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    MYCLOBUTANIL

    First draft prepared by M. Caris
    Bureau of Chemical Safety
    Health and Welfare Canada, Ottawa, Canada

    EXPLANATION

         Myclobutanil is a broad spectrum systemic fungicide of the
    substituted triazole chemical class of compounds. The mode of action
    of myclobutanil is by inhibition of sterol biosynthesis in fungi.
    Myclobutanil was considered for the first time by the present
    Meeting.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Biochemical aspects

    Absorption, distribution, and excretion

    Mice

         Male and female Crl:CD-1 mice were treated by gavage with a
    single oral dose of 2, 20 or 200 mg 14C-myclobutanil/kg bw,
    radiolabelled in the chlorophenyl ring, immediately following a 2-
    week pretreatment period with unlabelled myclobutanil (81.1% purity)
    at dietary levels of 10, 100 or 1000 ppm a.i. Excretion of
    radioactivity after 96 h accounted for 81-107% of the administered
    14C-radiolabel, with the majority of the radiolabel recovered
    within 24-48 h post-dosing. Comparable amounts of radiolabel were
    found in both the urine (with cage wash: 41-57%) and faeces (31-52%)
    with no significant differences with respect to sex or dose level.
    Orally administered 14C-myclobutanil was rapidly absorbed from the
    gastrointestinal tract, with peak blood concentrations occurring
    within 0.2-1 h post-dosing and with absorption half-lives of 0.04-
    0.3 h. Elimination was generally biphasic with half-lives of 0.6-0.9
    and 6.0-30.1 h, respectively. The single exception to the
    elimination profile was the high-dose (200 mg/kg bw) treated male
    group, where only one phase of elimination (half-life: 6.2 h) was
    observed. Presence of 14C-radiolabel in groups of mice killed one-
    hour after dosing revealed similar dose-related concentrations in
    whole blood and plasma. Concentrations of radiolabel in the liver
    were 4 to 11-fold higher than in the blood, although the ratio of
    liver to blood 14C-concentration was shown to decrease with
    increasing dose (Steigerwalt  et al., 1986a).

    Rats

         Four Sprague Dawley rats/sex were administered a single oral
    dose of 150 mg 14C-myclobutanil/kg bw, radiolabelled at the 3 and
    5 carbons of the triazole ring and suspended in aqueous 0.5% methyl
    cellulose. The major routes of elimination of the administered
    radiolabel, as determined 7 days post-dosing, were via the urine
    (48% males, 37% females) and faeces (51% males, 63% females). The
    level of radioactivity in expired CO2 was minimal (0.01-0.02%).
    Residual tissue levels were higher in males than in females,
    representing 0.2-0.5% of the radiolabel, with highest concentrations
    present in the intestine, liver and kidney (Streelman, 1984).

         Myclobutanil 14C-labelled in the chlorophenyl ring was
    administered by gavage to groups of male and female Crl:CD(SD)BR
    rats as a single oral dose of 1 or 100 mg/kg bw or as a single high-
    dose of 100 mg/kg bw preceded by a 14-day repeated exposure to

    unlabelled myclobutanil (81.1% purity) at a dietary level of 1000
    ppm a.i. An additional group of rats received a single intravenous
    dose of 1 mg 14C-myclobutanil/kg bw. Total radioactive recovery
    after 96 h, upon oral dosing ranged from 82-97% and following
    intravenous administration was 77% in males and 82% in females. The
    majority of the radiolabel was excreted via the urine (35-48%) and
    faeces (32-46% of the radiolabel dose), regardless of route of
    administration. 14C-myclobutanil was well absorbed (89-115%)
    following oral administration as determined by the ratio of the
    percentage dose excreted in the urine after oral and intravenous
    dosing. Peak blood and tissue levels as studied at the high-dose of
    100 mg/kg bw, occurred within 1 hour post-dosing. Elimination
    kinetics of 14C-radiolabel was biphasic after a single oral dose
    of 100 mg/kg bw alone (plasma half-lives of 5.3 and 25.7 h) or after
    dietary pretreatment (plasma half-lives of 2.0 and 31.5 h). Residual
    tissue levels in orally treated rats after 96 hours were generally
    less than 1% of the dose, with highest concentrations present in the
    liver, kidneys, adrenals, whole blood, thyroids and bone marrow
    (Steigerwalt  et al., 1986b).

    Biotransformation

    Mice

         Metabolic profiles from the urine and faeces of mice treated
    with 14C-myclobutanil (labelled in the chlorophenyl ring) at 2, 20
    or 200 mg/kg bw following 2-week dietary pretreatment (Steigerwalt
     et al., 1986a) revealed no significant sex or dose-related
    differences in 14C-metabolite patterns. Myclobutanil was
    extensively metabolized to more polar compounds. Unchanged parent
    detected in the excreta accounted for 0.7 to 7.2% of the
    administered dose. No further metabolic characterization was
    performed.

    Rats

         The proposed metabolic pathway in rats is depicted in Figure 1.

         14C-Myclobutanil (labelled in the 3 and 5 carbons of the
    triazole ring) was extensively metabolized when administered to SD
    rats as a single oral dose of 150 mg/kg bw (Streelman, 1984).
    Unchanged parent myclobutanil was estimated to represent only 2-3%
    of the excreted dose. The predominant pathway of metabolism has been
    suggested to be through a variety of oxygenation reactions of the
    butyl group. The major polar metabolites identified including a
    lactone, ketone, alcohol, carboxylate, dialcohol and sulfate
    conjugate, were distributed uniformly in the urine and faeces of
    both sexes. Quantitative differences in the metabolic profiles were
    apparent most notably with the major metabolite, the sulfate
    conjugate of RH-9090, which in females accounted for 75% and in
    males for only 15% of the total.

    FIGURE 1

         Myclobutanil, 14C-radiolabelled in the chlorophenyl ring was
    extensively metabolized to more polar metabolites when administered
    by gavage to groups of male and female rats as a single oral dose of
    1 or 100 mg/kg bw (Steigerwalt  et al., 1986b). 14C-Metabolites
    of myclobutanil were qualitatively similar with respect to sex and
    dose. Unchanged parent compound represented only 1-4% of the
    excreted dose. In males, five fractions with more than 10% of the
    excreted 14C were identified in the excreta compared with a single
    major fraction in females, which accounted for 53-61% of the
    radiolabel.

    Effects on enzymes and other biochemical parameters

    Mice

         Sections of liver were taken from 4 randomly selected mice/sex
    from the 6 highest dietary levels: 30, 100, 300, 1000, 3000 or 10
    000 ppm a.i. of the 3-month dietary toxicity study with myclobutanil
    in mice (Goldman  et al., 1986) for determination of hepatic MFO
    activity. Liver sections were analyzed for MFO activity using
    aminopyrine and benzphetamine N-demethylation assays. MFO activity
    per gram of liver as estimated by N-demethylation of benzphetamine
    was significantly increased in males (2.1 to 3.3-fold) at 1000 ppm
    and above, and in females (1.7 to 2.2-fold) at dietary levels of
    3000 ppm and higher. Enzyme activity as measured by N-demethylation
    of amino-pyrine revealed increased levels in males (2.2 to 2.3-fold)
    at 3000 ppm and higher, and in females at 10 000 ppm (1.7-fold).
    Hepatic microsomal protein was increased in both sexes at dietary
    levels of 3000 ppm and higher.

         Liver microsomal suspensions were prepared from 6 randomly
    selected mice/sex treated with myclobutanil at 0, 20, 100 and 500
    ppm a.i. as part of the long-term carcinogenicity study (Goldman &
    Harris, 1986a). The microsomal suspensions were assayed for MFO
    activity using aminopyrine N-demethylation after 3, 6 or 12 months
    of treatment. Hepatic MFO activity per gram of liver was increased
    after 3 months in females (1.3-fold) at 100 ppm, and in both sexes
    (1.5 to 2-fold) at 500 ppm; after 6 months in both sexes at 100 ppm
    (1.3 to 1.4-fold) and 500 ppm (2.8 to 2.9-fold); and after 12 months
    in females (1.5-fold) at 100 ppm, and in both sexes (1.6 to 3.7-
    fold) at 500 ppm. There were no treatment-related effects on hepatic
    microsomal protein concentration after 3, 6 or 12 months.

         Additional liver samples taken at 12 months were analyzed for
    hepatic peroxisomal -oxidation activity by measuring the conversion
    of acid insoluble 14C-palmitoyl-CoA (substrate) to the acid
    soluble acetyl CoA by hepatic acyl-CoA oxidase. There were no
    increases in peroxisomal 14C-palmitoyl-CoA oxidase activity
    indicative of peroxisomal proliferation (Goldman & Harris, 1986a).

    Rats

         Mixed function oxidase activity was measured by aminopyrine and
    benzphetamine N-demethylation assays on sections of liver taken from
    3 randomly selected rats/sex from the control group and from dietary
    level groups fed myclobutanil at 100 ppm and higher as part of a 3-
    month study (O'Hara & DiDonato, 1984). Significant, dose-related
    increases in MFO activity per gram of liver were recorded at dietary
    levels of 300 ppm in males only (1.7 to 1.9-fold), and in both sexes
    at 1000 ppm (1.8 to 2.3-fold), 3000 ppm (3 to 5.1-fold) and 10 000
    ppm (4.1 to 8-fold higher). Microsomal protein was increased in both
    sexes at 10 000 ppm and in males at 3000 ppm.

         Livers from 6 randomly selected rats/sex/group were collected
    from interim kills scheduled after 3, 6 or 12 months of treatment
    with myclobutanil at dietary levels of 0, 50, 200 or 800 ppm as a
    component of the long-term study in rats (Shellenberger  et al.,
    1986). Hepatic MFO analyzed by aminopyrine N-demethylation revealed
    increased activity per gram of liver after 3 months at 200 ppm in
    females (1.6-fold) and in both sexes at 800 ppm (1.5 to 1.8-fold),
    and after 6 months in males at 800 ppm (1.5-fold). Increases in MFO
    activity in females at 6 months and in both sexes at 12 months of
    study were not significantly different from the controls. The
    microsomal protein concentration in the treated groups was not
    markedly different from the controls.

         For determination of hepatic peroxisomal -oxidation activity,
    additional sections of liver were obtained from 6 rats/sex/group at
    the 12-month interim kill. There was no effect of myclobutanil
    treatment on hepatic peroxisomal -oxidation activity at dietary
    levels as high as 800 ppm (Shellenberger  et al., 1986).

    Toxicological studies

    Acute toxicity studies

         Acute toxicity studies with technical myclobutanil have been
    performed in several animal species, the results of which are
    summarized in Table 1. Myclobutanil, upon oral administration was
    only slightly toxic to the mouse and rat with LD50 values of 1.36
    to > 4.42 g/kg bw and 1.6 to 2.71 g/kg bw, respectively.
    Myclobutanil was practically non-toxic by the inhalation and dermal
    routes with a LC50 value greater than 5.1 mg/L in rats and LD50
    values greater than 5 g/kg bw in the rabbit, respectively.

        Table 1. Acute toxicity of technical myclobutanil
                                                                                               

    Species        Sex  Route       Vehicle   LD50        Puritya  Reference
    (strain)                                  (g/kg bw)
                                                                                               

    Mouse          M    oral        corn oil  3.23        81.1%    Krzywicki & Krajewski (1983)
    (CRCD-1)

    Mouse          M    oral        corn oil  > 4.42      91.9%    Morrison  et al. (1984a)
    (CRCD-1)

    Mouse          M+F  oral        corn oil  M: 1.91     91.9%    Morrison  et al. (1986b)
    (CRCD-1)                                  F: 1.84

    Mouse          F    oral        corn oil  1.36        91.9%    Romanello  et al. (1986a)
    (CRCD-1)

    Mouse          M+F  oral        corn oil  M: 2.27     91.4%    Shimizu (1987a)
    (CRJ: CD-1                                F: 2.44
    (ICR))

    Rat            M+F  oral        corn oil  M: 1.75     84.5%    Krzywicki (1983)
    (CR(CD)SD)                                F: 1.80

    Rat            M+F  oral        corn oil  M: 1.6      91.9%    Krzywicki & Morrison (1984a)
    (CR(CD)SD)                                F: 2.29

    Rat            M+F  oral        corn oil  M: 2.62     91.4%    Shimizu (1987b)
    (CRJ: CD-1                                F: 2.71
    (SD))

    Rat            M+F  inhalation  -         LC50:       91.4%    Fisher  et al. (1987)
    (Crl:CDBR)                                > 5.1 mg/L
                                              air

    Rabbit (NZW)   M+F  dermal      -         > 5.0       84.5%    Krzywicki (1983)

    Rabbit         M+F  dermal      -         > 5.0       91.9%    Krzywicki & Bonin (1984)
    (NZW)
                                                                                               

    a = active ingredient (a.i.)
    
    Short-term toxicity studies

    Mice

         Myclobutanil (81.1% purity) was administered daily for a period
    of 3 months to groups of 10 Crl:CD-1 (ICR) BR mice/sex at dietary

    levels of 0, 3, 10, 30, 100, 300, 1000, 3000 or 10 000 ppm a.i.
    (equal to 0, 0.4, 1.5, 4.8, 14.1, 42.1, 132, 542 or 2035 mg a.i./kg
    bw/day in males and 0, 0.6, 2.1, 6.9, 22.9, 65.5, 232, 710 and 2027
    mg a.i./kg bw/day in females, respectively). A NOAEL of 300 ppm,
    equal to 42.1 mg/kg bw/day, was indicated based on treatment-related
    hepatic alterations at dietary levels of 1000 ppm and higher,
    manifested histomorphologically as hepatocytic inflammation,
    centrilobular hypertrophy, vacuolation, and necrosis. Associated
    hepatic changes were increases in liver weight, accentuated liver
    lobular architecture, increased MFO activity, increased ALAT levels
    and decreased cholesterol. Other microscopic treatment-related
    changes pertained to increased cytoplasmic eosinophilia (> 1000
    ppm males, > 3000 females) and hypertrophy of the  zona
     fasciculata cells of the adrenal gland (3000 ppm and higher in
    both sexes). Treatment-related effects observed at dietary levels of
    3000 ppm and above were decreased body-weights, increased ASAT,
    decreased glucose, increased pigmentation of the liver Kupffer cells
    and macrophages of the spleen, and a slight increase in lymphoid
    necrosis of the spleen. Additional effects of treatment observed
    only at the highest dietary level of 10 000 ppm were related to
    scant faecal droppings and fluctuations in haematological (both
    sexes: decrease HCT, decrease MCV, decrease MCH, increase MCHC;
    males: decrease WBC, decrease lymphocytes, increase segmented
    neutrophils; females: decrease haemoglobin and increase platelets)
    and blood biochemical parameters (increased ALP, GGT and BUN).
    Histopathological changes at 10 000 ppm were noted as bile duct
    proliferation, slight increase in pigmentation of renal cortical
    tubular cells, lymphoid necrosis of the thymus and mesenteric lymph
    nodes, increased myeloid/erythroid ratio of bone marrow, immaturity
    of the uterus and absence of corpora lutea in ovaries as well as
    increased mononuclear cell infiltration of the skin (Goldman  et
     al., 1986).

    Rats

         Groups of 10 COBS-CD(SD) BR rats/sex were fed myclobutanil
    (81.1% purity) for 3 months at dietary levels of 0, 10, 30, 100,
    300, 1000, 3000, 10 000 or 30 000 ppm a.i. equal to 0, 0.5, 1.6,
    5.2, 15.3, 51.5, 158, 585 or 1730 mg a.i./kg bw/day in males and 0,
    0.7, 2.0, 6.9, 19.7, 65.8, 195, 665 or 1811 mg a.i./kg bw/day in
    females, respectively. A NOAEL for this study was 100 ppm, equal to
    5.2 mg/kg bw/day, as revealed by increased MFO activity in males at
    300 ppm and in both sexes at higher dietary levels. Increased liver
    weights and accentuated hepatic lobular architecture were observed
    at 1000 ppm and above. Treatment-related changes introduced at
    dietary levels of 3000 ppm were decreased body-weights, increased
    cholesterol, hepatic centrilobular hypertrophy and necrosis,
    increased kidney weights associated with renal congestion and
    pigmentation of the convoluted tubular epithelium, and
    histopathological alterations of the adrenal (increased cortical
    vacuolization), ovary (congestion), thyroid (increase in small
    follicles) and thymus (congestion). Additional effects of treatment

    exhibited at 10 000 ppm were decreased food consumption, slight
    haematological changes (decrease HCT, decrease Hb, decrease MCV and
    increase RBC, increase platelets), increased GGT, pigmentation of
    the liver Kupffer cells, hepatocytic vacuolation and coagulative
    necrosis, increased pigmentation in the red pulp of the spleen, and
    chronic pulmonary alveolitis. Treatment of rats with myclobutanil at
    the highest dietary level of 30 000 ppm resulted in 100% mortality.
    No effects of treatment were reported with respect to urinalysis and
    ophthalmology (O'Hara & DiDonato, 1984).

         A 13-week feeding study conducted with groups of 10 Crj:CD SD
    rats/sex given myclobutanil (91.4% purity) in the diet at levels of
    0, 100, 300 or 3000 ppm equal to 0, 6.2, 18.8 or 192 mg/kg bw/day in
    males and 0, 6.9, 19.6 or 225 mg/kg bw/day in females respectively,
    demonstrated a NOAEL of 300 ppm, equal to 18.8 mg/kg bw/day.
    Treatment with myclobutanil at the highest dietary level of 3000 ppm
    culminated in histomorphological alterations of the liver, kidney
    and adrenal glands. Specific organ changes were characterized in the
    liver as slight to moderate hepatocytic hypertrophy (10/10 males,
    8/10 females) and in the kidney as slight vacuolar degeneration of
    the renal tubular epithelium (7/10 males). Alterations of the
    adrenal were described as vacuolization of the cortical cells (7/10
    males), atrophy of the  zona fasciculata (5/10 males) and fine
    vacuolization of the  zona glomerulosa (1/10 males). A single male
    at 3000 ppm exhibited changes in the reproductive organs depicted in
    the testes as moderate atrophy of the seminiferous tubule(s) and
    giant cell-like changes with absence of sperm cells in the
    epididymis. Other effects of treatment with myclobutanil were
    decreased body-weight, blood chemistry changes (decreased bilirubin,
    glucose and triglycerides), increased liver and kidney weights,
    decreased adrenal weights and a slight increase in the number of
    males with round cells in the urine. Decreased food intake was
    observed in males during the first week of treatment. There were no
    treatment-related effects on haematology or ophthalmoscopy (Shimizu,
    1987c).

    Dogs

         A range-finding study was undertaken with 2 beagle dogs/sex per
    group fed myclobutanil (84.5% purity) for a period of 4 weeks at
    dietary levels of 0, 50, 250, 1000 or 4000 ppm a.i. equal to 0, 2.2,
    10.5, 45.3 or 45 mg a.i./kg bw/day in males and 0, 2.0, 10.6, 39.3
    or 47 mg a.i./kg bw/day in females respectively. Dogs treated at the
    highest dietary level of 4000 ppm were sacrificed after 2 weeks of
    treatment due to severely depressed food intake. The NOAEL for the
    study was determined to be 250 ppm, equal to 10.5 mg/kg bw/day,
    based on slightly decreased body-weight and food consumption
    recorded during the first week of treatment in females at 1000 ppm.
    There were no treatment-related consequences on clinical signs,
    haematological and blood chemistry investigations (12 and 28 days),
    or gross pathological examination (Goldman & Emmons, 1986).

         Myclobutanil (81.1% purity) was administered to groups of 4
    beagle dogs/sex for a period of 3 months at dietary levels of 0, 10,
    200, 800 or 1600 ppm a.i. equal to 0, 0.3, 7.3, 29.1 or 56.8 mg
    a.i./kg bw/day in males and 0, 0.4, 7.9, 32.4 or 58 mg a.i./kg
    bw/day in females respectively. The NOAEL was determined to be 10
    ppm, equal to 0.3 mg/kg bw/day, based on the dose-related incidence
    of centrilobular or midzonal hepatocellular hypertrophy noted at 200
    ppm (3/4 males) and in all animals of both sexes at 800 and 1600
    ppm. Periportal hepatocytes were enlarged in a few of the more
    severely affected livers. Liver weights were increased in males at
    800 ppm and in both sexes at 1600 ppm. Treatment-related effects on
    the kidney, evident as an increased incidence and severity of
    unilateral chronic nephritis were observed in males at 800 ppm and
    higher. Additional effects of treatment unveiled at the highest
    level of 1600 ppm were related to decreased body-weights and food
    consumption, due possibly to palatability of the diet. Slight
    changes in haematological and blood chemistry values were within
    range of normal variability and were not considered of toxicological
    consequence. There were no effects of treatment on ophthalmology.
    Increases in the ovarian weights at 800 and 1600 ppm were attributed
    to estrus (McLaughlin & DiDonato, 1984).

         Groups of 6 beagle dogs/sex were administered myclobutanil
    (91.4% purity) daily for 12 months in the diet at levels of 0, 10,
    100, 400 or 1600 ppm a.i. equal to 0, 0.3, 3.1, 14.3 or 54.2 mg
    a.i./kg bw/day in males and 0, 0.4, 3.8, 15.7 or 58.2 mg a.i./kg
    bw/day in females, respectively. The principal target organ was the
    liver, demonstrating a NOAEL of 100 ppm, equal to 3.1 mg/kg bw/day,
    based on hepatocellular hypertrophy, increased liver weights and
    increased serum alkaline phosphatase levels at dietary
    concentrations of 400 ppm and higher. At the highest level of 1600
    ppm, livers displayed accentuated lobular architecture and in 4 of 6
    females, the hepatocytes were expanded with large clear cytoplasmic
    spaces. Other effects observed at 1600 ppm were evident as decreased
    body-weight and food consumption, as well as changes in haematology
    (decrease RBC, increase platelet count) and blood chemistry
    (increase phosphorus, increase ALAT, increase GGT, decrease
    albumin). Treatment with myclobutanil failed to elicit any adverse
    effects on clinical signs, ophthalmological examination or
    urinalysis (Goldman & Harris, 1986b).

    Long-term toxicity/carcinogenicity studies

    Mice

         A 2-year study was conducted with Crl:CD-1 (ICR)BR mice fed
    diets containing myclobutanil (90.4% purity) at levels of 0, 20, 100
    or 500 ppm a.i. equal to 0, 2.7, 13.7 or 70.2 mg a.i./kg bw/day in
    males and 0, 3.2, 16.5 or 85.2 mg a.i./kg bw/day in females,
    respectively. A total of 110 males and 110 females per group were
    assigned to the chronic phase of this bioassay. Interim sacrifices

    were scheduled after 3 (10 mice/sex/group), 6 (10/sex/group) and 12
    months (20/sex/group) of study. All surviving animals (70/sex/group,
    maximum) were sacrificed after 24 months of continuous treatment.
    Treatment with myclobutanil resulted in a NOAEL for in-life
    parameters of 20 ppm, equal to 2.7 mg/kg bw/day, established on the
    basis of increased MFO activity at 3, 6 and 12 months at dietary
    levels of 100 and 500 ppm. At 500 ppm, target effects of treatment,
    primarily on the liver were demonstrated by increased ALAT activity
    (females, 3 months), increased liver weights (both sexes, 3 months),
    and histopathological alterations comprising centrilobular
    hypertrophy (males, 3/6/12 months), periportal vacuolation (males,
    3/6/12 months; both sexes, 24 months), Kupffer cell pigmentation
    (males, 6/12 months), hepatocellular necrosis (males, 12 months) and
    hepatocellular alteration (tinctorial and dimensional properties:
    both sexes, 24 months). There were no changes attributed to
    treatment with myclobutanil with regard to survival, clinical signs,
    body-weight, food consumption, ophthalmoscopy, haematology or
    urinalysis. Myclobutanil was not oncogenic when administered to mice
    for 2 years at dietary levels up to 500 ppm (Goldman & Harris,
    1986a).

    Rats

         Groups of 110 Charles River Sprague Dawley rats/sex were
    treated with myclobutanil (90.4% & 91.4% purity) for a period of up
    to 24 months at dietary levels of 0, 50, 200 or 800 ppm a.i. equal
    to 0, 2.5, 9.8 or 39.2 mg a.i./kg bw/day in males and 0, 3.2, 12.9
    or 52.3 mg a.i./kg bw/day in females, respectively. Interim
    sacrifices were performed after 3 and 6 months (10 rats/sex/group),
    12 months (20/sex/group) and 17 months of study (18 males and 10
    females/group). The survivors (52 male, 60 female/group, maximum)
    were sacrificed after 24 months of treatment. Treatment with
    myclobutanil indicated a NOAEL for in-life parameters of 50 ppm,
    equal to 2.5 mg/kg bw/day. Effects of treatment at 200 ppm were
    observed as decreased testes weights in association with slight
    testicular atrophy at 24 months. At the highest dietary level of 800
    ppm, testes weights were decreased at 12 and 24 months with slight
    to moderate increases in the incidence of testicular atrophy. (The
    seminiferous tubules were reported frequently found to be devoid of
    spermatid formation and germinal epithelial cells; the tubules
    appeared smaller than normal. In severe cases only Sertoli cells
    remained). Increased ovary weights in females treated at 800 ppm and
    sacrificed at 12 months were not correlated with any
    histomorphological changes. Other treatment-related effects noted at
    800 ppm were decreased body-weights (both sexes) and food
    consumption (males), and increased liver weights (females).
    Increased MFO activity was recorded at 200 ppm in females (3 months)
    and at 800 ppm in both sexes (3 months) and in males (6 months).
    There were no treatment-related effects on survival, clinical
    signs, ophthalmoscopy, haematology, blood chemistry or urinalysis.
    Treatment with myclobutanil at dietary levels up to 800 ppm failed

    to uncover any evidence of carcinogenic potential (Shellenberger  et
     al., 1986).

    Reproduction studies

    Rats

         A two-generation (two litter per generation) reproduction study
    was conducted with groups of 25 CRI:CD(SD)BR rats/sex fed
    myclobutanil (84.5% purity) at dietary levels of 0, 50, 200 or 1000
    ppm a.i. equal to 0, 3.6, 14.7 or 73.6 mg a.i./kg bw/day in males
    and 0, 4.3, 17.4 or 87 mg a.i./kg bw/day in females respectively. In
    the F0 generation, treatment with myclobutanil commenced 8 weeks
    before mating. In the F1 generation, the F1a litter-derived
    parental animals were exposed to the test material throughout
    weaning and for a minimum period of 8 weeks post-weaning. In both
    generations, treatment continued throughout the reproductive phases.
    Treatment-related effects on reproduction were denoted at the
    highest dietary level of 1000 ppm by a decreased number of females
    delivering litters (F0F1a and both matings from second
    generation), decreased mean number of pups per litter (first mating
    of second generation) and an increased number of stillborn pups (all
    matings of both generations). An increase, albeit minimal in the
    proportion of dead pups in both matings of the first generation was
    similarly recorded at 200 ppm when compared to the controls. Effects
    on the reproductive organs were evident in the second generation
    F1 males treated at 1000 ppm as multifocal or diffuse atrophy of
    the testes, decreased spermatozoa and/or necrotic spermatocytes of
    the epididymides, as well as atrophy of the prostate. Systemic
    toxicity was observed at 200 ppm as increased liver weights in males
    of both parental generations in association with centrilobular
    hepatocytic hypertrophy in the F1 generation males. At 1000 ppm,
    increased liver weights and hepatocytic hypertrophy were observed in
    males and females of both generations. Decreased body-weight in
    males and depressed food intake in both sexes were recorded at 1000
    ppm in both parental generations. Body-weights were similarly
    decreased at 1000 ppm in both sexes of all filial generations. The
    NOAEL for this study was determined to be 50 ppm, equal to 3.6 mg/kg
    bw/day, for systemic and reproductive effects (Costlow & Harris,
    1985).

    Special studies on teratogenicity

    Rats

         A range-finding study was performed with groups of 8 mated
    female Crl:CD(SD)BR rats treated orally by gavage with myclobutanil
    (81.1% purity) at 0 (vehicle, corn oil), 32, 68, 100, 215, 464 or
    700 mg a.i./kg bw/day on days 6 to 15 of gestation. Day 0 of
    gestation was considered the day sperm were evident in the vaginal
    smear. All surviving dams were killed on day 20 of gestation. The

    NOAEL for maternal toxicity was 215 mg/kg bw/day. At dose levels of
    464 and 700 mg/kg bw/day, treatment with myclobutanil resulted in
    mortality (25% and 100%, respectively), decreased body-weights and
    clinical signs of toxicity manifest as scant faeces,
    chromodacryorrhea, red exudate around mouth, rough and urine-stained
    hair coat, and salivation. Embryofetal toxicity was expressed at
    levels of 68 mg/kg bw/day and higher as increased resorptions and
    decreased viability indices (viable fetuses/implantation sites).
    Decreased fetal weights were recorded at 464 mg/kg bw/day. In the
    absence of detailed visceral and skeletal examinations of the
    fetuses, the NOAEL for developmental toxicity was 32 mg/kg bw/day
    (Costlow & Kane, 1984a).

         Myclobutanil (84.5% purity) was administered orally by gavage
    at 0 (vehicle, corn oil), 31, 94, 310 or 470 mg a.i./kg bw/day to
    groups of 25 presumed pregnant Crl:CD(SD)BR rats from days 6 to 15
    of gestation. The day on which sperm were found in the vaginal smear
    was considered day 0 of gestation. All surviving dams were killed on
    day 20 of gestation. A NOAEL for maternal toxicity was indicated at
    94 mg/kg bw/day based on clinical signs of toxicity (rough hair
    coat, desquamation and salivation) at doses of 310 mg/kg bw/day and
    higher. At 470 mg/kg bw/day, red exudate around the mouth, scant
    faeces and decreased body-weights were also observed. Decreased
    viability indices (viable fetuses /implantation sites) and a slight
    trend toward increasing resorption rate were recorded at 94 mg/kg
    bw/day and higher resulting in a NOAEL for embryofetal toxicity of
    31 mg/kg bw/day. An increased incidence of skeletal variations of
    the ribs (7th cervical and 14th rudimentary ribs) was observed at
    310 mg/kg bw/day and higher. Treatment with myclobutanil failed to
    reveal any evidence of teratogenic potential (Costlow & Kane,
    1984b).

    Rabbits

         A range-finding study was conducted with myclobutanil (84.5%
    purity) administered orally by gavage at 0 (vehicle, 1% methyl
    cellulose), 10, 31.6, 100, 215, 464 or 700 mg a.i./kg bw/day to
    groups of 6 artificially inseminated New Zeeland white rabbits on
    days 7 through 19 of gestation. The day of artificial insemination
    was designated as day 0 of gestation. All surviving animals were
    killed on day 29. Myclobutanil was lethal at doses of 464 mg/kg
    bw/day and higher resulting in 100% mortality. Maternal toxicity
    represented by clinical signs (irregular faeces and red-stained
    urine) and decreased body-weights was noted at 215 mg/kg bw/day. An
    increased incidence of resorptions and decreased litter size (viable
    fetuses/litter) was observed at 215 mg/kg bw/day, resulting in an
    overall NOAEL of 100 mg/kg bw/day. Viable fetuses from surviving
    rabbits appeared normal upon gross examination (Costlow & Kane,
    1984c).

         Groups of 18 artificially inseminated female New Zeeland white
    rabbits were administered myclobutanil (90.4% purity) at 0
    (distilled water control), 0 (vehicle, 1% methyl cellulose), 20, 60
    or 200 mg a.i./kg bw/day orally by gavage from day 7 through 19 of
    gestation. The day of insemination was designated as day 0 of
    gestation. All surviving rabbits were killed on day 29. The NOAEL
    for maternal toxicity was 20 mg/kg bw/day, based on minimal,
    transient body-weight loss at 60 mg/kg bw/day. Maternal animals at
    the high-dose of 200 mg/kg bw/day experienced body-weight loss and
    clinical signs reported as irregular faeces and blood-stained urine.
    Embryofetal toxicity was manifest at 200 mg/kg bw/day as an
    increased frequency of abortion and total litter resorption, an
    increased incidence of litters with resorptions as well as reduced
    litter size and fetal weight. Myclobutanil was not teratogenic when
    administered to pregnant rabbits at dose levels of up to 200 mg/kg
    bw/day (Costlow & Kane, 1984d).

    Special studies on genotoxicity

         Myclobutanil did not reveal any evidence of genotoxic potential
    when investigated in a battery of assays specific for gene mutation
    in microbial and mammalian cells or for detection of chromosomal
    aberrations in cytogenetics studies. Myclobutanil did not induce
    unscheduled DNA synthesis in isolated rat hepatocytes and was
    negative in a DNA repair test with  Bacillus subtilis. A dominant
    lethal study in rats was also negative. The results of the
    genotoxicity studies are presented in Table 2.

    Special studies on irritation and sensitization

         Eye irritation potential was studied in 9 male New Zeeland
    white rabbits given technical myclobutanil with a purity of 78.4%
    (Krzywicki, 1983) or 91.9% (Krzywicki & Bonin, 1984). Treatment of
    the eyes with higher purity technical material produced both corneal
    and conjunctival effects suggestive of moderate to severe irritating
    potential. Myclobutanil with a purity of 78.4% was only slightly
    irritating to the eyes of rabbits resulting in reversible
    conjunctival effects.

         A 0.5 ml aliquot of technical myclobutanil was applied dermally
    to the shaved backs of 6 male New Zeeland rabbits under occluded
    conditions for a 4-hour exposure period. Myclobutanil with a purity
    of 78.4% (Krzywicki, 1983) or 91.9% (Krzywicki & Bonin, 1984) was
    practically non-irritating to the skin of male rabbits.


    
    Table 2.  Genotoxicity of technical myclobutanil
                                                                                                                                               

    Test              Test system                         Concentration (vehicle)                 Puritya  Results    Reference
                                                                                                                                               

    Reverse           S. typhimurium                                                              99%      negative   Byers & Lohse (1983)
    Mutation          TA 98, 100,                         75, 250, 750, 2500, 7500 g/plate                1., 2.
    (in vitro)        TA 1535,                            75, 250, 500, 1000, 5000 g/plate
                      TA 1537                             250, 750, 1500, 2500, 7500 g/plate
                                                          (DMSO)

                      S. typhimurium                                                              84.5%    negative   Byers & Chism (1983a)
                      TA 98, 100, 1535, 1537              75 - 7500 g/plate (DMSO)                        1., 2.

                      S. typhimurium                                                              90.4%    negative   Byers & Chism (1983b)
                      TA 98, 100, 1535, 1537              75, 250, 750, 2500, 7500 g/plate                1., 2.
                                                          (DMSO)
                      S. typhimurium                                                              91.4%    negative   Sutou (1987)
                      TA 98, 100, 1535, 1537              125, 250, 500, 1000, 2000 ug/plate               1., 2.

                      E. coli WP2 uvrA-                   (DMSO)

    Point mutation    Chinese hamster ovary, K1BH4 cell   1. 120-175 g/ml                        81.1%    negative   O'Neill  et al. (1984)
    (in vitro)        line - HGPRT locus                  2. 25-90 g/ml                                   1., 2.
                                                          (DMSO)

    Chromosome        Chinese hamster ovary WB1 cells     1. 20, 30, 40, 50 g/ml                 91.9%    negative   Ivett (1985)
    aberration                                            2. 25, 50, 75 g/ml                              1., 2.
    (in vitro)                                            (DMSO)

    Chromosome        Mouse (male CR CD-1)                0, 65, 260, 650 mg/kg bw                81.1%    negative   McLeod & McCarthy (1984)
    aberration        bone marrow                         (corn oil)
    (in vivo)

                      Mouse                               0, 117, 585, 1170 mg/kg bw              91.4%    negative   Sames & Frank (1987)
                      (male and female                    (corn oil)
                      Crl:CD-1(ICR)), 
                      bone marrow

    Table 2 (cont'd)
                                                                                                                                               

    Test              Test system                         Concentration (vehicle)                 Puritya  Results    Reference
                                                                                                                                               

    DNA repair        Bacillus subtilis                   312.5, 625, 1250, 2500, 5000 ug/plate   91.4%    negative   Sutou (1987)
    (in vitro)        H17, M45                                                                             1., 2.

    Unscheduled DNA   Rat                                 0.1 - 1000 ug/ml                        91.9%    negative   Muller (1986)
    synthesis         (CRCD, Crl:CDBR male)               (DMSO)
    (in vitro)        hepatocytes

    Dominant          Rat, male                           0, 10, 100, 735 mg/kg bw                91.4%    negative   Dearlove  et al. (1986)
    lethal            Crl:COBS(SD)BR                      (corn oil)
    (in vivo)
                                                                                                                                               

    DMSO = dimethyl sulfoxide

    1. = in the presence of metabolic activation
    2. = in the absence of metabolic activation

    a  = active ingredient (a.i.)


    
         The potential of technical myclobutanil to induce delayed
    contact hypersensitivity in the Hartley guinea-pig using a modified
    Buehler procedure, could not unequivocally be ascertained due to a
    low incidence of animals with erythema response following challenge
    doses with the test material (Bonin & Hazelton, 1987a). Technical
    myclobutanil, when administered to guinea-pigs according to the
    method of Magnusson and Kligman did not produce delayed contact
    hypersensitivity (Kreuzmann, 1989).

    Observations in humans

         No information was available.

    COMMENTS

         Myclobutanil was rapidly absorbed when administered orally to
    rats and mice. The principal routes of excretion were via the urine
    and faeces, with no significant residual tissue accumulation. The
    toxicokinetic model in the rodent did not differ markedly with
    respect to species, sex, single versus repeated exposures or doses.

         In both the rat and mouse, myclobutanil was extensively
    metabolized to more polar compounds. The proposed metabolic pathway
    of myclobutanil in the rat was through oxidation of the butyl group.
    The major excretory metabolites were qualitatively similar with
    respect to sex and dose.

         Myclobutanil was only slightly toxic upon acute oral
    administration to rats and mice. WHO has classified myclobutanil as
    slightly hazardous (WHO, 1992).

         The primary target organ upon repeated dietary exposure to
    myclobutanil was the liver. Histomorphological changes,
    characterized predominantly by centrilobular hepatocytic hypertrophy
    in association with increased liver weights, were observed in all
    species investigated. Microscopically, there was accentuated lobular
    architecture, hepatocytic vacuolation, inflammation, necrosis, and
    pigmentation of the Kupffer cells. Increased hepatic enzyme activity
    in serum (ALAT, ASAT, GGT, ALP) were also observed. Hepatic
    microsomal MFO activities in rats and mice were increased
    correspondingly. There were no similar increases in hepatic
    peroxisomal -oxidation activity that would have suggested
    peroxisomal proliferation.

         A three-month dietary study with myclobutanil in the mouse at
    levels of 0, 3, 10, 30, 100, 300, 1000, 3000 or 10 000 ppm revealed
    hepatic alterations at dietary levels of 1000 ppm and higher,
    resulting in a NOAEL of 300 ppm, equal to 42.1 mg/kg bw/day.

         Two 3-month studies in rats fed myclobutanil at levels of 0,
    10, 30, 100, 300, 1000, 3000, 10 000, or 30 000 ppm and 0, 100, 300,
    or 3000 ppm indicated a NOAEL of 100 ppm, equal to 5.2 mg/kg bw/day,
    based on treatment-related hepatic effects.

         The NOAEL for myclobutanil-related liver effects in dogs
    treated for 3 months at 0, 10, 200, 800 or 1600 ppm was 10 ppm,
    equal to 0.3 mg/kg bw/day. Treatment of dogs with myclobutanil for
    12 months at dietary levels of 0, 10, 100, 400, or 1600 ppm resulted
    in a NOAEL for hepatic effects of 100 ppm, equal to 3.1 mg/kg
    bw/day. Myclobutanil administered to two dogs per sex at dietary
    levels of up to 1000 ppm, equal to 39 mg/kg bw/day, did not produce
    any hepatic changes after a period of 4 weeks.

         Long-term dietary treatment of mice with myclobutanil for two
    years at 0, 20, 100 or 500 ppm revealed a NOAEL of 20 ppm, equal to
    2.7 mg/kg bw/day, based on increased MFO activity at 100 ppm as well
    as more pronounced liver toxicity at 500 ppm and above. Myclobutanil
    was not carcinogenic in mice.

         A 24-month long-term toxicity/carcinogenicity study in rats at
    dietary concentrations of 0, 50, 200 or 800 ppm revealed a NOAEL of
    50 ppm, equal to 2.5 mg/kg bw/day, based on findings of testicular
    atrophy and increased MFO activity at 200 ppm and above.
    Myclobutanil was not carcinogenic in rats.

         A two-generation reproduction study in rats at dietary
    concentrations of 0, 50, 200 or 1000 ppm revealed a NOAEL of 50 ppm,
    equal to 3.6 mg/kg bw/day, based on increased liver weights and an
    increase in numbers of stillborn pups at 200 ppm and above. At 1000
    ppm atrophy of the testes and prostate were observed.

         An oral teratogenicity study in rats at gavage doses of 0, 31,
    94, 310, or 470 mg/kg bw/day demonstrated clinical signs of toxicity
    at 310 mg/kg bw/day and above, indicating a NOAEL of 94 mg/kg
    bw/day. The NOAEL for embryofetal toxicity was 31 mg/kg bw/day.
    There was no evidence of teratogenicity at doses up to 470 mg/kg
    bw/day.

         Myclobutanil was not teratogenic when administered to the
    rabbit at gavage doses of 20, 60 or 200 mg/kg bw/day. A NOAEL for
    maternal toxicity was 20 mg/kg bw/day, based on decreased body-
    weight at 60 mg/kg bw/day and above. Embryofetal toxicity was
    evident at 200 mg/kg bw/day.

         After reviewing the available genotoxicity data, the Meeting
    concluded that myclobutanil was not genotoxic.

         An ADI was allocated on the basis of NOAELs in two-year feeding
    studies in mice and rats, a reproduction study in rats and a one-
    year study in dogs, using a 100-fold safety factor.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

         Mouse:    20 ppm, equal to 2.7 mg/kg bw/day (two-year study)

         Rat:      50 ppm, equal to 2.5 mg/kg bw/day (two-year study)
                   50 ppm, equal to 3.6 mg/kg bw/day (two-generation
                        reproduction study)

         Dog:      100 ppm, equal to 3.1 mg/kg bw/day (one-year study)

    Estimate of acceptable daily intake for humans

         0 - 0.03 mg/kg bw

    Studies which will provide information valuable in the continued
    evaluation of the compound

         1.   Results of ongoing long-term studies in mice and rats
              known to be in progress.

         2.   If the results of (1) show a carcinogenic response,
              studies (a) to determine whether myclobutanil acts as a
              tumour promoter in the two-stage rat liver bioassay and
              (b) whether it causes inhibition of intercellular
              communications.

         3.   Observations in humans.

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    Byers, M.J. & Chism, E.M. (1983a). Microbial mutagen assay. Report
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    Spring House, PA, USA.

    Steigerwalt, R.B., Udinsky, J.R. & Longacre, S.L. (1986b). RH-3866
    kinetic and metabolism study in rats. Report No. 83R-144.
    Unpublished report dated August 29, 1986 from Rohm and Haas Company,
    Spring House, PA, USA. Submitted to WHO by Rohm and Haas Company,
    Spring House, PA, USA.

    Streelman D.R. (1984). Material balance and metabolism study of
    14C-RH-3866 in rats. Report No. TR 310-84-16. Unpublished report
    dated June 22, 1984 from Rohm and Haas Company, Spring House, PA,
    USA. Submitted to WHO by Rohm and Haas Company, Spring House, PA,
    USA.

    Sutou, S. (1987). Mutagenicity testing of RH-53,866 technical with
    bacteria. Report No. NRILS 86-2112; R & H Report No. 87RC-037.
    Unpublished report dated August 21, 1987 from NRI Life Science,
    Kanagawa, Japan. Submitted to WHO by Rohm and Haas Company, Spring
    House, PA, USA.

    WHO (1992). The WHO recommended classification of pesticides by
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    Available from the International Programme on Chemical Safety, World
    Health Organization, Geneva, Switzerland.


    See Also:
       Toxicological Abbreviations