIGURE 1
    New data submitted to the meeting with respect to long-term
    carcinogenesis studies in two species was found to be inadequate to
    meet the current criteria for appropriate carcinogenicity studies. 
    The survival rate of rats for 24 months was poor, and a carcinogenic
    evaluation could not be made with this species.  In contrast to the
    rat study however there was a significant number of survivors in the
    mouse study.

    The metabolic profile of imazalil especially with respect to salt
    formulations appears to be well defined.  Imazalil did not affect
    reproduction in rats.

    A temporary acceptable daily intake for man was reaffirmed on the
    basis of studies in rat and dog.  In several respects the long-term
    studies reported to the meeting have been inadequate to evaluate fully
    the toxicological profile.  The Meeting felt that while no immediate
    problems exist, a further adequately performed long-term study is
    required.  Additionally, short-term mutagenicity tests should be
    performed.

    Additional information has now been provided on the metabolic fate of
    3H-labelled imazalil on banana plants grown in a greenhouse, and on
    the results of supervised trials.

    The major part of the radioactivity (95.2-100%) recovered in the
    plants was present on the surface and in the upper layers of the
    leaves.  Translocation of the radioactivity from the sprayed areas to
    the rest of the plant was minimal, since the rhizome, roots and new
    leaves that emerged after the last treatment showed very little or no
    radioactivity.  The parent compound formed 15% of the total
    radioactivity recovered in the leaves of plants treated nine times,
    while the single major metabolite,
    alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol accounted for 25%.
    The rest of the radioactivity was due to a large number of
    unidentified minor metabolites, each representing at most only a few
    percent of the radioactivity.  A considerable part of the unidentified
    material might consist of degradation products formed during the
    analytical procedure.

    The results of the metabolism studies and the supervised trials are in
    agreement with the findings discussed in the 1977 evaluation, and
    support the recommendations made then.

    Level causing no toxicological effect

    Rat:  5 mg/kg bw/day
    Dog:  1.24 mg/kg bw/day

    Estimate of temporary acceptable daily intake for man

    0-0.01 mg/kg bw/day

    FURTHER WORK OR INFORMATION

    Required (by 1984)

    1.  An adequate long-term study on rats to define fully a no-effect
    level.
    2.  Short-term tests to evaluate the mutagenic potential.


    REFERENCES

    Heykants, J.J.P. On the metabolism of imazalil and related compounds
    in animals and man. A review. (1978) Unpublished summary review of
    published and unpublished metabolism data from Janssen Pharmaceutica
    submitted to the World Health Organization by Janssen Pharmaceutica.

    Marsboom, H. Oral three-generation study in Wistar rats. (1978)
    Unpublished report from Janssen Pharmaceutica submitted to the World
    Health Organization by Janssen Pharmaceutica.

    Marsboom, R. and Herin, V. Oral carcinogenicity study in Wistar rats.
    (1979a) Unpublished report (No. 667) from Janssen Pharmaceutica
    submitted to the World Health Organization by Janssen Pharmaceutica.

    Marsboom, R. and Herin, V. Oral carcinogenicity study in albino Swiss
    mice. (1979b) Unpublished report (No. 666) from Janssen Pharmaceutica,
    submitted to the World Organization by Janssen Pharmaceutica.

    Meuldermans, W., Swijsen, E., Hendricks, J., Wolstenborghs, R.,
    Lauwers, W. and Heykants, J. (1979) Unpublished report from Janssen
    Pharmaceutica submitted to the World Health Organization by Janssen
    Pharmaceutica.

    Meuldermans, W. et al. The metabolic fate of imazalil on banana
    plants. Janssen Research Products Int. Service. R 23979/21.

    Niemegears, C.J.E. Comparative acute oral toxicity studies of the
    different salts of imazalil in rats. (1979) Unpublished report from
    Janssen Pharmaceutica, submitted to the World Health Organization by
    Janssen Pharmaceutica.

    


    See Also:
       Toxicological Abbreviations
       Imazalil (ICSC)
       Imazalil (Pesticide residues in food: 1977 evaluations)
       Imazalil (Pesticide residues in food: 1984 evaluations)
       Imazalil (Pesticide residues in food: 1984 evaluations)
       Imazalil (Pesticide residues in food: 1985 evaluations Part II Toxicology)
       Imazalil (Pesticide residues in food: 1986 evaluations Part II Toxicology)
       Imazalil (Pesticide residues in food: 1991 evaluations Part II Toxicology)
       Imazalil (JMPR Evaluations 2000 Part II Toxicological)
       Imazalil (JMPR Evaluations 2001 Part II Toxicological)
       Imazalil (JMPR Evaluations 2005 Part II Toxicological)