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    FLUSILAZOLE

    First draft prepared by
    S. Ma,

    Health Evaluation Division, Pest Management Regulatory Agency, Health
    Canada, Ottawa, Canada

    Explanation
    Evaluation for acceptable daily intake
         Biochemical aspects
              Absorption, distribution, and excretion
              Biotransformation
         Toxicological studies
              Acute toxicity
              Short-term toxicity
              Long-term toxicity and carcinogenicity
              Reproductive toxicity
              Developmental toxicity
              Genotoxicity
              Special studies
                   Dermal and ocular irritation and dermal sensitization
                   Mechanisms of Leydig-cell tumour induction
              Comments
              Toxicological evaluation
    References

    Explanation

         Flusilazole was previously evaluated by the Joint Meeting in 1989
    (Annex I, reference 56). An ADI of 0-0.001 mg/kg bw was allocated on
    the basis of an NOAEL of 0.14 mg/kg bw per day (5 ppm) for liver
    toxicity in a one-year feeding study in dogs. The compound was
    reexamined at the present Meeting in response to a request from the
    manufacturer. This monograph summarizes pertinent new (since 1989)
    data as well as relevant data from the previous monograph.

    Evaluation for acceptable daily intake

    1.  Biochemical aspects

         The toxicokinetics of technical-grade flusilazole has been
    studied after oral administration of radiolabelled test material in
    rats. Summaries of the relevant data are presented below.

    (a)  Absorption, distribution, and excretion

         14C-Flusilazole (uniformly phenyl-labelled) was administered
    orally to groups of two male and two female Charles River CD rats by
    one of three regimens: a single low dose of about 8 mg/kg bw; a single
    low dose of about 8 mg/kg bw after pretreatment for 21 days with a
    diet containing 100 ppm unlabelled flusilazole; or a single high dose
    of about 200 mg/kg bw. By 96 h (first regimen) or 168 h (other two
    regimens), about 90% of the administered radiolabel had been
    eliminated in the urine and faeces, with an excretion half-life of
    about 34 h. No significant amount of radiolabel was recovered in the
    expired air as carbon dioxide or volatile metabolites. The faeces was
    the main route of elimination, and there was an apparent sex
    difference in the excretion pattern: in males, 87 and 8% of the dose
    were eliminated in faeces and urine, respectively, while in females
    the corresponding figures were 59 and 23%. Pretreatment with non-
    radiolabelled flusilazole did not affect excretion. Tissue retention
    of the radiolabel was very low, with total residues accounting for
    < 2.5% of the administered dose. The largest amounts of radiolabel
    were found in the carcass, gastrointestinal tract, and liver (means,
    < 1% of dose). The tissue concentrations were proportional to the
    amount of flusilazole administered. In the same study, an additional
    group of one male and one female rat was given a single oral dose of
    14C-flusilazole (triazole-3-labelled) of about 8 mg/kg bw. As with
    the phenyl-labelled flusilazole, 88% of the administered radiolabel
    was recovered in urine and faeces within 96 h of treatment; however,
    urine was the predominant route of excretion. In males, urinary and
    faecal radiolabel accounted for 78 and 11% of the dose, respectively,
    while in females the corresponding figures were 59 and 26%. Tissue
    retention of radiolabel was low, total residues accounting for about
    3% of the administered dose. The highest concentrations of label were
    found in the carcass (about 2% of dose), skin, gastrointestinal tract,
    and liver (means, < 0.5% of dose) (Anderson  et al., 1986).

         14C-Flusilazole (triazole-3-labelled) was administered orally to
    groups of five male and five female Charles River Crl:CD(SD)BR rats by
    one of three regimens: a single dose of about 8 mg/kg bw; a single
    dose of about 8 mg/kg bw after pretreatment with about 8 mg/kg bw per
    day of non-radiolabelled flusilazole by gavage for 14 days; or a
    single dose of about 224 mg/kg bw. At 96 h (first regimen) or 120 h
    (other two regimens), the total recovery of radiolabel was 92.6-99.2%,
    with about 90% eliminated within the first 48 h of treatment. Urine

    was the primary route of excretion,, accounting for about 72% of the
    administered dose, while faecal excretion accounted for about 17%. No
    differences according to sex or regimen were observed. Tissue
    retention of the radiolabel was low: the carcass accounted for < 3%
    of dose and the remaining tissues for < 0.2.% (Cheng, 1986).

    (b)  Biotransformation

         14C-Labelled flusilazole was extensively metabolized after oral
    administration to Charles River CD rats. Recovered parent compound
    accounted for only 2-11% of the dose in all animals, regardless of
    dose or pretreatment with unlabelled compound, and was found
    predominantly in the faeces; the urinary levels represented < 1% of
    the dose. After absorption, flusilazole was cleaved at the triazole
    ring. With phenyl-labelled material, the major faecal metabolites
    identified were: [bis(4-fluorophenyl)methyl] silanol (about 30% of the
    dose in males, about 19% in females); [bis(4-fluorophenyl)methylsilyl]
    methanol (about 9% of the dose in animals of each sex); the fatty acid
    conjugates of [bis(4-fluorophenyl)methylsilyl] methanol (19% of the
    dose in males, 10% in females); and disiloxane (about 11% in males,
    about 7% in females). Except for the fatty acid conjugates, the same
    metabolites were found in urine. In males, all three urinary
    metabolites represented < 1% of dose; in females, [bis(4-
    fluorophenyl)methyl] silanol, [bis(4-fluorophenyl)methylsilyl]
    methanol, and siloxane represented 7.5, 2.2, and 1.9% of the
    administered dose, respectively. With triazole-labelled material, the
    main metabolite identified was 1 H-1,2,4-triazole, which was found
    predominantly in urine as 63.8% of the dose in males and 51.6% in
    females. Faeces contained only a minor amount of the metabolite (4% of
    the dose in males, 17% in females). A metabolic pathway for
    flusilazole in rats was proposed on the basis of these results 
    (Figure 1) (Anderson  et al., 1986).

    2.  Toxicological studies

    (a)  Acute toxicity

         The results of studies of the acute toxicity of technical-grade
    flusilazole are summarized in Table 1.

         Flusilazole is moderately toxic to mice, rats, and rabbits when
    given orally and minimally toxic to rats and rabbits when administered
    dermally or by inhalation. Symptoms of toxicity after oral
    administration included weight loss, weakness, lethargy, and, at
    higher doses, prostration, salivation, laboured breathing,
    convulsions, and loss of righting reflex. Dermal administration
    resulted only in mild erythema at the site of application. The effects
    of inhalation were mainly laboured breathing and lung sounds.

    CHEMICAL STRUCTURE 1

        Table 1.  Acute toxicity of flusilazole
                                                                                                   

    Species      Sex                Route             LD50 or LC50        Reference
                                                      (mg/kg bw or
                                                      mg/litre air)
                                                                                                   

    Mouse        Male               Oral                   680            Wylie et al. (1985)
                 female                                   1000
    Rat          Male               Oral                  1500            Wylie et al. (1983)
                 Male               Oral                  1110            Wylie et al. (1984a)
                 Female                                    674
                 Male               Inhalation             2.7            Poindexter et al. (1984)
                 Female                                    3.7
                 Male, female       Inhalation         6.8-7.7            Turner et al. (1985)
    Rabbit       Male, female       Oral                   450            Redgate et al. (1985)
                 Male, female       Dermal              > 2000            Gargus & Sutherland (1983)
                                                                                                   
        (b)  Short-term toxicity

    Mice

         Groups of 20 male and 20 female Crl:CD-1 mice were given
    technical-grade flusilazole (purity, 96.7%) at dietary levels of 0,
    25, 75, 225, 500, or 1000 ppm, equal to 0, 4, 12, 36, 82, or 164 mg/kg
    bw per day for males and 0, 5, 15, 43, 92, or 222 mg/kg bw per day for
    females, for up to 90 days. Ten mice of each sex per group were
    sacrificed after four weeks of treatment; the remaining mice were
    killed at the end of the study. No histopathological examination was
    conducted on animals killed at four weeks. There were no clinical
    symptoms of toxicity and no treatment-related effects on body weight
    or food consumption in animals sacrificed at either interval. The
    target organs of toxicity were liver and urinary bladder; females were
    slightly more sensitive to flusilazole than males. No treatment-
    related effects were observed at the lowest dose. At 75 ppm, increased
    absolute and relative liver weights and an increased incidence (1/10)
    of hepatocellular vacuolar cytoplasmic changes were observed in
    females. At > 225 ppm, increased liver weight, dose-related
    increases in the incidence of hepatocellular vacuolar cytoplasmic
    changes, hepatocellular hypertrophy, and urinary bladder urothelial
    cell hyperplasia were evident in animals of each sex. At the highest
    dose, absolute and relative kidney weights were decreased in males,
    but no treatment-related renal lesions were observed. In addition,
    slight decreases in erythroid parameters (haemoglobin, haematocrit,
    and erythrocyte counts) were noted in animals of each sex. The NOAEL
    was 4 mg/kg bw per day (Pastoor  et al., 1984).

         Groups of 16 male and 16 female Crl:CD-1(ICR)BR mice were fed
    diets containing technical-grade flusilazole (purity, 94%) at doses of
    0, 1000, 2500, or 5000 ppm, equal to 0, 161, 436, and 1004 mg/kg bw
    per day for males and 0, 239, 601, and 1414 mg/kg bw per day for
    females, for at least 90 days. An additional six mice of each sex per
    group were assessed for cellular proliferation in the liver and
    urinary bladder on days 14 and 106. At the lowest dose, treatment-
    related effects included reduced mean body weight and food efficiency
    (males only), increased absolute and relative liver weights (animals
    of each sex), decreased absolute and relative kidney weights (females
    only), and hypertrophy or hyperplasia, cytoplasmic vacuolation, and
    inflammation of the liver and urinary bladder (animals of each sex).
    At the mid-dose, increased cellular proliferation of the urinary
    bladder was also observed in animals of each sex. At the highest dose,
    severe body-weight loss occurred in males and reduced body weight and
    food efficiency were noted in females. The males at this dose were
    sacrificed on day 44 owing to excess mortality and moribund condition.
    There was no NOAEL (Keller, 1990).

    Rats

         Groups of six male Crl:CD rats were given technical-grade
    flusilazole (purity, 95.5%) by gavage in corn oil at a dose of 0 or
    300 mg/kg bw per day, five days per week for two weeks. Three rats per
    group were killed at the end of the treatment period, and the
    remaining rats were sacrificed after a two-week recovery period. All
    animals were examined histopathologically. One treated animal died
    after the fifth dose on day 7. Clinical signs of toxicity (lowered
    body weight, alopecia, diarrhoea, stained or wet perineal area,
    salivation, and hypersensitivity) were observed in four rats during
    the treatment period. Treatment-related histopathological changes were
    observed in liver, kidney, urinary bladder, and testis; the lesions
    appeared to be less severe in rats killed after the recovery period.
    Remarkable histopathological findings in treated rats included
    hepatocellular vacuolation (six rats), hyperplasia and vacuolation of
    the urinary bladder transitional epithelium (six rats) and renal
    pelvis urothelium (two rats), and necrosis and cellular degeneration
    of the germinal epithelium of the seminiferous tubules (two rats)
    (Wylie  et al., 1984b).

         Four groups of 10 male and 10 female Charles River CD rats were
    fed diets containing technical-grade flusilazole (purity, 96.7%) at
    doses of 0, 25, 125, 375, or 750 ppm, equal to 0, 2, 9, 27, and
    55 mg/kg bw per day for males and 0, 2, 11, 31, and 70 mg/kg bw per
    day for females, for 90 days. No treatment-related effects were
    oberved at the two lower doses. At 375 ppm, an increased serum
    cholesterol level was seen in animals of each sex and an increased
    incidence of mild bladder urothelial hyperplasia in one male and four
    females. At the highest dose, bladder urothelial hyperplasia was seen
    in five males and eight females. Additional treatment-related changes
    included decreased body weight in females, increased absolute and
    relative liver weights in animals of each sex, and remarkable
    histopathological findings (hepatocellular hypertrophy, mild fatty
    changes, and hepatocytolysis) in the livers of five males. The NOAEL
    was 9 mg/kg bw per day (Pastoor  et al., 1983).

         Groups of 52 male and 52 female Crl:CD BR rats were fed diets
    containing technical-grade flusilazole (purity, 95%) at doses of 0,
    10, 125, 375, or 750 ppm, equal to 0, 0.58, 7.27, 22.1, and 44.7 mg/kg
    bw per day for males and 0, 0.74, 9.40, 27.6, and 59.0 mg/kg bw per
    day for females, for up to 91 days. Each group was divided into three
    subgroups of 20 rats of each sex in order to study possible mechanisms
    of action of toxicity in the liver and urinary bladder. Five rats of
    each sex were sacrificed on days 7/8, 14, 46, and 91 for assessment of
    cellular proliferation and histopathology. For evaluation of
    cytochrome P450 and peroxisome proliferation, five rats of each sex
    were sacrificed on days 14 and 90 (males) or 15 and 91 (females).
    Serum levels of testosterone, estradiol, and luteinizing hormone were
    determined in all males sacrificed on days 14 and 90. No adverse

    treatment-related effects were observed at the two lower doses. At
    125 ppm, absolute and relative liver weights were increased; in the
    absence of histopathological changes or any other symptoms of
    toxicity, the finding was not considered to be toxicologically
    significant. At the two highest doses, liver hypertrophy was observed.
    The response was sex-dependent: lamellar bodies and periportal
    hypertrophy occurred in males and centrilobular hypertrophy without
    lamellar bodies was seen in females. In the urinary bladder,
    transitional-cell necrosis, exfoliation and hyperplasia were noted.
    Hepatic cytochrome P450 levels were higher than control values, but
    there was no evidence of peroxisome proliferation in the livers of
    treated rats. No significant changes in the serum levels of
    testosterone, estradiol, or luteinizing hormone were found in any of
    the males examined. The NOAEL was 7.27 mg/kg bw per day (Keller,
    1992a).

    Rabbits

         Groups of five male and five female New Zealand white rabbits
    received dermal applications of technical-grade flusilazole (purity,
    94.9%) at doses of 0, 1, 5, 25, or 200 mg/kg bw per day for 21 days.
    The test material was applied daily as a paste in distilled water; the
    exposure sites were occluded for 6 h and then washed with water. No
    evidence of systemic toxicity was observed. The NOAEL for dermal
    toxicity was 5 mg/kg bw per day, based on diffuse epidermal
    hyperplasia or thickening (slight to mild) at > 25 mg/kg bw per
    day. At the highest dose, mild erythema was seen on days 6-12. The
    NOAEL for systemic toxicity was > 200 mg/kg bw per day (Sarver
     et al., 1986).

    Dogs

         Groups of four male and four female beagle dogs were fed diets
    containing technical-grade flusilazole (purity, 93%) at concentrations
    of 0, 25, 125, or 750/500 ppm, equal to 0.9, 4.3, and 13.4 mg/kg bw
    per day for males and 0, 0.9, 4.3, and 14.2 mg/kg bw per day for
    females, for three months. The high-dose group received 750 ppm
    flusilazole in the diet for the initial three weeks, then control diet
    for one week, and 500 ppm flusilazole in the diet for the remaining
    period. The dose was reduced because of marked body-weight loss and
    decreased food intake at 750 ppm. At the lowest dose, there was a
    treatment-related increase in the incidence of hyperplasia of the
    lymphoid follicles in the pyloric glandular mucosa of the stomach in
    males: 0/4, 3/4 3/4, and 4/4 at 0, 25, 125, and 750/500 ppm,
    respectively. At 125 ppm, an increased incidence of pyloric granular
    mucosa hyperplasia was also observed in females: 0/4, 0/4, 3/4, and
    4/4 at 0, 25, 125, and 750/500 ppm, respectively. Higher levels (in
    comparison with control values) of alanine aminotransferase activity
    and an increased incidence of mild urinary bladder mucosal hyperplasia
    were observed in males at 125 ppm and in animals of each sex at the

    highest dose. Additional treatment-related effects seen at the highest
    dose included clinical signs of toxicity (weakness or tremors) in
    animals of each sex, reduced mean body-weight gain in males,
    body-weight loss in females, decreased mean food consumption in
    animals of each sex, a slight increase in leukocyte and monocyte
    counts in males, decreased plasma levels of cholesterol, total
    protein, and albumin in animals of each sex, and increased absolute
    and relative liver weights in animals of each sex. There was no NOAEL
    (Rickard  et al., 1983).

         Groups of five male and five female beagle dogs were fed
    technical-grade flusilazole (purity, 95.8%) in the diet at doses of 0,
    5, 20, or 75 ppm, equal to 0, 0.14, 0.7, and 2.4 mg/kg bw per day for
    males and 0, 0.14, 0.7, and 2.6 mg/kg bw per day for females, for one
    year. No treatment-related effects were observed at the lowest dose.
    At the next dose, 20 ppm, the serum albumin level was decreased in
    males and there was a dose-related increase in the incidence of
    centrilobular hepatocellular hypertrophy in both males (0/5, 0/5, 4/5,
    and 5/5) and females (0/5, 0/5, 2/5, and 5/5). More severe gastric
    mucosal lymphoid hyperplasia, from minimal at 0 or 5 ppm to mild at 
    20 ppm, was also noted in males. At the highest dose, additional
    treatment-related effects included higher leukocyte counts in animals
    of each sex; elevated alkaline phosphatase and lowered cholesterol,
    total protein, and albumin levels in males only; and increases in
    relative liver (animals of each sex) and kidney weights (females). All
    high-dose animals exhibited a greater degree of hepatic centrilobular
    inflammatory infiltration, and distinct centrilobular hepatocellular
    vacuolation was observed in three males. The lymphoid hyperplasia in
    the gastric mucosa was of moderate severity in both males and females.
    The NOAEL was 0.14 mg/kg bw per day (O'Neal  et al., 1985).

    (c)  Long-term toxicity and carcinogenicity

    Mice

         Groups of 80 male and 80 female Crl:CD-1(ICR)BR mice received
    technical-grade flusilazole (purity, 96.5%) in the diet at doses of 0,
    5, 25, or 200 ppm, equal to 0, 0.66, 3.4, and 27 mg/kg bw per day for
    males and 0, 0.92, 4.6, and 36 mg/kg bw per day for females, for 18
    months. Each group included 10 mice of each sex that were killed at
    six months. There were no treatment-related effects on mortality
    (terminal survival rates were 76-86% in males and 57-80% in females),
    body weight, food consumption, haematology, or serum chemistry at any
    dose. At the highest dose, a toxicologically significant increase in
    the incidence of hepatocellular fatty changes was observed, with
    terminal incidences of 4/80, 3/80, 10/80, and 40/80 males and 2/80,
    4/80, 3/80, and 24/80 females at 0, 5, 25, and 200 ppm. In addition,
    there was an increase in absolute and relative liver weights in
    animals of each sex, a decrease in absolute kidney weight in females,
    and an increase in lymphocytic infiltration in the lung and urinary

    bladder in males. There was no treatment-related increase in the
    incidence of any tumour type at doses > 27 mg/kg bw per day, the
    highest dose tested. The NOAEL for systemic toxicity was 3.4 mg/kg bw
    per day (Brock  et al., 1985).

         Groups of 100 male and 100 female Crl:CD-1(ICR)BR mice received
    diets containing technical-grade flusilazole (purity, 94%) at doses of
    0, 100, 500, or 1000 ppm in males, equal to 0, 14.3, 73.1, and
    144 mg/kg bw per day, and 0, 100, 1000, or 2000 ppm in females, equal
    to 0, 19.4, 200, and 384 mg/kg bw per day, for 18 months. An
    additional group of 100 mice of each sex was fed a diet containing
    25 ppm technical-grade flusilazole (equal to 3.51 mg/kg bw per day for
    males and 4.38 mg/kg bw per day for females) and was used to study
    cell proliferation. The doses were selected on the basis of the
    results of a 90-day study that indicated greater sensitivity of males
    to the test compound. At the lowest dose tested, there was a slight
    decrease in absolute kidney weight, an increase in focal necrosis of
    the liver, and urinary bladder hyperplasia in males. No remarkable
    changes were seen in females at this dose. At the two highest doses,
    females had increased cellular hyperplasia in the urinary bladder and
    urethra, but no focal necrosis of the liver. At these two doses,
    additional treatment-related systemic effects included increased
    absolute and relative liver weights, lower absolute and relative
    kidney weights, increased numbers of foci of hepatocellular
    alteration, and an increased incidence of hepatic vesicular or
    vacuolar changes with cellular hypertrophy. Significantly higher
    mortality occurred at 1000 ppm in males and 2000 ppm in females. In
    addition, increased cellular proliferation in the urinary bladder (but
    not in the liver) was observed in females at the highest dose. At
    terminal sacrifice, an increased incidence of hepatocellular adenomas
    and carcinomas was observed in males at all doses (13/80, 23/79,
    20/80, and 18/78 at 0, 100, 500, and 1000 ppm, respectively;
    historical control range, 6.3-13.8%) and in females at the two highest
    doses (1/79, 3/80, 11/77, and 43/76 at 0, 100, 1000, and 2000 ppm,
    respectively; historical control range, 0-2.6%). The increase in
    tumour incidence was dose-related and statistically significant in
    females but not in males. There was no NOAEL for systemic toxicity or
    oncogenicity (Keller, 1992b).

    Rats

         Groups of 70 male and 70 female rats (Crl:CD(SD)BR strain)
    received technical-grade flusilazole (purity, 95.6%) in the diet at
    doses of 0, 10, 50, or 250 ppm, equal to 0, 0.4, 2.0, and 10 mg/kg bw
    per day for males and 0, 0.5, 2.6, and 13 mg/kg bw per day for
    females, for two years. Each group included two groups of 10 rats of
    each sex that were killed at six months and one year. About 100 days
    after the beginning of treatment, 20 rats of each sex in each group
    were mated and were returned to the study after weaning of the second
    litter. The rats killed at six months were examined for bladder

    lesions only. No treatment-related effect on mortality was observed;
    the survival rate was > 50% in all groups until week 98. No signs of
    systemic toxicity was seen at the low dose. At 50 ppm, there was a
    dose-related increase in the incidence of pyelonephritis in females at
    two years (3/66, 3/62, 8/67, and 10/65 at 0, 10, 50, and 250 ppm,
    respectively). Increases in relative liver weight in females and in
    the incidence of hydronephrosis in males were observed at interim
    sacrifice at one year. At the highest dose, additional treatment-
    related changes included an increased incidence of hepatic lesions
    (centrilobular hepatocellular hypertrophy and polyploidy) in females
    and hydronephrosis in males at one year and an increased incidence of
    acidophilic foci of hepatocellular alteration (13/65; 3/66 in
    controls) and hepatic diffuse fatty changes (23/65; 9/66 in controls)
    in females at two years. No hepatic lesions were observed in males
    during the study, and no lesions of the urinary bladder were seen in
    animals of either sex at either interim or terminal sacrifice. There
    was no treatment-related increase in the incidence of any tumour type
    at any dose. At 250 ppm, males had a slightly increased incidence of
    squamous-cell carcinomas of the oral and nasal cavities (0/66, 1/63,
    0/67, and 3/64 at 0, 10, 50, and 250 ppm, respectively). On the basis
    of historical control data, from five two-year feeding studies in rats
    with no tumours of this type and one study with an incidence of 2/60,
    the incidence of nasal tumours in this study was judged to be
    incidental. The NOAEL for systemic toxicity was 0.4 mg/kg bw per day;
    however, the effects reported were relatively mild, and higher doses
    might have been tolerated (Pastoor  et al., 1986).

         Groups of 65 male and 65 female Crl:CD(SD)BR rats received diets
    containing technical-grade flusilazole (purity, 95%) at doses of 0,
    125, 375, or 750 ppm, equal to 0, 5.03, 14.8, and 30.8 mg/kg bw per
    day for males and 0, 6.83, 20.5, and 45.6 mg/kg bw per day for
    females, for two years. Each group included 10 rats of each sex for
    interim sacrifice at one year. Dose-related increases in the incidence
    of hepatocellular hypertrophy were observed in animals of each sex at
    both interim (males: 0/10, 4/10, 8/10, and 8/10; females: 0/10, 7/10,
    10/10, and 10/10; at 0, 125, 375, and 750 ppm, respectively) and
    terminal sacrifice (males: 2/53, 2/51, 10/53, and 19/53; females:
    2/56, 4/55, 12/53, and 25/54). There was an apparent sex difference in
    the hepatic lesions: periportal hepatocellular hypertrophy with
    lamellar bodies was seen in males, while females showed centrilobular
    hepatocellular hypertrophy with eosinophilic cytoplasm but no lamellar
    bodies. At terminal sacrifice, there was also an increased incidence
    of mixed foci of cellular alteration in males (6/53, 14/51, 17/53, and
    19/53). At 375 and 750 ppm, there were also significant decreases in
    terminal body weight in females, increases in absolute and/or relative
    liver weights at interim and/or terminal sacrifice in males and/or
    females, and an increase in the incidence of urinary bladder
    transitional-cell hyperplasia at the end of the study in animals of
    each sex (3/46, 6/45, 27/47, and 42/51 in males and 5/47, 3/49, 15/49,
    and 33/53 in females). At 750 ppm, additional treatment-related

    changes included an increased incidence of hepatic fatty changes in
    males and increased cellular proliferation in the liver and urinary
    bladder. At the highest dose, a treatment-related increase in the
    incidence of urinary bladder transitional-cell papillomas and
    carcinomas was observed in animals of each sex at the end of the study
    (males: 0/46, 0/45, 1/47, and 5/51; and females: 0/47, 1/49, 0/49, and
    13/53; at doses of 0, 125, 375, and 750 ppm, respectively). The
    incidence of testicular interstitial-cell (Leydig-cell) tumours in
    males at the highest dose was also increased: 2/53, 4/51, 2/53, and
    9/53 at 0, 125, 375 and 750 ppm, respectively. There was no NOAEL for
    systemic toxicity; the NOAEL for oncogenicity was 14.8 mg/kg bw per
    day (Keller, 1992c).

    (d)  Reproductive toxicity

    Rats

         Groups of six male and six female Crl:CD(SD)BR rats from a 90-day
    feeding study were used in a one-generation study of reproductive
    toxicity. The rats were fed daily diets containing technical-grade
    flusilazole (purity, 96.7%) at doses of 0, 25, 125, or 375 ppm, equal
    to 0, 2, 9, and 27 mg/kg bw per day for males and 0, 2, 11, and
    31 mg/kg bw per day for females, for 90 days before mating. Males and
    females within the same dose group were mated for 15 days; females
    were examined daily for evidence of a copulation plug. After the
    mating period, females were housed individually. The fertility index
    was low in all groups and especially in the control group (67.7%);
    three to six females per group became pregnant. At the highest dose, a
    lower gestation index, a lower percentage of liveborn pups, and lower
    pup weight at day 4 were observed in comparison with controls (Pastoor
     et al., 1983). The small group size and the absence of individual
    data on some parameters limit the usefulness of this study for
    evaluation of reproductive toxicity.

         Groups of 20 male and 20 female Crl:CD(SD)BR rats from a two-year
    study of toxicity and oncogenicity were used in a two-generation,
    two-litter study. The rats were fed diets containing technical-grade
    flusilazole (purity, 95.6%) at doses of 0, 10, 50, or 250 ppm, equal
    to 0, 1, 3, and 18 mg/kg bw per day for males and 0, 1, 4, and
    20 mg/kg bw per day for females (premating intake) for at least 100
    days before mating. Males and females (F0) within the same dose group
    were mated 1:1 for 15 days; the females were examined daily for
    evidence of a copulation plug. After the mating period, F0 females
    were housed individually and allowed to give birth to F1a litters.
    About one week after the last F1a litter had been weaned, F0 females
    were mated with different F0 males of the same dose group to produce
    the F1b litters. After weaning of the F1b litters, 20 rats of each
    sex per group were selected as the F1 parents for the F2 generation.
    These rats were maintained on the same diets as their F0 parents for
    90 days before mating to produce the F2a and F2b litters. Sibling

    mating was avoided. The mean body weights of the F1b males at 250 ppm
    were decreased during the premating period. No other treatment-related
    systemic effect was observed in any F0 or F1 adult. There was no
    evidence of a treatment-related effect on mating or fertility.
    Treatment-related embryo-, feto-, and litter toxicity were observed at
    the mid- (50 ppm) and high doses (250 ppm). An increased number of
    stillborn pups and a decreased viability index (days 0-4) occurred in
    the F2a litters at 50 ppm and in all litters at 250 ppm. Litter
    survival after day 4 was similar in all groups in the F1 and F2
    generations. At 250 ppm, the mean weights of F1b and F2b pups of each
    sex at weaning were slightly reduced, and the absolute and relative
    liver weights of F2b weanling male pups were increased. An increased
    incidence of unilateral and/or bilateral hydronephrosis was noted in
    F2b female weanlings: 1/10, 4/10, 3/10, and 5/10 at 0, 10, 50, and
    250 ppm, respectively. Since neither the severity nor the incidence
    of the lesions showed a dose-response relationship and since
    hydronephrosis is a common lesion in weanling pups (historical control
    range in 13 in-house studies, 0-30%), the finding was judged to be
    toxicologically insignificant. The NOAEL was 1 mg/kg bw per day, based
    on embryo- and fetotoxicity and decreased viability of pups in the
    F2a litters at 50 ppm (Pastoor  et al., 1986).

         In a two-generation study with one or two litters per generation,
    groups of 30 male and 30 female Crl:CD(SD)BR rats were fed diets
    containing technical-grade flusilazole (purity, 94%) at doses of 0, 5,
    50, or 250 ppm, equal to 0, 0.34, 3.46, and 17.3 mg/kg bw per day for
    males and 0, 0.40, 4.04, and 19.6 mg/kg bw per day for females
    (premating intake), for 73 days (F0 rats) or 91 days (F1 parents)
    before mating. Males and females within the same dose group were
    randomly paired 1:1 for 15 days; females were examined daily for
    evidence of a copulation plug. One litter (F1a) was produced in the
    F0 generation and two litters (F2a and F2b) in the F1 generation.
    No treatment-related effects were seen in either the F0 or F1
    generation at the low dose. At the middle dose, increased smooth
    endoplasmic reticulum was seen in the hepatocytes of males and
    centrilobular hepatocellular hypertrophy in females. At the highest
    dose, F1 females had slightly but consistently lower body weights.
    Reproductive toxicity was indicated by higher mortality during
    parturition and increased gestational length of the F0 and F1 dams:
    mean, 22.9-23.2 days in comparison with 22.4-22.6 days in controls. No
    treatment-related effects on mating or fertility indices were
    observed. Embryo-, feto-, and litter toxicity were seen as reduced
    number of pups per litter and increased numbers of stillborn pups per
    litter in the F1a, F2a, and F2b litters and decreased mean pup
    weights on lactation days 14 and 21 (F2a litters only) at 250 ppm.
    The NOAEL for systemic toxicity was 0.34 mg/kg bw per day, on the
    basis of hepatic lesions at 50 ppm; the NOAEL for reproductive
    toxicity was 4.04 mg/kg bw per day, on the basis of treatment-related
    mortality during parturition, increased gestational length, and
    embryo-, feto-, and litter toxicity at 250 ppm (Mullin, 1990).

    (e)  Developmental toxicity

    Rats

         In a range-finding study, groups of seven pregnant rats received
    technical-grade flusilazole (purity, 99%) by gavage at doses of 0,
    100, or 300 mg/kg bw per day on days 7-16 of gestation. Flusilazole
    was maternally and embryotoxic at both doses, and the highest dose
    induced cleft palates in about 51% of the fetuses in each litter. In
    the main study of developmental toxicity, groups of 25 mated female
    Crl:CD(SD)BR rats received technical-grade flusilazole (purity, 95.6%)
    by gavage in corn oil at doses of 0, 10, 50, or 250 mg/kg bw per day
    on days 7-16 of gestation; the day a copulation plug was observed was
    designated as day 1 of gestation. On gestation day 21, all surviving
    dams were sacrificed and necropsied; fetuses were delivered by
    caesarean section and examined for external, visceral, and skeletal
    abnormalities. No treatment-related signs of maternal toxicity were
    seen at the lowest dose. At 50 mg/kg bw per day, slight decreases in
    body-weight gain and food consumption and a slight increase in the
    relative liver weight of dams were seen. At the highest dose,
    additional maternal toxic effects included higher mortality and
    clinical signs of toxicity (chromodacryorrhoea, chromorhinorrhoea, wet
    or stained perineal areas, red vaginal discharges or stains, and focal
    alopecia) in 23 animals. Fetuses had treatment-related increases in
    the incidence of skeletal anomalies (misaligned sternebrae, extra
    ossification centres in ribs, and delayed ossification in sternebrae)
    at all doses. At 50 mg/kg bw per day, there was a reduced mean number
    of liveborn fetuses per litter, a higher total number of stunted
    fetuses (0, 1, 4, and 3 at 0, 10, 50, and 250 mg/kg bw per day,
    respectively), and an increased incidence of rudimentary ribs. At the
    highest dose, additional signs of embryo- and fetotoxicity were an
    increased mean incidence of resorptions, reduced mean fetal weight per
    litter, and all increased incidence of extra ribs in the fetuses. An
    increased incidence of cleft palate (28/241; 0/331 in controls) and an
    absence of renal papillae (21/155; 0/175 in controls) were observed in
    fetuses of dams given 250 mg/kg bw per day but not at lower doses. An
    unusually high incidence of hydrocephalus and/or dilated lateral
    ventricles of the brain was also noted in all groups, including
    controls; the effect was not dose-related and was not considered to be
    related to treatment. The NOAEL for maternal toxicity was 10 mg/kg bw
    per day; there was no NOAEL for embryo- or fetotoxicity; and the NOAEL
    for teratogenicity was 50 mg/kg bw per day (Lamontia  et al., 1984a).

         Groups of 24 mated female Crl:CD(SD)BR rats received technical-
    grade flusilazole (purity, 95.6%) by gavage in corn oil at doses of 0,
    0.4, 2, 10, 50, or 250 (10 females only) mg/kg bw per day on days 7-16
    of gestation; the day a copulation plug was observed was designated
    day 1 of gestation. On day 21 of gestation, all surviving dams were
    sacrificed and necropsied; fetuses were delivered by caesarean section
    and examined for external, visceral, and skeletal abnormalities. No

    treatment-related signs of maternal toxicity were observed at doses
    > 10 mg/kg bw per day. Slightly reduced body-weight gain and food
    consumption during treatment and increased relative liver weights of
    dams were seen at 50 mg/kg bw per day. At the highest dose, clinical
    signs of maternal toxicity (alopecia, brown stains on the face and
    limbs, and stained perineal area) were also observed. No treatment-
    related embryo- or fetotoxic effects were seen at the two lowest
    doses. At > 10 mg/kg bw per day, an increased number of dams with
    median or late resorptions, an increased total number of stunted
    fetuses, a higher incidence of visceral (large renal pelvis and small
    renal papilla) and skeletal (rib) anomalies, and an increased
    incidence of delayed ossification (sternebrae and vertebral arches)
    were observed. An increased incidence of cleft palate (21/116; 0/291
    in controls) was observed in fetuses of dams receiving 250 mg/kg bw
    per day but not at lower doses. Hydrocephalus was not observed. The
    NOAEL for maternal toxicity was 10 mg/kg bw per day, that for embryo-
    and fetotoxicity was 2 mg/kg bw per day, and that for teratogenicity
    was 50 mg/kg bw per day (Lamontia  et al., 1984b).

         Groups of 24 mated female rats (Crl:CD (SD)BR strain) received
    diets containing technical-grade flusilazole (purity, 96.5%) at doses
    of 0, 50, 100, 300, or 900 ppm equal to 0, 4.6, 9.0, 26.6, and
    79.2 mg/kg bw per day) on days 7-16 of gestation; the day a copulation
    plug was observed was designated day 1 of gestation. On gestation day
    21, all surviving dams were sacrificed and necropsied; fetuses were
    delivered by caesarean section, weighed, sexed, and then examined for
    external, visceral, and skeletal abnormalities. No treatment-related
    signs of maternal toxicity were seen at doses > 100 ppm.
    Significant, dose-related reductions in body-weight gain and food
    consumption were observed during treatment at the two highest doses.
    Treatment-related embryo- and fetotoxic effects were noted at
    > 100 ppm, including an increased incidence of median or late
    resorptions, small litters (< 10 fetuses per litter), and significant
    dose-related increases in skeletal variations with extra ossification
    of the sternebrae. Additional symptoms of fetal toxicity seen at the
    two highest doses included an increased number of stunted fetuses, a
    higher incidence of rudimentary ribs, extra ossification in cervical
    ribs, and delayed ossification of the cervical vertebral arches. None
    of the fetuses had cleft palate, and there were no treatment-related
    malformations at any dose. The NOAEL for maternal toxicity was
    9.0 mg/kg bw per day, that for embryo- and fetotoxicity was 4.6 mg/kg
    bw per day, and that for teratogenicity was 79.2 mg/kg bw per day
    (Alvarez  et al., 1984).

         In a study of prenatal and postnatal toxicity in rats, groups of
    24 (phase I, prenatal study) or 22 (phase II, postnatal study) mated
    female rats (Crl:CD (SD)BR strain) received technical-grade
    flusilazole (purity, 96.5%) in 0.5% aqueous methylcellulose by gavage
    at doses of 0, 0.2, 0.4, 2, 10, or 100 mg/kg bw per day on days 7-16
    of gestation; the day a copulation plug was observed was designed day

    1 of gestation. In phase I, dams were sacrificed on gestation day 21
    for examination of the uterine contents. Additional control groups and
    animals at 100 mg/kg bw per day were killed on gestation day 22 to
    determine whether the absence of renal papillae was a compound-related
    effect or an anomaly. As the concentrations of the first few solutions
    used in phase I were found to be only 1-19% of the nominal
    concentrations and the next analysis which showed 75-110% of the
    nominal concentration, was done only on day 7, no definitive
    conclusions could be drawn from the results of this part of the study.
    Signs of maternal toxicity seen at the highest dose included clinical
    signs (chin or perinasal staining and/or wet perineum), reduced
    body-weight gain and food consumption during treatment, and increased
    absolute and relative liver weights. Embryo- and fetotoxicity were
    observed at > 10 mg/kg bw per day, manifested as increased numbers
    of stunted fetuses and a higher incidence of visceral anomalies (small
    renal papillae and distended ureter). Additional embryo- and
    fetotoxicity seen at the highest dose included an increased incidence
    of median or late resorptions and a decrease in the mean number of
    live fetuses per litter. Treatment-related malformations (absence of
    renal papillae) were observed in three fetuses from two litters of
    dams at 100 mg/kg bw per day. In phase II of the experiment, dams were
    permitted to deliver naturally and to raise their litters to weaning.
    All dams and pups were sacrificed on day 21 of lactation and subjected
    to gross necropsy. The solutions were found to be adequately prepared
    in this phase. No treatment-related maternal toxicity was evident at
    doses > 10 mg/kg bw per day. At the next dose, 100 mg/kg bw per
    day, increased mortality (5/22; 0/22 in controls), clinical signs of
    difficult parturition (pallor, bunching, weakness, and/or dystocia
    during parturition and lactation in four dams), reduced body-weight
    gain and food consumption during the early part of treatment, and
    increased liver weight were observed. No treatment-related embryo-
    or fetotoxicity was noted at doses > 2 mg/kg bw per day. At
    > 10 mg/kg bw per day, there were overt, dose-related increases in
    mean gestational length (22.8, 23.1, 22.9, 23.1, 23.5, and 24.7 days
    at 0, 0.2, 0.4, 2, 10, and 100 mg/kg bw per day, respectively),
    decreases in mean litter size and the number of liveborn fetuses per
    litter, an increased number of small litters (< 10 fetuses per
    litter), and a higher incidence of dilated renal pelvis and/or ureter
    in pups at weaning. Additional signs of treatment-related embryo- and
    fetotoxicity at the highest dose included a reduced total number of
    live litters, an increased mean number of dead fetuses per litter, and
    reduced viability indexes (days 0-4; 82%; 98-99% in control and lower
    dose groups). There were no treatment-related malformations in the
    live pups examined. Of the 42 pups found dead, 29 were in the group at
    100 mg/kg bw per day; two had no renal papillae and four had small
    papillae. The NOAEL for maternal toxicity was 10 mg/kg bw per day,
    that for embryo- and fetotoxicity was 2 mg/kg bw per day, and that for
    teratogenicity was 100 mg/kg bw per day, the highest dose tested
    (Alvarez  et al., 1985a).

    Rabbits

         Four groups of 18 artificially inseminated female New Zealand
    white rabbits received technical-grade flusilazole (purity, 96.5%) in
    corn oil by gavage at nominal doses of 0, 2, 5, or 12 mg/kg bw per
    day, equal to 0, 1.9, 4.8, and 10.1 mg/kg bw per day on the basis of
    analysis of the solutions, on days 7-19 of gestation; the day of
    insemination was designated gestation day 0. On gestation day 29, all
    surviving does were sacrificed and necropsied; fetuses were delivered
    by caesarean section and examined for external, visceral, and skeletal
    abnormalities. There was no treatment-related mortality, no clinical
    signs of maternal toxicity, and no disturbances of intrauterine
    development of the conceptuses at any dose up to and including
    12 mg/kg bw per day, the highest dose tested. All of the fetuses
    delivered showed normal development. No evidence of fetotoxicity and
    no treatment-related increases in malformations were observed in
    fetuses at any dose. The higher incidence of hydrocephalus observed in
    this study (one fetus in one litter, two in one litter, four in two
    litters, and four in three litters at 0, 2, 5, and 12 mg/kg bw per
    day, respectively), which was not confirmed in three subsequent
    studies (in only one fetus at 35 mg/kg bw per day and in none at lower
    doses or in controls), was not considered to be treatment-related. The
    NOAEL for maternal toxicity, embryo- and fetotoxicity and
    teratogenicity was 12 mg/kg bw per day (10.1 mg/kg bw per day by
    analysis) (Solomon  et al., 1984).

         Three groups of 20 artificially inseminated female New Zealand
    white rabbits received technical-grade flusilazole (purity, 96.5%) by
    gavage at nominal doses of 0, 12, or 35 mg/kg bw per day, equal to 0,
    11.2 and 31.5 mg/kg bw per day on the basis of analysis of the
    solutions, on days 7-19 of gestation; the day of insemination was
    designated gestation day 0. On gestation day 29, all surviving does
    were killed and necropsied; fetuses were delivered by caesarean
    section and examined by gross pathology. No evidence of maternal, or
    embryo- or fetotoxicity was observed at the low dose, and the
    incidence of malformations and fetal variations was not increased in
    comparison with the controls. At the high dose, there was an increased
    incidence of red vaginal discharge and stained tail and an increased
    incidence of periodic anorexia. Two of 13 treated does and 0/16
    controls aborted, and 10 of 13 had early total resorption in
    comparison with 1/16 controls. Teratogenicity could not be assessed at
    this dose because only one live litter was produced. The NOAEL for
    maternal, embryo-, and fetotoxicity was 12 mg/kg bw per day
    (11.2 mg/kg bw per day by analysis), and that for teratogenicity was
    > 12 mg/kg bw per day (11.2 mg/kg bw per day by analysis) (Zellers
     et al. 1985).

         In a range-finding study, four groups of seven artificially
    inseminated female New Zealand white rabbits were fed diets containing
    technical-grade flusilazole (purity, 94.8%) at doses of 0, 500, 1000,

    or 2000 ppm on days 7-19 of gestation. The pregnancy rate (3/7 per
    group) was low at 500 and 1000 ppm, and the incidence of mortality
     in utero was high at 2000 ppm (4/7 females with total resorptions).
    In the main study, dietary levels of 0, 300, 600, or 1200 ppm
    technical-grade flusilazole (purity, 94.8%), equal to 0, 8.9, 21.2,
    and 37.8 mg/kg bw per day, were given to groups of 20 artificially
    inseminated female New Zealand white rabbits on days 7-19 of
    gestation; the day of insemination was designated day 0 of gestation.
    All surviving does were sacrificed on gestation day 29 and necropsied;
    fetuses were delivered by caesarean section and examined for external,
    visceral, and skeletal abnormalities. During treatment with the
    highest dose, maternal body weight and food consumption were
    decreased. The pregnancy rate was reduced in all treated groups: 9/20,
    10/20, and 7/20 at 300, 600, and 1200 ppm, respectively. The number of
    does with total resorptions was increased at 600 and 1200 ppm. There
    were no treatment-related effects on the mean number of live fetuses
    per litter, the mean number of resorptions in dams with live fetuses,
    or fetal weight. The small number of litters available from dams at
    600 and 1200 ppm (three per group) precluded any definitive assessment
    of fetotoxicity or teratogenic potential at these doses. There were no
    apparent treatment-related effects at the low dose. In a supplementary
    study, dietary levels of 0, 30, 100, or 300 ppm, equal to 0, 0.81,
    2.84, and 8.32 mg/kg bw per day, of technical-grade flusilazole were
    administered to groups of 18 or 25 (300 ppm) inseminated female New
    Zealand white rabbits on days 7-19 of presumed gestation. The
    pregnancy rate was again low in all groups, including the controls
    (8/18). Total resorption occurred at 0 and 300 ppm in 25 and 29%,
    respectively, of the pregnant does, but not at the low or mid-dose.
    Because of the small number of live litters available for examination,
    the results could not be adequately assessed for embryo- or
    fetotoxicity or teratogenic potential. The NOAEL for maternal toxicity
    was 21.2 mg/kg bw per day, based on decreased body weight and food
    consumption at 1200 ppm. There was no NOAEL for embryo- or
    fetotoxicity or teratogenicity (Alvarez  et al., 1985b).

         Four groups of 18 artificially inseminated female New Zealand
    white rabbits received technical-grade flusilazole (purity, 93.8%) in
    0.5% methylcellulose by gavage at doses of 0, 7, 15, or 30 mg/kg bw
    per day on days 7-19 of gestation. The pregnancy rate was acceptable
    in all groups: 12/18, 14/18, 16/18, and 16/18 at 0, 7, 15, and
    30 mg/kg bw per day, respectively. No treatment-related maternal,
    embryo- or fetotoxicity was seen at the low dose. At the two higher
    doses, clinical signs of maternal toxicity (red discharge and
    brown-yellow-stained tail) and increased incidences of abortion (one
    dam per group) and total resorptions (4/16 and 12/16, respectively)
    were observed. At the highest dose, food consumption was also
    decreased. The mean numbers of liver fetuses per litter and of dead
    fetuses, mean fetal weight and mean male:female ratio were comparable
    in the control and treated groups. No treatment-related external,
    visceral, or skeletal malformation or variation was observed in

    fetuses of does at any dose; however, it should be noted that
    assessment of fetotoxicity and teratogenic potential at the highest
    dose was based on data for only three of 11-12 live litters in the
    control and lower dose groups. These limited data reduce confidence in
    the accuracy of any conclusions drawn on the basis of observations at
    this dose. The NOAEL for maternal, embryo-, and fetal toxicity was
    7 mg/kg bw per day, and that for teratogenicity was > 15 mg/kg bw
    per day (Alvarez, 1900).

    (f)  Genotoxicity

         A battery of studies on technical-grade flusilazole was conducted
    to assess its potential to induce gene mutation, chromosomal
    aberration, or unscheduled DNA synthesis. The results (summarized in
    Table 2) were clearly negative.

    (g)  Special studies

    (i)  Dermal and ocular irritation and dermal sensitization

         Two male New Zealand white rabbits received 0.01 ml of undiluted
    flusilazole (purity, 90%) into the right conjunctival sac; the left
    eye was not treated. The right eye of one rabbit was washed with
    100 ml of water after 20 sec of exposure. Irritation was graded
    according to the Draize scale 1, 4, 24, 48, and 72 h after
    instillation. Both animals had mild conjunctival redness and chemosis
    1-4 h after instillation. The washed eye also showed some discharge at
    1 h, but by 24 h these effects were no longer observed. The unwashed
    eye showed slight corneal opacity at 1-4 h, and biomicroscopic
    examination revealed slight cloudiness at 24-48 h; the eye was normal
    at 72 h. Flusilazole was thus minimally irritating to the eyes of male
    New Zealand white rabbits, causing transient, mild irritation.
    Flushing the eye with water after exposure eliminated the corneal but
    not the conjunctival effects (Hall  et al., 1984).

         In a study of primary skin irritation, six young male New Zealand
    white rabbits received a flusilazole formulation (Nustar 20DF,
    containing 21% technical-grade flusilazole and 79% unspecified, inert
    ingredients; undiluted, moistened with distilled water) topically on
    shaved skin sites on the back at a concentration of about 0.5 g. The
    test site was covered with a gauze patch and a sheet of rubber for
    4 h; the coverings were then removed and the site washed gently with
    warm water. Each site was evaluated for irritation potential according
    to the Draize scale 4, 24, 28, and 72 h after treatment. At 4 h, grade
    1 erythema was observed in four rabbits and grade 1 or 2 oedema in two
    rabbits. By 24 h, only two rabbits had erythema and no oedema was
    observed. No signs of dermal irritation were seen in any rabbit at
    72 h. The primary irritation index was 0.167. Flusilazole as
    formulated for this test was thus minimally irritating to the skin
    (Brock, 1988).

        Table 2.  Results of tests for the genotoxicity of flusilazole
                                                                                                                                             

    End-point                  Test system                      Concentration             Purity        Results        Reference
                                                                or dose                   (%)
                                                                                                                                             

     In vitro
    Reverse mutation           Salmonella typhimurium TA98,     1-250 g/plate            90.0          Negativea      Donovan & Irr (1982)
                               TA100, TA1535, TA1537
    Reverse mutation           Salmonella typhimurium TA97,     5-250 g/plate            97.7          Negativea      Arce et al. (1988)
                               TA98, TA100, TA1535
    Reverse mutation           Salmonella typhimurium TA97,     10-300 g/plate           61.7          Negativea      Reynolds (1991)
                               TA98, TA100, TA1535
    hprt forward mutation      Chinese hamster ovary cells      0.04-0.275 mmol/litre     95.5          Negative       McCooey et al. (1983)
                               (KI/BH4 clone)
    Chromosomal aberration     Human lymphocytes                1.7-100 g/ml             94.9          Negativea      Vlachos et al. ( 1986),
    Unscheduled DNA            Rat primary hepatocytes          1  10-5 - 1.1  102      95.5          Negative       Chromey et al. (1983)
    synthesis                                                   mmol/litre

     In vivo
    Chromosomal aberration     CrI:CD rat bone marrow           50-500 mg/kg bw           NR            Negative       Farrow et al. (1983)
                                                                (single dose by gavage)
    Micronucleus formation     CD-1 mouse bone marrow           375 mg/kg bw              95.5          Negative       Sorg et al. (1984)
                                                                (single dose by gavage)
                                                                                                                                             

    NR, not reported
    a  With and without exogenous metabolic activation
             A range-finding study in young adult Hartley guinea-pigs
    indicated mild erythema 24 h after dermal application of undiluted
    flusilazole (purity, 90%) in aliquots of 0.05 ml. No dermal irritation
    was observed with concentrations > 50%. In a study of primary skin
    irritation, flusilazole (purity, 90%) was applied topically to the
    skin at concentrations of 5 or 50% (w/v, solution in dimethyl
    phthalate), and two test sites per animal were scored for signs of
    irritation at 24 and 48 h. No dermal irritation was seen in any of the
    10 exposed animals. Flusilazole was thus not irritating to the skin of
    guinea-pigs (Wylie  el al., 1984c).

         In a study of the sensitization of young adult Hartley
    guinea-pigs, intradermal injections of a 1% solution (w/v) of
    flusilazole (purity, 90%) in methyl phthalate weekly for four weeks
    caused erythema and oedema with necrotic centres at the sites of
    injection by 24 h. Challenge with topical applications of 5 or 50%
    solutions to the skin did not induce sensitization (Wylie  el al.,
    1984c).

         In another study of sensitization, 10 male and 10 female young
    adult Duncan Hartley albino guinea-pigs were given three weekly dermal
    applications of 0.4 ml (equivalent to 0.192 g) flusilazole (purity,
    97.7%). The material, slightly moistened with dimethyl phthalate, was
    applied to the intact shaved skin on the back and covered with plastic
    wrap for 6 h, and dermal irritation was scored 24 and 48 h after
    treatment. None was observed. Two weeks after the last induction, the
    animals were challenged with a single dermal application of 0.4 ml of
    flusilazole (0.192 g) on an untreated site, which was then covered for
    6 h. The irritation response was again scored at 24 and 48 h. No signs
    of dermal irritation were observed. A positive control group treated
    with 1-chloro-2,4-dinitrobenzene showed severe erythema with necrosis
    24 h after the second and third inductions and severe erythema after
    the challenge dose two weeks later (Brock  et al., 1988).

    (ii)  Mechanisms of Leydig-cell tumour induction

         Groups of 10 Crl:CD BR male rats received technical-grade
    flusilazole (purity, 94%) in corn oil by subcutaneous injection at
    doses of 0, 20, 50, 150, or 250 mg/kg bw per day (given as two equal
    half-doses, twice daily) for 14 days. The control group and that at
    250 mg/kg bw per day each included an additional subgroup of 10 male
    rats which were treated with human chorionic gonadotropin (hCG) 1 h
    before sacrifice. Ketoconazole (a known inhibitor of 17-hydroxylase)
    was used as the positive control: Groups of 10 male rats were given
    subcutaneous injections of 0, 20, 50, 100, or 200 mg/kg bw per day
    ketoconazole in saline (as two equal half-doses, twice daily) for 14
    days. The control group and that at 200 mg/kg bw per day each included
    an additional subgroup of 10 male rats which were treated with hCG 1 h
    before sacrifice. All surviving animals were killed on day 15 of
    treatment and necropsied. Testicular interstitial fluid and serum

    samples were collected from rats that did not receive hCG; the
    interstitial fluid was analysed for testosterone and the serum samples
    for testosterone, estradiol, luteinizing hormone, and follicle-
    stimulating hormone. The serum samples collected from the hCG-treated
    rats were analysed for testosterone, androstenedione, 17-hydroxy-
    progesterone, and progesterone. Increased absolute and relative liver
    weights and a dose-related inhibition of serum testosterone levels
    (statistically significant at doses > 150 mg/kg bw per day) and
    estradiol (statistically significant at all doses) were observed at
    doses > 20 mg/kg bw per day. At the two highest doses, clinical
    symptoms of toxicity (sores, stained or wet fur, dehydration, and
    diarrhoea), decreased body weight and body-weight gain, and reduced
    food consumption were evident; eight of 10 rats at the highest dose
    died. The rats at 250 mg/kg bw per day that were treated with hCG had
    significantly lower serum testosterone levels than controls; no other
    significant differences in hormone levels were noted. The positive
    controls had significantly lower levels of testosterone,
    androstenedione, and 17-hydroxyprogesterone and a higher level of
    progesterone, indicating inhibition of 17-hydroxylase. There was no
    NOAEL (Cook, 1993).

         Leydig cells were collected from rat testes at termination of the
    above study and cultured in microplate wells; the cells were then
    incubated with either technical-grade flusilazole or ketoconazole at
    doses of 0.05-100 mol/litre in 70% ethanol for 2 h. The culture media
    were sampled and analysed for testosterone, androstenedione,
    17-hydroxyprogesterone, and progesterone. The results confirmed the
    hormonal changes observed  in vivo. Incubation of testicular Leydig
    cells with technical-grade flusilazole caused a dose-dependent
    lowering of testosterone and androstenedione levels, suggesting
    inhibition of the enzymes involved in steroid biosynthesis. The IC50
    for testosterone was 3.475  1.455 mol/litre without hCG and 2.774 
    0.646 mol/litre with hCG pretreatment. The positive control had an
    IC50 for testosterone of 0.97  0.83 mol/litre without hCG and 0.154
     0.065 mol/litre with hCG (Cook, 1993).

    Comments

         In rats, orally administered 14C-labelled flusilazole was
    readily absorbed and rapidly excreted in the urine and faeces, with
    little radiolabel recovered in expired air. Over 90% of the
    administered dose was eliminated within 96 h. 14C from phenyl-
    labelled material was excreted predominantly in the faeces (87% in
    males and 59% in females), while that from triazole-3-labelled
    material was recovered primarily in the urine (72% in each sex).
    Tissue retention of 14C was low (< 1%, excluding the carcass).
    14C-Flusilazole was extensively metabolized in rats. After
    absorption, it was cleaved at the triazole ring. Recovered parent
    compound accounted for only 2-11% of the administered dose and was
    found predominantly in faeces, the urinary level being < 1%.

         Flusilazole is slightly toxic to mice, rats, and rabbits when
    given as a single oral dose and is minimally toxic to rats and rabbits
    when given dermally or by inhalation. The oral LD50 in rats was
    > 500 mg/kg bw, the dermal LD50, in rabbits was > 2000 mg/kg bw,
    and the inhalation LC50 in rats was 6.8-7.7 mg/litre. Flusilazole was
    minimally irritating to the eyes and skin of rabbits; it was virtually
    not irritating to the skin and was not a dermal sensitizer in guinea-
    pigs. WHO has classified flusilazole as 'slightly hazardous'.

         Repeated oral administration of flusilazole to mice (90 days),
    rats (90 days), and dogs (90 days and one year) resulted primarily in
    lesions of the liver (hepatocellular hypertrophy, hyperplasia and
    vacuolation) and urinary bladder (urothelial hyperplasia and
    vacuolation). On the basis of the hepatic and/or urinary bladder
    changes, the NOAEL was 25 ppm (equal to 4 mg/kg bw per day) in mice,
    125 ppm (equal to a mean of 8.1 mg/kg bw per day in two 90-day
    studies) in rats, and 5 ppm (equal to 0.14 mg/kg bw per day) in dogs
    (one-year study). Repeated dermal application of flusilazole to
    rabbits for 21 days did not result in treatment-related systemic
    toxicity at doses up to and including 200 mg/kg bw per day.

         In two 18-month studies of carcinogenicity, flusilazole was
    administered at dietary concentrations of 0, 5, 25, or 200 ppm to mice
    of each sex in the first study and at 0, 100, 500, or 1000 ppm to
    males and 0, 100, 1000, or 2000 ppm to females in the second study.
    The overall NOAEL for the two studies was 25 ppm (equal to 3.4 mg/kg
    bw per day) on the basis of hepatotoxicity and urinary bladder
    hyperplasia in males at 100 ppm. In the second study, an increased
    incidence of hepatocellular adenomas and carcinomas was observed in
    males at > 100 ppm (equal to 14 mg/kg bw per day) and in females at
    > 1000 ppm (equal to 200 mg/kg bw per day), doses at which lesions
    of the liver (focal necrosis, hepatocellular hypertrophy and
    hyperplasia, and vacuolation) were seen. Flusilazole was carcinogenic
    in the second study.

         In two two-year studies of toxicity and carcinogenicity, rats
    received flusilazole at dietary concentrations of 0, 10, 50, or
    250 ppm (first study) or 0, 125, 375, or 750 ppm (second study). In
    the first study, the NOAEL was 10 ppm (equal to 0.4 mg/kg bw per day)
    on the basis of mild nephrotoxicity (pyelonephritis in females and
    hydronephrosis in males) at 50 ppm. In the second study, an increased
    incidence of urinary bladder transitional-cell papillomas and
    carcinomas in rats of each sex and testicular Leydig-cell tumours in
    males were observed at 750 ppm (equal to 31 mg/kg bw per day), doses
    at which lesions of the urinary bladder (urothelial necrosis,
    exfoliation, and hyperplasia) were clearly demonstrated. Flusilazole
    was carcinogenic in the second study.

         A special two-week study to investigate the possible mechanism
    by which testicular Leydig-cell tumours are induced was conducted
    in rats. Flusilazole caused a dose-dependent lowering of serum
    testosterone and estradiol levels at > 20 mg/kg bw per day  in
     vivo and a dose-related decrease in testosterone and androstenedione
    production in cultured testicular Leydig cells by inhibiting enzymes
    involved in steroid biosynthesis.

         In a two-generation study of reproductive toxicity, rats were fed
    diets containing flusilazole at concentrations of 0, 5, 50, or
    250 ppm. The NOAEL for parental systemic toxicity was 5 ppm (equal to
    0.34 mg/kg bw per day) on the basis of hepatic lesions at 50 ppm. The
    NOAEL for reproductive toxicity was 50 ppm (equal to 4.0 mg/kg bw per
    day) on the basis of treatment-related mortality during parturition,
    increased length of gestation, reduced numbers of liveborn pups per
    litter, and decreased pup growth at 250 ppm.

         Three studies of developmental toxicity were performed in rats
    with doses of 0, 10, 50, or 250 mg/kg bw per day in the first study,
    0, 0.4, 2, 10, 50, or 250 mg/kg bw per day in the second study, and 0,
    0.2, 0.4, 2, 10 or 100 mg/kg bw per day in the third study. A study of
    developmental toxicity was also conducted in which rats were fed
    flusilazole at dietary levels of 0, 50, 100, 300, or 900 ppm (equal to
    0, 4.6, 9, 27, and 79 mg/kg bw per day) on days 7-16 of gestation. The
    NOAEL for maternal toxicity was 10 mg/kg bw per day, on the basis of a
    slight reduction in body-weight gain and food consumption during
    treatment at > 27 mg/kg bw per day. The NOAEL for embryo- and
    fetotoxicity was 4.6 mg/kg bw per day on the basis of increased
    resorption, increased length of gestation, reduced litter size and a
    higher incidence of skeletal variations or anomalies at > 9 mg/kg
    bw per day. At 250 mg/kg bw per day, an increased incidence of cleft
    palate was observed. The NOAEL for teratogenicity was 100 mg/kg bw per
    day.

         In three studies of developmental toxicity in rabbits, animals
    were treated with flusilazole at 0, 2, 5, or 12 mg/kg bw per day
    (first study), 0, 12, or 35 mg/kg bw per day (second study), or 0, 7,

    15, or 30 mg/kg bw per day (third study) on days 7-19 of gestation.
    The NOAEL for maternal and embryo- or fetotoxicity was 12 mg/kg bw per
    day on the basis of clinical signs of toxicity and an increased
    incidence of abortion and total resorptions at > 15 mg/kg bw per
    day. There was no evidence of teratogenicity at doses up to and
    including 15 mg/kg bw per day, the highest dose at which an adequate
    number of live litters was available for an assessment of
    teratogenicity.

         Flusilazole has been adequately tested for genotoxicity in a
    series of assays  in vivo and  in vitro. The Meeting concluded that
    flusilazole is not genotoxic.

         An ADI of 0-0.001 mg/kg bw was established on the basis of the
    NOAEL of 5 ppm, equal to 0.14 mg/kg bw per day, in the one-year
    dietary study in dogs, and a safety factor of 100.

    Toxicological evaluation

     Levels that cause no toxic effect

    Mouse:    25 ppm, equal to 3.4 mg/kg bw per day (18-month study of
              toxicity and carcinogenicity)

    Rat:      10 ppm, equal to 0.4 mg/kg bw per day (two-year study of
              toxicity and carcinogenicity)

              5 ppm, equal to 0.34 mg/kg bw per day (maternal toxicity in
              a two-generation study of reproductive toxicity)

              50 ppm, equal to 4 mg/kg bw per day (two-generation study of
              reproductive toxicity)

              4.6 mg/kg bw per day (embryo- or fetotoxicity in a study of
              developmental toxicity)

              100 mg/kg bw per day (teratogenicity in a study of
              developmental toxicity)

    Rabbit:   12 mg/kg bw per day (maternal and embryo- or fetal toxicity
              in a study of developmental toxicity)

              15 mg/kg bw per day (teratogenicity in a study of
              developmental toxicity)

    Dog:      5 ppm, equal to 0.14 mg/kg bw per day (one-year study of
              toxicity)

     Estimate of acceptable daily in take for humans

         0-0.001 mg/kg bw

     Studies that would provide information useful for continued
     evaluation of the compound

         Observations in humans

        Toxicological criteria for setting guidance values for dietary and non-dietary exposure to flusilazole
                                                                                                                          

    Exposure                      Relevant route, study type, species               Result/remarks
                                                                                                                          

    Short-term (1-7 days)         Dermal, irritation, rabbit                        Minimally irritating
                                  Eye, irritation, rabbit                           Minimally irritating
                                  Skin, sensitization, guinea-pig                   Not a skin sensitizer
                                  Oral, toxicity, rat                               LD50 > 500 mg/kg bw
                                  Dermal, toxicity, rabbit                          LD50 > 2000 mg/kg bw
                                  Inhalation, 4 h, toxicity rat                     LC50 = 6.8-7.7 mg/litre

    Medium-term (1-26 weeks)      Repeated dermal, 21--day, toxicity, rabbit        NOAEL = 200 mg/kg bw per day, highest
                                                                                    dose tested for systemic toxicity
                                  Repeated dietary, reproductive toxicity, rat      NOAEL = 0.34 mg/kg bw per day; hepatic
                                                                                    toxicity
                                  Repeated gavage, developmental toxicity,          NOAEL = 7 mg/kg bw per day for
                                  rabbit                                            maternal, embryo-, or fetotoxicity;
                                                                                    NOAEL > 15 mg/kg bw per day for
                                                                                    teratogenicity

    Long-term (> one year)        Repeated dietary, one year, toxicity, dog         NOAEL = 0.14 mg/kg bw per day, primarily
                                                                                    for liver toxicity
                                                                                                                          

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         gavage on days 7-19 of gestation. Unpublished report No. HLR
         669-85, from Haskell Laboratory for Toxicology and Industrial
         Medicine, Wilmington, Delaware, USA. Submitted to WHO by E.I. du
         Pont de Nemours & Co., Inc., Wilmington, Delaware, USA.
    


    See Also:
       Toxicological Abbreviations
       Flusilazole (Pesticide residues in food: 1989 evaluations Part II Toxicology)