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    PESTICIDE RESIDUES IN FOOD - 1984


    Sponsored jointly by FAO and WHO






    EVALUATIONS 1984




    The monographs



    Data and recommendations of the joint meeting
    of the FAO Panel of Experts on Pesticide Residues
    in Food and the Environment and the
    WHO Expert Group on Pesticide Residues
    Rome, 24 September - 3 October 1984

    Food and Agriculture Organization of the United Nations
    Rome 1985

    AMITRAZ

    Explanation

         Amitraz was evaluated by the Joint Meeting in 1980 (FAO/WHO,
    1981) 1/ and a temporary ADI of 0-0.0005 mg/kg body weight was
    allocated. Further data required included a long-term carcinogenicity
    study in mice using Amitraz; a separate long-term carcinogenicity
    study using the metabolite N(2,4-dimethyl-phenyl)N-methylformamidine;
    and studies on the mutagenic activity of 2,4-xylidine, N,N'-bis
    (2,4-dimethylphenyl) formamidine and 2,4-dimethyl-formanilide. Since
    the previous evaluation (FAO/WHO, 1981) additional data have become
    available and are reviewed in this monograph addendum.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, distribution and excretion

         Male and female B6C3F1 mice, either untreated or pre-dosed for 3
    weeks with 400 ppm. Amitraz in the diet, were orally intubated with a
    single dose of 10 mg/kg bw 14C- Amitraz. In the first 24 h, 85.9
    percent of the radio-labelled dose was excreted and 62.3 percent was
    present in the urine. It was completely excreted by 96 h, 72.7 percent
    being in the urine. The route and rate of excretion was similar in
    both sexes, as well as in untreated and pre-dosed mice. The highest
    14C- Amitraz concentration was found in liver, adrenals and eyes,
    with the least activity being in bone and muscle (Campbell & Needham,
    1983).

         Following administration of a single oral dose of 14C- Amitraz
    to Sprague-Dawley CD strain rats at 1, 10, 50 and 100 mg/kg body
    weight, the compound was rapidly excreted and effectively metabolized,
    with no apparent sex differences identified. At all dose levels,
    4-formamido-3-methylbenzoic acid and 4-acetamido-3-methylbenzoic acid
    were the major metabolites, which together accounted for up to 32
    percent of the total urinary excretion. The excretion of the
    metabolite N(2,4-dimethylphenyl-N'-methylformamidine) was found to be
    dose-dependent. At 1 mg Amitraz/kg bw, approximately 4 percent was
    excreted; at 100 mg/kg bw, 23-38 percent of the dose was excreted as
    this metabolite. Minor metabolites identified accounted for
    approximately 2 percent of the total excretion and included
    2,4-dimethylformanilide and 4-amino-3-methylbenzoic acid (Campbell &
    Needham, 1984a).



              

    1/  See Annex 2 for FAO and WHO documentation

         In separate studies, beagles were dosed orally (via capsule) or
    dermally with 14C- Amitraz. Peak blood levels of radiolabel were
    observed in the first 8 h following oral administration. Approximately
    80 percent of the oral dose was excreted in the first 24 h and 100
    percent within 72 h, preferentially in the urine. After dermal
    treatment, peak blood levels occurred in the 24-72 h post-treatment
    interval, and only 25-40 percent of the dermal dose was recovered in
    urine and faeces over a 10-day collection period, thus demonstrating
    the poor dermal absorption of Amitraz. The metabolism of Amitraz by
    either oral or dermal administration was essentially the same with
    4-formamido-3-methylbenzoic acid, the predominant residue in both
    blood and urine. Parent compound and the first formed hydrolysis
    products (N-2,4-di-methylphenyl-N-methylformamidine and
    2,4-dimethylformanilide) were never observed at measurable levels in
    either blood or urine (Hornish & Nappier, 1983).

         Approximately 57-81 percent of the applied radioactivity was
    recovered in dermal washings 12 h after application of a diluted E.C.
    formulation of 14C-Amitraz (18 mg a.i.) to the shaved dorsal shin of
    pigs. Approximately 6.7 percent of the applied radioactivity was
    recovered in the excreta collected over 60 h post-application. Tissue
    residues were less than 0.05 ppm (Campbell, 1984a).

         14C-Amitraz, given orally to baboons at 10 mg/kg bw, was rapidly
    excreted in both sexes, mainly in urine (75-83 percent of the dose
    recovered within the first 24 h). Tissue residues were similar in both
    sexes and were highest in the liver and eyes and lowest in muscle
    (Campbell & Needham, 1984b).

         Two male human volunteers received a single oral dose of
    14C-Amitraz of 0.25 mg/kg bw. Approximately 62.9 percent was
    recovered in the urine within the first 24 h, and 87.9 percent within
    72 h. Urinary metabolites were the same as in other species examined
    (Campbell, 1984b,c).

    TOXICOLOGICAL STUDIES

    Special Studies on Hormone Levels

         The effect of technical Amitraz on the oestrus cycle and hormone
    levels was evaluated in groups of female B6C3F1 mice fed diets
    containing 0, 25, 100 and 400 ppm Amitraz for 28 weeks. Pro-oestrus
    was prolonged and a trend towards reduced duration of dioestrus was
    evident at 400 ppm on examination of vaginal smear data. There
    were lower blood levels of progesterone and higher levels of
    dehydroepiandrosterone sulphate in the 400 ppm groups, and to a lesser
    degree in the 100 ppm group, when compared to controls. Liver weights
    were increased at 400 and 100 ppm. There were no treatment-related
    effects at 25 ppm (Hounsell & Rush, 1984).

    Special Study on Carcinogenicity

         In a pilot study, groups of 20 male and female B6C3F1 mice were
    administered Amitraz in the diet at doses of 0, 100, 200, 400, 600 and
    800 ppm for 13 weeks. Aggressive behaviour was observed in males in
    the 400 to 800 ppm groups. Overall, body weight gain was reduced in
    males at >400 ppm and in females at >200 ppm. There were no
    significant differences in food consumption between control and
    treated groups. There was no microscopic evaluation of tissues (Colley
    et al., 1981).

         Groups of 75 male and 75 female B6C3F1 hybrid mice (33 days old)
    were administered diets containing 25, 100 and 400 ppm Amitraz for 104
    weeks. The untreated controls consisted of 100 male and 100 female
    B6C3F1 hybrid mice (33 days old). Clinical signs, food consumption and
    body weight were recorded throughout the study. Survivors were killed
    at the end of the 2-year dosing period and all animals were subjected
    to a complete gross autopsy. A complete collection of tissues was
    taken from all animals and preserved for future examination.
    Histopathological examination was performed on all tissues from the
    high dose group and controls. At the low and intermediate dosage
    levels, liver, pancreas, spleen, lung, stomach, pituitary, thyroid,
    adrenals, ovaries, uterus, sternum and all macroscopically abnormal
    tissues were examined microscopically.

         Males at 400 ppm exhibited hyperactivity and aggressive
    behaviour. The incidence of cutaneous ulceration and inflammation of
    the perigenital and perianal areas was greater at 100 and 400 ppm than
    at 25 ppm and in controls. The incidence of urogenital swelling was
    greater in all treatment groups than that observed for the controls.
    Incidences of gross adverse effects for treated females were
    comparable to control incidences. Mortality was increased in high dose
    group animals compared to other groups, including the control group.

         Mean body weight gain was reduced throughout the study for high
    dose animals, with a significant decrease in 100 ppm females over the
    last 74 weeks of the study. Food consumption was marginally reduced
    initially at 100 and 400 ppm but was comparable to controls from week
    25 to termination.

         An increase in liver and lymph node involvement was apparent for
    males and females fed 400 ppm. The incidence of preputial gland
    enlargement of 400 ppm males was greater than that observed for
    controls (20/75 vs 12/100). Prominence of the limiting ridge of the
    stomach was greater than that of controls for females at all levels
    and for males at 100 and 400 ppm. A decrease in the production of
    myeloid elements accompanied by an increase in erythroid elements
    resulted in a significant decrease in the M/E ratio for males fed
    400 ppm and females fed 100 and 400 ppm amitraz. The incidence of
    spleen haematopoiesis in males and stomach focal hyperkeratosis in
    males and females fed all three dosage levels were greater than that
    observed in the control groups.

         There is an apparent liver involvement in females with an
    increased incidence, compared to controls, in heptocellular tumours at
    the 400 ppm level (15/75 vs 2/100 hepatocarcinoma; 16/75 vs 4/100
    hepatocellular adenoma). This was accompanied by a dose-response
    increase in the incidence of hyperplastic nodules, telangiectatic and
    basophilic foci of the liver at 25, 100 and 400 ppm. Males at the
    400 ppm level exhibited an apparent increase, compared to controls, in
    the incidence of hyperplastic nodules, and telangiectatic and
    basophilic foci of liver (Colley et al., 1983).

    Special Studies on Mutagenicity

         See Tables 1 and 2 for a summary of the mutagenicity studies
    evaluated.

    COMMENTS

         Amitraz was first reviewed by the JMPR in 1980; a temporary ADI
    was allocated pending further toxicological information. New studies
    on metabolism have shown that after oral administration amitraz is
    rapidly metabolized and excreted, mainly in the urine; the pattern of
    metabolism is qualitatively similar in mouse, rat, dog, baboon and
    human. Mutagenicity studies were all negative, both in the presence
    and absence of metabolic activation.

         In the carcinogenicity study in B6/C3 F1 mice the only
    significant change in tumour incidence observed after treatment with
    amitraz for 104 weeks was the increased incidence of hepatocellular
    tumours in female mice at 400 ppm, which was not considered to be
    significant to human health (FAO/WHO 1983a).

         The request for a long-term carcinogenicity study of
    N-(2,4-dimethylphenyl)-N'-methylformamidine was considered, but was
    found to be clearly unnecessary as the new metabolic studies have
    indicated that this compound has been effectively examined. An ADI was
    estimated on the basis of the data submitted.

    TOXICOLOGICAL EVALUATION

    Level Causing no Toxicological Effect

    Mouse:    2.5 mg/kg bw/day

    Rat:      60 ppm in the diet equivalent to 3 mg/kg bw

    Dog:      0.25 mg/kg bw/day

    Estimate of Acceptable Daily Intake for Man

         0 - 0.003 mg/kg bw


        TABLE 1.  Special Studies on Mutagenicity (Amitraz)

                                                                                                                                      

    Test System                   Test               Concentrations       Purity             Results                   Reference
                                  Organism           of Amitraz used
                                                                                                                                      

    Ames' Test                    S. typhimurium     0, 33, 100, 333,     97-99%             Negative.                 McGregor &
    (with and without             TA 98              1000, 3300,                             Positive control          Prentice, 1983
    metabolic activation)         TA 100             10,000 g/plate                         gave the expected
                                  TA 1535                                                    response.
                                  TA 1537
                                  TA 1538

    Cell Transformation           C3H/10 T          Activated            97-99%             Negative                  McGregor & Riach,
    (with and without             clone 8 mouse      12.5, 25,                                                         1983a
    metabolic activation)         embryo             37.5 g/ml
                                  fibroblasts        Non-activated
                                                     5, 10, 15 /ml

    Unscheduled DNA               Human embryonic    20, 60, 100, 140,    97-99%             Negative.                 McGregor & Riach,
    Synthesis (with and           fibroblasts        180, 220, 260,                          Positive control          1983b
    without metabolic                                300 g/ml                               gave the expected
    activation)                                                                              response.

    Mouse Lymphoma                Mouse              Activated            97-99%             Negative                  McGregor & Riach,
    Forward Mutation              L5178Y TK+/-       Up to 33 /ml                                                     1984
    Assay                         phenotype cells    Non-activated
    (with and without                                Up to 20 g/ml
    metabolic activation)
                                                                                                                                      

    TABLE 2.  Special Studies on Mutagenicity (Metabolites of Amitraz)

                                                                                                                                             

    Test System                 Test               Concentration        Test                        Results                Reference
                                Organism           Used                 Material
                                                                                                                                             

    Ames' Test                  S. typhimurium     Up to 5000           N-(2,4-dimethyl-            Negative.              Richold et al.,
    (with and with-             TA 98              g/plate             phenyl)-N'-methyl-                                 1983a
    out metabolic               TA 100                                  formamidine and
    activation)                 TA 1535                                 2,4-dimethylformanilide     Negative.              Richold et al.,
                                TA 1537                                                                                    1983b.
                                TA 1538

    Micronucleus                Mouse, bone        56.3, 112.5 and      2,4-dimethylaniline         Negative.              Hounsell &
                                marrow             225 mg/kg, twice                                 No increase in         Walker, 1983
                                                   24 h apart                                       polychromatic
                                                                                                    erythrocytes
                                                                                                    containing
                                                                                                    micronuclei

    Mouse Lymphoma              Mouse              1, 3.3, 10,          2,4-dimethylaniline         Positive in            McGregor & Riach,
    Forward Mutation            L5178Y TK +/-      33.3, 100, 200,                                  activated              1983c
    Assay                       phenotype          300, 333.3,                                      systems at
    (with and with-                                400, 500,                                        3.3 g/ml and
    out metabolic                                  600 g/ml                                        above. Negative
    activation)                                                                                     in non-activated
                                                                                                    systems up to
                                                                                                    300 g/ml.

    Cell Transformation         C3H/10 T          Activated            2,4-dimethylaniline         Negative.              McGregor et al.,
    (with and                   clone 8 mouse      5, 10 and 20 g/ml                                                      1984.
    without metabolic           embyro             Non-activated
    activation)                 fibroblasts        100, 200 and
                                                   400 g/ml
                                                                                                                                             
    
    FURTHER WORK OR INFORMATION

    Desirable

         Further observations in humans.

    REFERENCES

    Campbell, J.K. Dermal absorption of 14C-amitraz in E.C. formulation by
    1984a     male and female pigs given a single topical application of
              18 mg A.I. Report submitted by FBC Limited to WHO.
              (Unpublished)

    Campbell, J.K. Urinary excretion of 14C-amitraz by two human males
    1984b     following a single oral dose of 0.25 mg/kg body weight.
              Report submitted by FBC Limited to WHO. (Unpublished)

    Campbell, J.K. A comparison of the metabolism of 14C-amitraz in rat,
    1984c     mouse, baboon and human. Report submitted by FBC Limited to
              WHO. (Unpublished)

    Campbell, J.K. & Needham, D. Excretion and tissue residues of
    1983      14C-amitraz in male and female mice given a single oral
              dose of 10 mg 14C-amitraz per kg body weight. Report
              submitted by FBC Limited to WHO. (Unpublished)

    Campbell, J.K. & Needham, D. The metabolism of 14C-amitraz by male and
    1984a     female rats. Report submitted by FBC Limited to WHO.
              (Unpublished)

    Campbell, J.K. & Needham, D. Excretion and tissue residues of
    1984b     14C-amitraz in a male and female baboon given a single oral
              dose of 10 mg 14C-amitraz per kg body weight. Report
              submitted by FBC Limited to WHO. (Unpublished)

    Colley, J., Dawe, S., Heywood, R., Prentice, D.E. & Gibson, W.A.
    1981      Amitraz toxicity by mice by dietary administration for 13
              weeks. Report from Huntingdon Research Center, Huntingdon,
              U.K., submitted by FBC Limited to WHO. (Unpublished)

    Colley, J., Dawe, S., Heywood, R., Street, A.E. & Gibson, W.A.
    1983      Amitraz: 104 week tumorigenicity in mice. Report from
              Huntingdon Research Centre, Huntingdon, U.K., submitted by
              FBC Limited to WHO. (Unpublished)

    Hornish, R.E. & Nappier, J.M. The absorption, metabolism of mitaban
    1983      (amitraz) in the dog from oral and dermal exposure. Report
              from Agricultural Research and Development Laboratories, The
              Upjohn Company, U.S., submitted by FBC Limited to WHO.
              (Unpublished)

    Hounsell, I.A.G. & Walker, A.K. A micronucleus study in mice using BTS
    1983      24868 (2,4-dimethylaniline). Report submitted by FBC Limited
              to WHO. (Unpublished)

    McGregor, D.B. & Prentice, R.D. Technical amitraz: Ames bacterial
    1983      mutagenicity test. Report submitted by FBC Limited to WHO.
              (Unpublished)

    McGregor, D.B. & Riach, G.G. Technical amitraz: Induction of
    1983a     morphological transformation in C3H10T1/2  cells. Report
              submitted by FBC Limited to WHO. (Unpublished)

    McGregor, D.B. & Riach, G.G. Technical amitraz: Unscheduled DNA
    1983b     synthesis in human embryonic cells. Report submitted by FBC
              Limited to WHO. (Unpublished)

    McGregor, D.B. & Riach, G.G. Technical BTS 24868: Mouse lymphoma
    1983c     mutation assay. Report submitted by FBC Limited to WHO.
              (Unpublished)

    McGregor, D.B. & Riach, G.G. Technical amitraz. Mouse lymphoma
    1984      mutation assay. Report submitted by FBC Limited to WHO.
              (Unpublished)

    McGregor, D.B., Brown, A.G. & Riach, G.G. Technical BTS 24868:
    1984      Induction of cell transformation in C3H10T1/2 cells. Report
              submitted by FBC Limited to WHO. (Unpublished)

    Richold, M., Jones, E. & Fenner, L.A. Technical BTS 27271: Ames
    1983a     bacterial mutagenicity test. Report submitted by FBC Limited
              to WHO. (Unpublished)

    Richold, M., Jones, E. & Fenner, L.A. Technical BTS 27919: Ames
    1983b     bacterial mutagenicity test. Report submitted by FBC Limited
              to WHO. (Unpublished)
    


    See Also:
       Toxicological Abbreviations
       Amitraz (ICSC)
       Amitraz (Pesticide residues in food: 1980 evaluations)
       Amitraz (Pesticide residues in food: 1983 evaluations)
       Amitraz (Pesticide residues in food: 1984 evaluations)
       Amitraz (JMPR Evaluations 1998 Part II Toxicological)