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    PESTICIDE RESIDUES IN FOOD - 1997


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    with the support of the International Programme
    on Chemical Safety (IPCS)




    TOXICOLOGICAL AND ENVIRONMENTAL
    EVALUATIONS 1994




    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Core Assessment Group 

    Lyon 22 September - 1 October 1997



    The summaries and evaluations contained in this book are, in most
    cases, based on unpublished proprietary data submitted for the purpose
    of the JMPR assessment. A registration authority should not grant a
    registration on the basis of an evaluation unless it has first
    received authorization for such use from the owner who submitted the
    data for JMPR review or has received the data on which the summaries
    are based, either from the owner of the data or from a second party
    that has obtained permission from the owner of the data for this
    purpose.



    FENBUCONAZOLE

    First draft prepared by
    M. Watson
    Ricerca Inc., Cleveland, Ohio, USA

         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 
                   Genotoxicity 
                   Reproductive toxicity 
                        Multigeneration reproductive toxicity
                        Developmental toxicity 
                   Special studies 
                        Dermal and ocular irritation and dermal
                        sensitization 
                        Effects on thyroid function and the liver
         Comments 
         Toxicological evaluation 
         References 

    Explanation

         Fenbuconazole is the common name for 4-(4-chlorophenyl)-2-phenyl-
    2-(1 H-1,2,4-triazol-1-ylmethyl)butyronitrile. It is a triazole
    fungicide intended for use as an agricultural and horticultural
    fungicide spray for the control of leaf spot, yellow and brown rust,
    powdery mildew, and net blotch on wheat and barley and apple scab,
    pear scab, and apple powdery mildew on apples and pears. Fenbuconazole
    was considered for the first time at the present Meeting.

    Evaluation for acceptable daily intake

    1.  Biochemical aspects

    (a)  Absorption, distribution, and excretion

         Groups of four Crl:CD-BR rats were given uniformly
    phenyl-ring-labelled 14C-fenbuconazole (radiochemical purity, > 99%)
    by gavage in a 0.5% suspension of methyl cellulose at 100 mg/kg bw and
    were killed seven days later. Whole-blood samples were taken from the
    four males in the first group for liquid scintillation counting 0.25,
    1, 3, 6, 24, 48, 72, 96, and 168 h after treatment. In the second
    group of four males, radiolabel was measured in urine and faeces
    collected 0, 6, 24, 48, 72, 96, and 168 h after treatment, in expired
    carbon dioxide, and in selected tissues and organs. Urine and faeces

    were collected at the same intervals from four males and four females
    but were frozen over liquid nitrogen for analysis of metabolites.

         The average total recovery was 67% of the administered dose,
    predominantly in faeces (62%) with about 4% in urine; radiolabel in
    expired carbon dioxide accounted for only 0.05% of the administered
    dose. Most of the excretion had occurred by 48 h. Peak blood and
    plasma radiolabel levels were detected at 6 h. A biphasic elimination
    pattern was seen, with a rapid alpha-phase (half-life, 7 h) followed
    by a slower ß-phase (half-life, about 50 h for plasma and about 187 h
    for whole blood). After seven days, < 0.5% of the administered dose
    was detected in tissues, the levels in liver being the highest: 2.5
    ppm (0.13% of the dose). Of the average total 0.53% of the radiolabel
    found in the bodies after seven days, 0.24% occurred in the carcass
    (Anderson et al., 1988).

         Groups of four male and four female Crl:CD-BR rats were given
    uniformly phenyl-ring-labelled 14C-fenbuconazole (radiochemical
    purity, 99.4%) by gavage in a 0.5% suspension of methyl cellulose or
    intravenously in dimethyl sulfoxide. Groups received an oral or an
    intravenous dose of 1 mg/kg bw, an oral dose of 100 mg/kg bw, or an
    oral (pulse) dose of 1 mg/kg bw after 14 daily doses of 10 ppm
    unlabelled compound in the diet; samples of excreta were taken for
    analysis at 0, 24, 48, 72, and 96 h, when the animals were killed for
    analyses of radiolabel in various tissues. Two further groups received
    oral doses of 1 or 100 mg/kg bw, and blood samples were taken for
    analysis at 0.5, 1, 3, 6, 24, 48, 72, and 96 h, when the animals were
    killed. The final group received single oral doses of 100 mg/kg bw,
    and three animals each were killed at 1, 6, 24, and 48 h for analysis
    of radiolabel in tissues.

         After intravenous treatment, 88% of the dose was detected in
    females and 98% in males; 64-79% was detected in faeces within 24 h,
    and the percentage continued to decrease up to 96 h. Urinary and
    faecal excretion in these animals over 96 h was 7 and 91% in males and
    10 and 77% in females, respectively. Similar profiles and rates of
    excretion were seen in animals treated orally at 1 mg/kg bw (both
    single and pulse treatments), with 6-10% in urine and 79-84% in
    faeces. Total excretion after the 100 mg/kg bw dose was similar to
    that at the low doses -- 5-13% in urine and 76-77% in faeces -- but
    excretion was slightly slower, proportionately more being seen at
    24-48 h and females showed a slight trend to increased urinary
    excretion.

         At the dose of 1 mg/kg bw, radiolabel was detected only between 1
    and 6 h (not at 0.5 or > 24 h), while at 100 mg/kg bw various
    levels of radiolabel were detected at 0.5-96 h. On the basis of the
    limited data for the lower dose and more information for the higher
    dose, the maximum level in blood and plasma was reached at 3-6 h. The
    maximum levels detected after the high dose were 8.99-9.99 g
    equivalents per g blood and 13.1-13.5 g equivalents per g plasma; the
    half-life for elimination of radiolabel at this dose was 10-20 h for
    both male and female rats.

         Radiolabel was generally undetectable in most tissues after the
    low oral dose, apart from the liver and kidneys, in which levels
    < 0.1 g equivalents per g were found. Similar levels were seen
    after intravenous treatment. At the higher dose, radiolabel was
    detected in most tissues at 96 h, the highest levels occurring in the
    liver in animals of each sex (3.6-5.0 g equivalents per g), in the
    adrenals (0.6-2.1 g equivalents per g) in females, and in the kidneys
    (0.7-1.2 g equivalents per g) in males. By 96 h, < 1% of the dose
    remained in the carcass. In the animals at 100 mg/kg bw that were
    killed serially, the tissue levels increased after 1 h to maximum
    levels at 6 h of 75-95 g equivalents per g in the liver, followed by
    the adrenals and fat. The levels in all tissues then continued to
    decline for 24-48 h, with no evidence of retention (LeVan, 1990).

         Groups of four male Crl:CD-BR rats received 14C-fenbuconazole
    (specific activity, 20.8 mCi/g and radiopurity, 95.7%) topically after
    a preliminary study in which groups of four rats received topical
    applications of 0.1% or undiluted material to determine application
    and washing techniques. About 100 µl of an aqueous suspension was
    applied in plastic skin enclosures to shaven areas (2.5 × 5 cm) on the
    back and covered with a nonocclusive dressing. Four animals were given
    the 0.1% dilution at 2 g/cm2, equivalent to 0.1 mg/kg bw; the site
    was washed with soap after 10 h, and excreta were collected until the
    rats were killed seven days later. Concentrations of 0.1 or 1%
    fenbuconazole in acetone and carboxymethyl cellulose or 250 mg/ml in a
    '2F' formulation were applied to provide doses equal to 0.1, 1.5, or
    110 mg/kg bw, respectively. Four animals in each group were killed
    after 0.5, 1, 2, 4, 10, and 24 h for analysis of radiolabel in
    residual urine in the urinary bladder, blood, plasma, faeces, skin
    (after washing), and carcass by liquid scintillation counting; two
    further animals received the formulation without radiolabel and were
    killed after 24 h for determination of the background levels of
    radioactivity.

         The average recovery of radiolabel was > 98%. No clinical
    symptoms of toxicity were seen during the study. In the preliminary
    study, in which animals were killed 4 h after treatment, 80-85% of the
    dose (diluted and undiluted material, respectively) was found in the
    skin wash, < 0.1% in excreta, 0.15-1.5% in the skin of animals washed
    before death, and 1.0-13% in animals washed after death. Skin was
    therefore subsequently washed before killing. After 168 h, the mean
    recovery was 73%, with 69% in the skin wash, 3.2% in faeces, 0.19% in
    urine, and none detected in the carcass. In animals killed after 24 h,
    the total recoveries ranged from 92% at 0.1 mg/kg bw to 105% at 111
    mg/kg bw. There was significant variation in the total recovery and in
    the recovery in skin washes among individuals in both groups treated
    at the lowest dose, which was attributed by the author to difficulty
    in maintaining a homogeneous suspension. By the end of the study,
    77-110% of the dose had been detected in the skin washes and 0.13-12%
    was determined to have been absorbed, since it was detected in faeces,
    urine, carcass, and skin. The proportion of the dose absorbed from the
    acetone, carboxymethyl cellulose and '2F' formulations after 10 h was
    4.3% at 0.1 mg/kg bw, 2.1% at 1.5 mg/kg bw, and 0.5% at 110 mg/kg bw;

    by 24 h, 12, 5.3, and 1.6% had been absorbed, respectively. These
    figures compare well with the absorption calculated by subtracting the
    totals in skin washes from the total dose: 13, 6.4, and 2%,
    respectively (Cheng, 1990).

         In a further study, groups of male and female Crl:CD-BR rats
    received pretreatment with 10 ppm fenbuconazole in the diet and then
    radiolabelled material at 1 or 100 mg/kg bw. In addition, rats with
    bile-duct cannulae received 1 mg/kg bw, and bile and excreta were
    collected over three days. The results were generally similar to those
    seen in the two previous studies. Overall, 88-91% of the oral dose of
    1 mg/kg bw was absorbed systemically. In the rats with bile-duct
    cannulae, 79-87% of the dose was detected in bile (DiDonato &
    Hazelton, 1993).

    (b)  Biotransformation

         In the studies conducted by Anderson et al. and DiDonato &
    Hazleton, frozen samples of urine and faeces were investigated for
    metabolites. Conjugates were investigated after acid hydrolysis, and
    sulfate conjugates were determined directly. A total of 19 metabolites
    were either identified or predicted, including compounds in the
    following 10 classes: lactones, iminolactone alpha-alcohol, phenols,
    phenol lactones, keto acid, sulfates, triazole, and triazole cleavage
    product. The parent compound, 12 metabolites, and conjugates were
    identified. The major metabolites resulted from enzymic oxidation of
    the first carbon a atom to the chlorophenol ring or to the 3- or
    4-position of the phenol ring. Further cyclization of the alcohol with
    the adjacent nitrile group, followed by hydrolysis, led to
    iminolactones and further hydrolysis to lactones. Iminolactones and
    lactones were isolated in both A and B diasteromeric forms. Cleavage
    to the triazole was identified as a minor pathway. The metabolic
    profiles in males and females were similar, although some quantitative
    diiferences were seen. Conjugation of the hydroxyl groups was also
    identified, as sulfate or, predominantly, the glucuronide. A proposed
    scheme of the metabolism of fenbuconazole in rats is shown in Figure
    1.

    2.  Toxicological studies

    (a)  Acute toxicity

         The results of studies of the acute toxicity of fenbuconazole are
    summarized in Table 1. The clinical signs of toxicity after treatment
    with fenbuconazole were generally nonspecific. Oral administration of
    2 or 5 mg/kg bw produced signs which indicated effects on the central
    nervous system (passivity, ataxia, tremors, prostration, and arched
    back), on the autonomic or peripheral nervous system (lachrymation and
    salivation), on the respiratory system, and on the gastrointestinal
    tract. Necropsy of decedents and survivors revealed no remarkable
    changes.

    FIGURE 1


        Table 1. Acute toxicity of fenbuconazole

                                                                                                                

    Species, strain       Sex     Route and vehicle         LD50/LC50      Purity    Reference
                                                            (mg/kg bw      (%)
                                                            or mg/L air)
                                                                                                                

    Rat, Crl:CD(SD)BR     M       Oral; aqueous methocel    > 2000         96.4      Lampé et al. (1987a)
    Rat, Crl:CD(SD)BR     M,F     Oral; aqueous methocel    > 2000         96.7      Krajewski et al. (1988a)
                                                            < 5000
    Rat, Crl:CD(SD)BR     M,F     Oral; aqueous methocel    > 5000         97.1      Lutz & Parno (1994)
    Rat, Crl:CD(SD)BR     M       Dermal, 0.85% saline      > 5000         96.4      Lampé et al. (1987b)
    Rat, Crl:CD(SD)BR     M,F     Dermal, 0.85% saline      > 5000         96.7      Krajewski et al. (1988b)
    Rat, Crl:CD(SD)BR     M,F     Inhalation                > 2.1          96.7      Duchosal & Thevenaz (1989)
                                                                                                                
    

    (b)  Short-term toxicity

     Mice

         In a two-week range-finding study, groups of five male and five
    female Crl:CD-1(ICR)BR mice were given diets containing fenbuconazole
    (purity, 98%) at doses of 0, 100, 250, 500, or 1000 ppm. There were no
    deaths, no clinical signs of reaction to treatment, and no
    treatment-related effect on weight gain or food intake. The weights of
    the livers of animals treated with doses of > 250 ppm were
    increased, and there was histopatholgical evidence of hepatotoxicity
    at the two highest doses. The NOAEL was 100 ppm, equal to 20 mg/kg bw
    per day (Morrison & Hazleton, 1986a).

         Groups of Crl:CD-1(ICR)BR mice were given diets containing
    fenbuconazole (purity, 96.4%) at 0, 20, 60, 180, or 540 ppm for three
    months. The only treatment-related effect observed was evidence of
    hepatotoxicity at the two higher doses. At 180 ppm, relatively mild
    effects were seen, including slightly increased liver weight in males,
    hepatocellular hypertrophy in animals of each sex, single-cell
    necrosis in one male, and increased aspartate aminotransferase
    activity in males. At 540 ppm, these effects were more pronounced and
    were seen in both males and females, in addition to hepatocyte
    vacuolation and increased alanine aminotransferase activity. The NOAEL
    was 60 ppm, equal to 11-18 mg/kg bw per day (Harris & Hazelton, 1988).

         Groups of 10 male and 10 female Crl:CD-1(ICR)BR mice were given
    fenbuconazole (purity, 96.7%) in the diet at 0, 540, 1000, 3000, or 10
    000 ppm for three months. Treatment-related effects were observed in
    all treated animals. At 540 ppm, the effects were similar to those
    observed in the preceding study, i.e. increased liver weights,
    hepatocellular hypertrophy, hepatocellular vacuolation, and
    single-cell or focal necrosis, in animals of each sex. The effects on
    the liver were more pronounced at higher doses, and the incidence
    and/or severity was greater in males than in females. At the higher
    doses, decreased renal weights were seen in males and decreased
    body-weight gain and food intake and changes in clinical chemical
    parameters in animals of each sex. At 10 000 ppm, 80-100% of the
    animals died within three weeks (Wolfe, 1989).

     Rats

         In a two-week range-finding study, groups of five male and five
    female Crl:CD-BR rats were given diets containing fenbuconazole
    (purity, 98%) at 0, 100, 300, 1000, or 3000 ppm. There were no deaths
    and no clinical signs of reaction to treatment. The rats receiving
    1000 or 3000 ppm had lower weight gain and food intake than controls.
    The liver weights of animals treated with doses > 300 ppm and of
    males at 100 ppm were increased; histopathological evidence of
    hepatotoxicity was seen at 1000 and 3000 ppm, and treatment-related
    changes in hepatic mixed-function oxidase activity were seen at all
    doses. There was no NOAEL (Morrison & Hazleton, 1986b).

         Groups of 10 male and 10 female CRL:CD-BR rats were given
    fenbuconazole (purity, 96.4%) in the diet at 0, 20, 80, 400, or 1600
    ppm for three months. No deaths or clinical symptoms of toxicity were
    seen during the study. Both food consumption and body-weight gain were
    significantly reduced in animals of each sex at 1600 ppm, although the
    magnitude of these effects declined towards the end of the study, with
    comparable or increased food consumption from week 9 onwards. No
    treatment-related ophthalmoscopic effects were seen, and no effects
    were seen in urinalyses. There were no treatment-related effects on
    either erythrocyte parameters or differential leukocyte counts. Serum
    g-glutamyl transferase activity was increased in females at 1600 ppm,
    and decreased triglycerides and increased cholesterol were seen in
    animals of each sex at this dose. Gross pathological examination
    revealed increased lobulization of the liver in animals of each sex at
    1600 ppm and dark-brown discolouration of the liver in four females at
    this dose. Adrenal weights were increased in animals of each sex
    (relative weight only in females) at 1600 ppm, but no histopatholgical
    correlate was seen in either sex. Increased ovarian weights seen at
    1600 ppm were of questionable toxicological significant, given that no
    histopathological effects occurred. Other increases in relative organ
    weights at this dose were considered to be related to decreased
    terminal body weight. A dose-related increase in the incidence and
    magnitude of the hepatic histopathological effects was seen.
    Hepatocellular hypertrophy was seen in one male at 80 ppm and most
    rats at doses > 400 ppm; it was mainly centrilobular with
    eosinophilic cells, some of which had basophilic nuclei. Mid-zonal
    vacuolation was seen in none of the controls, two rats at 80 ppm, four
    at 400 ppm, and six at 1600 ppm, while mid-zonal and periportal or
    perilobular vacuolation were seen in females at doses > 400 ppm.
    Centrilobular necrosis was seen in two males at 400 ppm. Thyroid
    follicular epithelial hyperplasia was seen in nine males and two
    females at 400 ppm and in eight males and 10 females at 1600 ppm. The
    NOAEL was 20 ppm, equal to 1.3 mg/kg bw per day, on the basis of a
    slight increase in the incidence of hepatic effects at 80 ppm (Bemacki
    & Hazelton, 1988).

         Groups of six male and six female Crl:CD-BR rats received
    technical-grade fenbuconazole (purity, 97.1%) moistened with saline
    topically on clipped areas of the back at doses of 0 or 1000 mg/kg bw
    per day or a water-dispersible formulation of fenbuconazole at 62.5,
    250, or 1000 mg/kg bw per day. Exposure was maintained with a patch of
    absorbent gauze under an occlusive dressing for 6 h per day, five days
    per week for 21-22 days; due to logistical constraints, half the
    animals of each sex at each dose were killed 24 h apart.

         No deaths or treatment-related clinical symptoms of toxicity were
    seen, and food consumption and body-weight gain were unaffected by
    treatment. No irritation was seen with the active ingredient alone.
    None-to-moderate erythema was seen in animals of each sex at the
    highest dose of the formulation, from day 19 in males and day 12 in
    females; females receiving the formulation control also showed
    erythema from day 11. Desiccation, reddened areas, and scabs were seen
    in these animals at necropsy. There were no treatment-related effects

    on clinical chemical, haematological, or urinary parameters. No gross
    or histopathological effects were seen in association with
    treatment-related systemic toxicity. The only apparent effect on organ
    weight was an increase in relative liver weights at 1000 mg/kg bw per
    day of the active ingredient in formulation in females and with the
    technical-grade material in males, which is of questionable
    toxicological significance and possibly related to the slightly
    lowered terminal body weights. Increased incidences of acanthosis,
    parakeratosis, eschar, or superficial exudate and necrosis of the
    epidermis were seen both with the formulation and formulation blank.
    The NOAEL for systemic toxicity was > 1000 mg/kg bw per day. Repeated
    dermal application of relatively high doses of the fenbuconazole
    co-formulants produced evidence of dermal irritation (Lampé et al.,
    1991).

     Dogs

         In a four-week range-finding study, groups of one or two beagle
    dogs of each sex were fed diets containing 0, 200, 400, 800, 1600, or
    3200 ppm fenbuconazole (purity, 96.4%). The only effect seen at 800
    ppm was increased serum alkaline phosphatase activity. Adverse effects
    seen at 1600 ppm included decreased food intake and weight gain,
    increased alanine aminotransferase activity, and decreased cholesterol
    level. At 3200 ppm, the effects on weight gain and food intake were
    incompatible with survival, and treatment was limited to two weeks.
    The NOAEL was 400 ppm, equivalent to 10 mg/kg bw per day (O'Hara et
    al., 1987).

         In a second four-week range-finding study, two male and two
    female beagles were given fenbuconazole (purity, 96.7%) in the diet at
    levels of 0, 100, 1600, or 3200 ppm. No deaths occurred, and there
    were no treatment-related clinical signs of toxicity. Decreased body
    weight was seen at 1600 and 3200 ppm, especially during the first two
    weeks of treatment, correlated with decreased food consumption at 1600
    and especially 3200 ppm. Gross examination showed no treatment-related
    effects. The NOAEL in this limited study was 100 ppm, equal to 3.6
    mg/kg bw per day, on the basis of effects on food consumption and body
    weight at 1600 ppm (Richards, 1991).

         Groups of four male and four female beagles were given
    fenbuconazole (purity, 96.4%) in the diet at levels of 0, 30, 100,
    400, or 1600 ppm for three months. No deaths or clinical symptoms of
    toxicity were seen. Body weights were decreased in animals of each sex
    at 1600 ppm up to two weeks of the study, correlated with a
    significant reduction in food consumption (23-41%). Body-weight gain
    was comparable at 2-13 weeks, although the cumulative weight gain at
    2-8 and 8-13 weeks was lower at 1600 ppm, especially in females. Food
    consumption was generally slightly lower in animals of each sex at
    1600 ppm, but the decrease was statistically significant only at 0-2
    weeks. By three months, a statistically significant reduction in
    erythrocyte count and decreased haematocrit and haemoglobin levels
    were seen in females at 1600 ppm, whereas the platelet counts were
    increased at both one and three months. Mean haemoglobin and cell

    volume were increased in animals of each sex at 1600 ppm. These
    effects were only slight, not related to dose, and of questionable
    toxicological significance in males at 100 and 400 ppm.

         Clinical chemical tests showed significant increases in alkaline
    phosphatase activity in animals of each sex at 1600 ppm and in females
    at 400 ppm at one and three months. Females at the highest dose also
    showed raised serum alanine aminotransferase and g-glutamyltransferase
    activity at these intervals. Cholesterol levels were slightly raised
    in females at 400 ppm but were lower at 1600 ppm at one and three
    months in females at 1600 ppm. The other effects were unremarkable and
    not clearly related to treatment. No effects were seen in urinalyses
    or ophthalmoscopic investigations. A dose-related trend to increased
    liver weight (absolute and relative) was noted at doses > 400 ppm
    in animals of each sex at three months. Hepatocytic hypertrophy was
    seen, with eosinophilia at 400 and 1600 ppm and minimal-to-slight
    multifocal vacuolation in animals of each sex at 1600 ppm. The NOAEL
    was 100 ppm, equal to 3.3 mg/kg bw per day, on the basis of hepatic
    hypertrophy with clinical chemical effects in animals of each sex at
    higher doses (Hazelton & Shade, 1988). 

         Fenbuconazole (purity, 96.7%) was administered in the diet to
    four male and four female beagles at concentrations of 0, 15, 150, or
    1200 ppm for one year. No treatment-related effects on survival were
    seen, nor were any clinical symptoms of toxicity or ophthalmoscopic
    effects. Body-weight gain was reduced consistently in females at 1200
    ppm and also at 150 ppm, predominantly during weeks 41-52; no clear
    treatment-related effects were seen in males. Food consumption was
    unaffected by treatment, except during the first few weeks at the
    highest dose. No treatment-related haematological effects were seen at
    12 or 26 weeks; however, at 52 weeks two males at 1200 ppm had
    creneated erythrocytes, and one also had Burr cells. At 1200 ppm,
    alkaline phosphatase activity was consistently increased in animals of
    each sex at 13, 26, and 52 weeks; alanine aminotransferase activity
    was increased consistently in one female at all three times and in
    males at 52 weeks. No treatment-related effects were seen in
    urinalyses. Total protein was reduced in females at 1200 ppm at 26
    weeks and slightly reduced in males at this dose at 13, 26, and 52
    weeks. These males also had reduced albumin levels at 26 and 52 weeks.
    Triglyceride levels were consistently increased in animals of each sex
    at 1200 ppm at all three times. Cholesterol levels were often slightly
    lower at this dose, mainly in males, but with no dose-response
    relationship; the only statistically significant result was seen in
    females at 26 weeks. The level of total bilirubin was raised in two of
    four males at 1200 ppm. The absolute and relative weights of the liver
    and adrenals and the relative renal weight were increased in animals
    of each sex at 1200 ppm. Histopathological examination showed
    eosinophilic hypertrophic hepatocytes (mainly mid-zonal) in all
    animals at 1200 ppm but not in other animals. Hepatocyte pigmentation
    reported to be consistent with lipofuscin (slight to moderate) was
    also seen in these animals. The NOAEL was 150 ppm, equal to 5.2 mg/kg
    bw per day, as the effects on body weight in females at 150 ppm were
    seen only towards the end of the study, with no evidence of systemic

    toxicity. Consistent reductions in body, weight, increased liver
    weight with histopathological changes, and possibly associated changes
    in clinical chemistry were, however, seen at 1200 ppm (Morgan, 1990).

    (c)  Long-term toxicity and carcinogenicity

     Mice

         Groups of 60 male and 60 female CD-1 mice were given
    fenbuconazole (purity, 96.7%) in the diet for 78 weeks. On the basis
    of calculations of the maximum tolerated dose in previous studies,
    males were treated at 0, 10, 200, or 650 ppm and females at 0, 10,
    650, or 1300 ppm. Ten mice of each sex at each dose were killed at 52
    weeks. No treatment-related effects on survival were seen, the rate
    being > 70% at the end of the study. The body-weight gain of males
    was reduced and was 13% less than that of controls at the end of the
    study. No treatment-related effects on food consumption were seen. No
    remarkable effects were seen on differential leukocyte counts, cell
    morphology, nucleated erythrocyte counts, or myeloid:erythroid ratio.
    Gross pathological examination showed an increase incidence of
    enlarged livers in both surviving and dead animals of each sex at the
    highest dose. The relative and absolute weights of the livers were
    increased at 52 weeks in males and females at 650 ppm and in females
    at 1300 ppm. Similar effects were seen at the time of the terminal
    kill, and increased weights were also seen in males at 200 ppm.
    Histopathological examination showed centrilobular to mid-zonal
    hepatocyte hypertrophy and vacuolation with some hyperplasia. The
    incidence of hepatocellular adenomas and carcinomas combined (8.3%)
    was significantly increased in females at 1300 ppm, although the
    control value was low (0); the incidence of hepatic tumours was very
    slightly greater than the range in historical controls (0-6.1%). The
    incidence of carcinomas in males at 200-650 ppm showed a
    nonsignificant trend when considered in isolation. The effects of
    fenbuconazole on the liver in this study are summarized in Table 2. No
    other treatment-related pathological effects were seen. The NOAEL was
    10 ppm, equal to 1.3 mg/kg bw per day, on the basis of hepatic effects
    at higher doses. Only equivocal evidence of hepatocellular
    tumorigenicity was seen, which was statistically significant only in
    females at a dose equivalent to 209 mg/kg bw per day (Wolfe, 1991a).

     Rats

         Groups of 70 male and 70 female Sprague-Dawley rats were fed
    diets containing fenbuconazole (purity, 96.7%) at concentrations of 0,
    8, 80, or 800 ppm for two years; lower levels (4, 40, or 400 ppm) had
    been fed up to week 2, then 6, 60, and 600 ppm up to week 4. Ten rats
    of each sex in each group were killed at 52 weeks. No
    treatment-related effects on survival were seen and no clinical
    symptoms of toxicity or ophthalmoscopic effects. Body-weight gain was
    consistently, significantly reduced in females at 800 ppm.
    Haematological analyses showed no consistent, dose-related effects in
    erythrocyte or leukocyte parameters. Females at 800 ppm had raised
    cholesterol levels, but the other effects were transient and not


        Table 2. Incidences of histopathological changes in the livers of CD-1 mice fed fenbuconazole for 52 or 78 weeks

                                                                                                                  
                                  Dose (ppm)
                                                                                                                  
                                  Males                                   Females
                                                                                                                  
                                  0         10        200       650       0         10        650       1300
                                                                                                                  

    No. examined                  60        59        60        60        58        60        57        60
    Hepatocellular effect
       Hypertrophy                4         4         22        55        1         1         34        49
       Vacuolation                2         1         11        31        4         1         20        31
       Hyperplasia                3         0         1         7         0         1         0         3
       Adenoma                    8         1         8         6         0         0         0         4
       Carcinoma                  1         1         3         5         0         1         0         1
       Adenoma and/or carcinoma   9         2         10        10        0         1         0         5
                                                                                                                  
    

    related to dose. The results of urinalyses were similarly
    unremarkable. No treatment-related gross pathological effects were
    seen; however, at 800 ppm, significantly increased liver weights
    (absolute and relative in males and relative in females) were seen at
    the time of the interim sacrifice, and both absolute and relative
    weights were increased in animals of each sex at the terminal kill.
    Thyroid/parathyroid weights were also increased at the terminal kill
    in animals at 800 ppm (absolute and relative in males and relative in
    females). The only treatment-related histopathological effects seen in
    mice that died during the study and at the interim kill were
    slight-to-moderate centrilobular to mid-zonal hepatocyte hypertrophy
    and vacuolization in animals of each sex; similar effects were seen at
    the terminal kill in animals at this dose. The incidence of focal
    cystic thyroid hyperplasia was increased in males at 800 ppm (12/70;
    1/70 in controls), and the incidence of thyroid follicular adenoma and
    carcinoma was also increased in these animals (6/70 and 4/70 in eight
    rats; one adenoma in control males). No other treatment-related
    effects or tumorigenicity were seen. The NOAEL was 80 ppm, equal to
    3.0 mg/kg bw per day, on the basis of effects on body weight, effects
    associated with hepatic hypertrophy (weight change, cholesterol
    levels, and histopathological effects), and histopathological effects
    in the thyroid at 800 ppm. The NOAEL for thyroid tumorigenicity (seen
    only in males) was also 80 ppm (Wolfe, 1990).

         Groups of 60 male Sprague-Dawley rats were given fenbuconazole
    (purity, 96.7%) in the diet for two years in order to ensure that the
    male rats in the previous study had been treated at the maximum
    tolerated dose. The dietary concentrations were altered to accommodate
    increasing body weights and food consumption at weeks 2 and 4, so that
    the animals received 0, (400, 600) then 800 ppm to term or (800, 1200)
    then 1600 ppm to term. Ten rats in each group were killed at 52 weeks.
    No treatment-related effects on survival were seen, nor were there any
    clinical symptoms of toxicity or ophthalmoscopic effects. Body-weight
    gain was increasingly reduced during the study in rats at 1600 ppm. No
    consistent effects on the results of haematology or clinical chemistry
    were seen, and no gross pathological effects were seen. At the interim
    kill, the liver weights (absolute and relative) were increased in
    animals at 1600 ppm, and the relative liver weights were increased at
    800 ppm. At the terminal kill, only the relative liver weights were
    higher (with lower body weights) at 1600 ppm. The absolute and
    relative thyroid and parathyroid weights were also increased at 800
    and 1600 ppm at the interim kill. These effects correlated with
    histopathological findings, with slight-to-moderate centrilobular to
    mid-zonal vacuolization in 7/10 animals at 800 ppm and all animals at
    1600 ppm at the interim kill. In 8/10 animals at 1600 ppm,
    minimal-to-slight thyroid follicular-cell hypertrophy was also seen.
    Dose-related effects on the degree of hypertrophy of livers were seen,
    with occasional vacuolation in animals at doses > 800 ppm at the
    terminal kill. These effects were seen in all surviving animals and in
    most of those that died during the study. A trend in the severity of
    the effects on the liver was seen, from minimal to moderate effects at
    800 ppm to moderate to moderately severe hypertrophy at 1600 ppm.
    Slight thyroid follicular hypertrophy occurred in 12/27 animals at

    1600 ppm. An apparent dose-response relationship in the incidence of
    thyroid follicular adenomas (2/60, 5/60, and 9/60) was seen at doses
    of 0, 800, and 1600 ppm, respectively; two carcinomas were seen at 0
    and 1600 ppm. No NOAEL was identified (Wolfe, 1991b).

    (d)  Genotoxicity

         The results of tests for the genotoxicity of fenbuconazole are
    summarized in Table 2.

    (e) Reproductive toxicity

    (i)  Multigeneration reproductive toxicity

     Rats

         In a two-generation study, 25 male and 25 female Crl:CD-BR rats
    (21 of each sex of the second parental group at 800 ppm) were given
    fenbuconazole (purity, 96.7%) in the diet at 0, 8, 80, or 800 ppm. The
    parental animals were fed for a minimum of 10 weeks before mating then
    throughout mating, gestation, and lactation. Only one litter per
    generation was produced. When the youngest litter reached 25 days, one
    animal (F1) per sex per litter was randomly selected to serve as the
    parents to produce the F2 litter; however, F0 males at 0 and 800 ppm
    were subsequently mated with untreated females. Males were killed and
    necropsied after the lactation period. Four days  post partum, the
    litters were culled randomly to eight (four of each sex when
    possible), and dams that had not delivered were killed. No deaths or
    symptoms of toxicity were seen during treatment, but F0 and F1 dams
    at 800 ppm showed increased mortality during delivery, with 13/25 and
    5/21 surviving, respectively. Reduced body-weight gain was seen in F0
    dams and F1 parental males and females at 800 ppm before mating, and
    these reductions were maintained in the females during gestation and
    lactation. The effects correlated with reduced food consumption in
    these animals. On necropsy, increased liver weights were seen in F0
    and F1 parental males and females at 800 ppm. A slight, equivocal
    increase in liver weight was seen at 80 ppm only in F1 dams, with no
    evidence of associated histopathological changes. Thyroid weights were
    increased F0 and F1 parental males; relative adrenal weights were
    increased in 15-19 F0 and F1 dams at 800 ppm and in only about seven
    at 0-80 ppm. Histopathological examination confirmed the hypertrophic
    effects in these three organs at 800 ppm, with centrilobular to
    mid-zonal hepatocyte hypertrophy and vacuolation, follicular-cell
    hypertrophy, and hypertrophy of the zona glomerulosa, respectively.
    Three F0 and four F1 dams at 800 ppm also had centrilobular
    hepatocellular necrosis No treatment-related effects on fertility were
    seen in males at doses up to 800 ppm; however, the numbers of F0 and
    F1 dams at this dose that delivered live young was reduced to 10/25
    and 4/21, respectively, and the number of stillborn pups was
    increased, so that the total and mean numbers of pups were reduced.
    The viability index (survival on day 4) was also reduced, from about
    97% in the other groups to 85% at 800 ppm, and pup weight was


        Table 2. Results of assays for genotoxicity with fenbuconazole

                                                                                                                       

    Test system         Test object          Concentration/         Purity        Results       References
                                             dose                   (%)
                                                                                                                       

    In vitro
    Reverse mutation    S. typhimurium       < 7500 mg/plate        Reportedly    Negativea     Chism (1984)
                        TA98, TA100,                                100
                        TA1535, TA1537

    Reverse mutation    S. typhimurium       < 5000 mg/plate        96.4          Negativea     Sames & Frank (1987)
                        TA98, TA100,
                        TA1535, TA1537

    Reverse mutation    S. typhimurium       < 5000 mg/plate        96.7          Negativea     Sames & Frank (1988)
                        TA98, TA100,
                        TA1535, TA1537

    Reverse mutation    S. typhimurium       < 2000 mg/plate        98            Negativea     Sames & Ella (1993)
                        TA98, TA100,
                        TA1535, TA1537

    Gene mutation       Chinese hamster      < 60 mg/ml             96.7          Negativea     Thilagar (1988a)
                        ovary cells, hprt
                        locus

    Chromosomal         Chinese hamster      < 30 mg/ml             96.7          Negativea     Thilagar (1990)
    aberration          ovary cells

    Unscheduled         Rat hepatocytes      < 15 mg/ml             96.7          Negative      Thilagar (1988b)
                        DNA synthesis

    DNA repair          B. subtilis          < 2000 mg/disc         97.1          Negative      Sarwar & Suzuki
    (1994)
                        M45, H17

    In vivo
    Chromosomal         Rat bone marrow      < 1 × 2500             96.7          Negative      Thilagar (1988c)
    aberration                               mg/kg bw
                                                                                                                       

    a With and without metabolic activation
    

    consistently lower. No treatment-related gross pathological effects
    were seen in the offspring. The NOAEL was 80 ppm, equivalent to 4
    mg/kg bw per day, on the basis of liver hypertrophy and maternal
    toxicity and fetotoxicity at 800 ppm (Solomon & Kulwich, 1990).

    (ii)  Developmental toxicity

     Rats

         Groups of 12 mated Sprague-Dawley rats received fenbuconazole
    (purity, 96.7%) at doses of 0, 50, 100, or 150 mg/kg bw per day by
    gavage on days 6-15  post coitum. Maternal and fetal toxicity
    occurred at the highest dose. The incidences of individual visceral
    and skeletal alterations at this dose were similar to those in
    controls, except that the total number of affected litters was higher
    (Solomon & Lutz, 1987).

         Fenbuconazole (purity, 96.4%) in an aqueous suspension of 0.5%
    methylcellulose was administered at 0, 30, 75, or 150 mg/kg bw per day
    to groups of 25 mated Sprague-Dawley rats by gavage on days 6-15 
     post coitum. The animals were killed on day 20. No treatment-related
    mortality was seen, although one animal at 150 mg/kg bw per day died,
    reportedly due to an intubation error. Animals at doses > 75 mg/kg
    bw per day had alopecia and few faeces, and the mean body weights were
    reduced during days 6-8, such that overall body-weight gain was lower
    by the end of the study. Food consumption was apparently not recorded.
    The numbers of animals that did not become pregnant (1, 3, 2, and 2 at
    the four doses, respectively) was acceptable. At 150 mg/kg bw per day,
    increased early, late, and total (one animal) resorptions were seen,
    with a corresponding reduction in the average number of fetuses per
    litter; average fetal weight was also reduced. No treatment-related
    fetal malformations were seen. A dose-related trend in the number of
    litters in which fetuses had partially or unossified sternebrae was
    seen at doses > 75 mg/kg bw per day, with a corresponding increase
    in the number of fetuses affected. In addition, an increased incidence
    of rudimentary 14th ribs and partially or unossified pubic bones
    occurred at 150 mg/kg bw per day, resulting in an overall increase in
    the number of fetuses per litter with developmental effects. The NOAEL
    for maternal toxicity was 30 mg/kg bw per day on the basis of reduced
    body-weight gain and clinical symptoms at doses > 75 mg/kg bw per
    day. The NOAEL for fetotoxicity was also 30 mg/kg bw per day on the
    basis of an increased incidence of partially or unossified sternebrae
    at the next dose. No evidence of teratogenicity was seen (Solomon &
    Lutz, 1988).

     Rabbits

         Fenbuconazole (purity, 96.7%) in an aqueous suspension of
    methylcellulose was administered to groups of 21 mated New Zealand
    white rabbits at 0, 10, 30, or 60 mg/kg bw per day by gavage on days
    7-20  post coitum. The animals were killed on day 29. One animal at
    60 mg/kg bw per day died and one was killed  in extremis after an
    early abortion; a further animal died due to an intubation error.

    Reduced food consumption and soft or few faeces were seen at 30-60
    mg/kg bw per day. At 60 mg/kg bw per day, only 1/19 pregnant does
    produced a viable litter, with 10 total resorptions and a total of six
    abortions. At this dose, only eight fetuses were available for
    examination. No treatment-related effects were seen in reproductive
    parameters at doses of 10-30 mg/kg bw per day. The incidences of
    retarded development and malformation were not increased in animals at
    these doses. The NOAEL for maternal toxicity was 10 mg/kg bw per day
    on the basis of clinical symptoms of toxicity at higher doses. The
    NOAEL for fetotoxicity was 30 mg/kg bw per day, as postimplantation
    losses and abortion were seen at 60 mg/kg bw per day, with no evidence
    of teratogenicity (Soloman & Lutz, 1989).

    (f)  Special studies

    (i)  Dermal and ocular irritation and dermal sensitization

         Six New Zealand white rabbits received 0.5 g fenbuconazole
    (purity, 96.4%) topically as a paste with 1 ml of saline onto clipped
    areas of the back. Exposure was maintained for 4 h under a
    semi-occlusive dressing. No erythema or oedema was observed in any of
    the animals after 24, 48, or 72 h (Lampé et al., 1987c).

         Nine male New Zealand white rabbits received 0.1 g fenbuconazole
    (purity, 96.4%) into one conjunctival sac. The eyes of six animals
    were left unirrigated, while those of an additional three animals were
    rinsed for 1 min about 30 s after administration. No corneal, iridial,
    or conjunctival reactions were seen at 24, 48, or 72 h (Lampé et al.,
    1987d).

         Fenbuconazole (purity, 96.7%) was administered topically to 10
    male and 10 female Hartley guinea-pigs, and their reactions were
    compared with those of five positive controls of each sex treated with
    1-chloro-2,4-dinitrobenzene at 1600 ppm in 80% aqueous ethanol and of
    five negative controls of each sex. Fenbuconazole was applied for 6 h
    once a week for three weeks as a 25% w/v solution in acetone that had
    been shown to cause only slight erythema in a range-finding study.
    Exposure to 0.4 ml of the test materials on clipped areas of the flank
    was maintained under an occlusive dressing; negative controls were
    shaved and received the dressing. A topical challenge exposure was
    given two weeks after the third induction, in which negative controls
    and fenbuconazole-treated animals received 0.4 ml of a 20% w/v
    solution in acetone, and positive controls received 0.4 ml of an
    800-ppm solution in acetone. The animals' backs were depilated 19-22 h
    later with hair remover, and any erythema scored 24 h after removal of
    the challenge patch. One negative control animal had erythema, but
    none reacted to the challenge with fenbuconazole. All positive
    controls reacted to challenge with 1-chloro-2,4-dinitrobenzene.
    Fenbuconazole thus showed no potential for skin sensitization in this
    Buehler test (Bonin et al., 1988).

         The ability of fenbuconazole to produce delayed contact
    hypersensitivity in guinea-pigs was tested with the maximization
    technique of Magnusson and Kligman. After initial screening tests, the
    animals were induced by intradermal injection of a 10% formulation of
    fenbuconazole in acetone and by topical application of a 25%
    formulation in the same solvent; they were challenged with a 10%
    formulation in acetone. A sensitization rate of 10% was observed after
    induction with fenbuconazole, but a 40% sensitization rate was
    elicited in the controls receiving acetone alone. After a second
    challenge with fenbuconazole as a 10% formulation in diethyl
    phthalate, the sensitization rate was 16% with fenbuconazole and 10%
    with the vehicle. The positive control substance, 85%
    hexylcinnamaldehyde, induced 100% sensitization. The results indicate
    that fenbuconazole has weak sensitizing potential (Morris, 1994). 

    (ii)  Effects on thyroid function and the liver

         Thyroid function and hepatic clearance of tetraiodothyronine
    (thyroxine; T4) were investigated in male Crl:CD-BR rats given
    fenbuconazole (purity, 97.1%) at concentrations of 8 or 800 ppm (10
    rats) or 0, 1600, or 3200 ppm (20 rats) for four or 13 weeks; a
    further two groups were treated with 1600 or 3200 ppm for four weeks
    before receiving control diet for nine weeks. Serum thyroid hormone
    levels were investigated in 10 rats per group at four and 13 weeks,
    and liver microsomal UDP-glucuronsyltransferase and biliary excretion
    of 125I-L-T4 were investigated in animals at 0 and 3200 ppm at four
    and 13 weeks and at four weeks plus recovery.

         No treatment-related deaths were seen. The only clinical symptom
    apparently associated with treatment was squinting in animals at 3200
    ppm for 13 weeks. The body weights of animals fed 1600 or 3200 ppm for
    13 weeks were reduced but not those of rats fed identical levels of
    treated diet followed by control diet. Corresponding effects on food
    consumption was seen, being reduced in rats fed 1600 or 3200 ppm for
    13 weeks and increased in the group allowed to recover. At 1600 and
    3200 ppm, there were no treatment-related effects on serum aspartate
    or alanine aminotransferase activities. Gross pathological examination
    revealed only increased weights of the liver and thyroid. After four
    weeks, the relative and absolute thyroid weights were increased by
    about 35% at 1600 ppm and the relative weights by about 50% at 3200
    ppm. Dose-related increases in relative and absolute liver weights
    were seen after four weeks, by about 20 and 30% at both 800 and 1600
    ppm and by about 85 and 40% at 3200 ppm, respectively. After 13 weeks
    of treatment, the absolute and relative thyroid weights were increased
    by 30-31% at 800 ppm, 41-47% at 1600 ppm, and 34-67% at 3200 ppm; the
    absolute and relative liver weights were increased by 21% at 800 ppm,
    45-51% at 1600 ppm, and 53-92% at 3200 ppm. After four weeks'
    treatment at 1600 or 3200 ppm and nine weeks' recovery, the weights of
    both the liver and thyroid had recovered and were comparable to those
    of controls.

         Histopathological examination of the thyroid after four weeks
    showed dose-related increases in the incidence and severity of diffuse
    follicular hypertrophy and hyperplasia in 4/10 animals at 800 ppm,
    9/10 at 1600 ppm, and 10/10 at 3200 ppm, with focal hyperplasia in one
    animal each at 1600 and 3200 ppm. Diffuse hypertrophy and hyperplasia
    were also seen at dose-related severity after 13 weeks in 9/10 rats at
    800 ppm and all rats at 1600 or 3200 ppm, with focal hyperplasia in
    one rat at 3200 ppm. These hypertrophic effects were reversible after
    nine weeks, the effects being of comparable severity in 6/10 controls,
    6/10 at 1600 ppm, and 8/10 at 3200 ppm. Focal hyperplasia was still
    present in one animal at 3200 ppm.

         After four weeks, the average thyroid-stimulating hormone (TSH)
    concentrations were increased in a dose-related trend, by about 79% at
    800 ppm, 83% at 1600 ppm, and 105% at 3200 ppm. The L-T4 and reverse
    L-triiodothyronine (rT3) levels were reduced to about 50% of the
    control levels at 3200 ppm, while the T3 levels were unaffected. After
    13 weeks, statistically significantly increased TSH levels were seen
    at 3200 ppm (63%) and reduced L-T4 levels at 1600 or 3200 ppm (by 53
    and 105%, respectively); however dose-related increases in TSH levels
    were seen at 800-3200 ppm (13, 58, and 63%, respectively), and rT3
    levels were reduced by about 49% at 3200 ppm. In the animals that were
    allowed to recover, the hormone levels were comparable with those in
    control animals, with the exception of rT3 in rats at 3200 ppm
    (Hazelton et al., 1991).

         Biliary excretion of L-T4 was investigated over 4 h in
    bile-cannulated rats at 0 or 3200 ppm after four weeks and 13 weeks
    and in the group allowed to recover after receiving 3200 ppm after
    intravenous injection of 125I-L-T4. In treated rats, the biliary
    clearance rate was increased by 165-281% at four weeks and 220-336% at
    13 weeks; 14.7-14.9% of the administered radiolabel was excreted by
    these animals in comparison to 7.2-7.5% by controls. About 70 and 85%
    of the increased biliary excretion correlated to increased excretion
    of L-T4-glucuronide at four and 13 weeks, respectively. By 13 weeks,
    these parameters were lower in animals allowed to recover and were
    comparable to those in control animals. In addition, a corresponding
    increase in the activity of UDP-glucuronosyltransferase was seen in
    these animals at four weeks, by 54% when expressed per mg microsomal
    protein, 187% per g liver, and 337% per liver; at 13 weeks, the
    respective activities were increased by 25, 144, and 300%, while the
    activity in animals allowed to recover was comparable to that of
    controls. Therefore, in rats at relatively high dietary doses, hepatic
    metabolism and biliary excretion of T4 were increased, and TSH levels
    were correspondingly increased, with hypertrophy and hyperplasia of
    this gland. The NOAEL for these effects was 8 ppm, equal to 1 mg/kg bw
    per day over 13 weeks (Hazelton et al., 1991).

         Six groups of female CD-1 mice received fenbuconazole in the diet
    at 0, 20, 60, 180, or 1300 ppm or 1000 ppm phenobarbital for one and
    four weeks. Groups of male CD rats received diets containing 1600 ppm
    fenbuconazole or 1000 ppm phenobarbital for four weeks. In addition,
    three groups of mice and rats received 1300 or 3200 ppm fenbuconazole

    or 1000 ppm phenobarbital for four weeks, followed by control diet for
    nine weeks. 

         No treatment-related effects on the liver were seen in mice at
    < 60 ppm. At 180 ppm, the effects on the liver included increased
    activities of cytochrome P450, cytochrome b5, and 7-pentoxyresorufin
     O-deethylase. The increase in cytochrome P450 was due to an increase
    in the phenobarbital-inducible form of the enzyme. At 1300 ppm, the
    effects were more pronounced and included hepatic enlargement,
    hepatocellular hypertrophy, and hepatocellular proliferation, as
    determined by bromodeoxyuridine immunohistochemistry. In rats,
    fenbuconazole increased liver weights and induced hepatocellular
    hypertrophy and cytochrome P450 activity, again due to an increase in
    the phenobarbital-inducible form of the enzyme. In both species, the
    effects on the liver were similar to phenobarbital-induced toxicity at
    1000 ppm and were reversible after cessation of treatment with
    fenbuconazole or phenobarbital (Hazelton et al., 1995).

         The synthesis of T4 and T3 in the thyroid depends on a dietary
    supply of iodine, which is taken up in the thyroid follicular cells,
    oxidized by thyroid peroxidase to iodine, and bound at the apical
    membrane to tyrosyl residues on thyroglobulin, either as
    monoiodotyrosine or diiodotyrosine, which are coupled to produce T3
    and T4, which remain part of thyroglobulin. This protein is secreted
    into the follicular lumen and taken up by the follicular cells by
    pinocytosis, where monoiodotyrosine, diiodotyrosine, T3, and T4 are
    released. While T3 and T4 pass into the circulation, mono- and
    diiodotyrosine remain in the follicular cells, where they are
    deiodinated; the iodine is used to produce more hormone.

         The level of circulating T4 is monitored by thyrotrophs in the
    anterior pituitary, which are responsible for the production of TSH,
    the major thyrotrophic hormone. In the thyrotrophs, T4 is deiodinized
    in the outer ring by 5'-deiodinase II, to give T3, which then binds to
    nuclear receptors in the cell. A decrease in occupancy of T3 receptors
    results in increased synthesis of TSH. Greater control is exercised by
    the hypothalamus, by the secretion of throtrophin-releasing hormone,
    which also stimulates the release of TSH from thyrotrophs. Thus, any
    reduction in the level of circulating T4 will result in an increased
    level of TSH. In humans, thyroid-binding globulin is the main plasma
    protein that binds thyroid hormones, with a greater affinity for T4
    than T3. In rats, thyroid hormones are bound mainly and with lower
    affinity to albumin, and only low levels of a protein that has 70%
    homology with human thyroid-binding globulin are present (Imamura et
    al., 1991).

         Circulating T4 is taken up by various organs, but most is
    metabolized in the liver by 5'-deiodinase I. This enzyme can catalyse
    deiodination of both the outer ring to give T3, the more active
    thyroid hormone, and the inner ring to give rT3, which has no known
    function. Deiodination is the main route of catabolism in humans,
    finally resulting in thyronine production. Thyroid hormones are also

    sulfated and glucuronidated; the latter pathway is of little
    importance in humans but the major route in rats. 

         Broadly speaking, five categories of xenobiotic influence thyroid
    hormone homeostasis: 

         -    directly acting substances that inhibit either iodine uptake
              by the thyroid or thyroid peroxidase activity; e.g.
              aminosalicylic acid, propylthiourea, and resorcinol;

         -    substances that stimulate T4 clearance, predominantly
              through effects on the liver, by inducing microsomal enzymes
              (resulting in increased biliary clearance; e.g.
              phenobarbital) or by affecting hepatic transport of thyroid
              hormones;

         -    substances that influence deiodination, either by
              stimulating 5'-deiodinase I (e.g. phenobarbital) or by
              inhibiting 5'-deiodinase II (e.g. iopanoic acid);

         -    substances that affect plasma binding of thyroid hormones
              (e.g. salicylates); and

         -    substances that interact with receptors of neurotransmitters
              such as dopamine that have been implicated in the control of
              TSH output by thyrotrophin-releasing hormone (e.g.
              clomiphene).

         Thus, studies of the effects of fenbuconazole on the liver in
    male mice and rats show that the microsomal enzymes cytochrome b5 and
    7-pentoxyresorufin- O-deethylase (a marker of the cytochrome P450 2B
    subfamily) are induced in both species (Hazelton et al., 1995). More
    significantly, UDP-glucuronosyltransferase activity and biliary
    clearance of 125I-thyroxine metabolites, including T4-glucuronide,
    were increased after dietary administration, although the only dose
    tested was 3200 ppm (Hazelton et al., 1991). In studies by dietary
    administration to rats for up to 13 weeks, the plasma levels of TSH
    were increased at doses > 800 ppm, while those of T4 and rT3 were
    decreased and those of T3 unaltered; these changes correlated to
    thyroid follicular hypertrophy and hyperplasia and increased thyroid
    weight (Hazelton et al., 1991). None of these effects was seen at 8
    ppm (Wolfe, 1990). A dietary concentration of 800 ppm was the lowest
    at which an increased incidence of adenomas and carcinomas of thyroid
    follicular cells was seen in rats treated for up to two years (Wolfe,
    1991b). Thus, the basis for the possible carcinogenicity of
    fenbuconazole is chronic stimulation of the thyroid by TSH with
    reduced plasma T4 concentrations. The reason for the reduced T4 levels
    at all relevant doses of fenbuconazole has not been found, but the
    observation of increased biliary clearance of thyroid hormone
    metabolites at a high dose (the only one tested) indicates another
    component of the mechanism of carcinogenesis.

    Comments

         Fenbuconazole is rapidly absorbed and eliminated, mainly in the
    faeces through significant biliary excretion; there was no evidence of
    significant retention in tissues. The compound was also extensively
    metabolized by phase-I oxidation or hydroxylation at a number of sites
    in the molecule, followed by phase-II sulfate and glucuronide
    conjugation (predominantly glucuronidation). Dermal absorption of
    fenbuconazole (technical material and a formulation) constituted 2-13%
    of an administered dose over 24 h, the absorption over 10 h being
    < 5%.

         Fenbuconazole was of low acute toxicity when administered orally
    (LD50 > 2000 mg/kg bw), dermally (LD50 > 5000 mg/kg bw), or by
    inhalation (LC50 > 2.1 mg/L air). It was not irritating to the skin
    or eyes and was not a sensitizer in a Buehler test, but was a weak
    sensitizer in a maximization test. WHO has not yet classified
    fenbuconazole for acute toxicity.

         After dietary administration, hepatomegaly with associated
    effects on clinical chemistry, such as changes in cholesterol and
    triglyceride levels and increases in the serum activity of hepatic
    enzymes, were seen in mice, rats, and dogs. In a 13-week study of
    toxicity in mice with dietary levels of 0, 20, 60, 180, or 540 ppm the
    NOAEL was 60 ppm (equal to 11 mg/kg bw per day) on the basis of
    hepatic effects at higher doses. In a three-month study of toxicity in
    rats with dietary levels of 0, 20, 80, 400, or 1600 ppm, the NOAEL was
    20 ppm (equal to 1.3 mg/kg bw per day) on the basis of hepatic effects
    and hypertrophy of thyroid gland follicular cells at higher doses. In
    a 13-week study of toxicity in dogs with dietary levels of 0, 30, 100,
    400, or 1600 ppm, the NOAEL was 100 ppm (equal to 3.3 mg/kg bw per
    day). In a one-year study in dogs with dietary levels of 0, 15, 150,
    or 1200 ppm, the NOAEL was 150 ppm (equal to 5.2 mg/kg bw per day).
    The NOAELs in the studies in dogs were based on decreased body-weight
    gain and increased incidences of hepatic hypertrophy with associated
    effects on clinical chemistry at higher doses.

         In a 78-week study of toxicity and carcinogenicity in mice, with
    dietary levels of 0, 10, 200, or 650 ppm in males and 0, 10, 650, or
    1300 ppm in females, there was clear evidence of treatment-related
    hepatomegaly, with dose-related hepatocytic hypertrophy and
    vacuolation, and limited evidence of treatment-related hyperplasia and
    tumorigenicity in the liver at the highest dose. The NOAEL was 10 ppm
    (equal to 1.3 mg/kg bw per day). In a two-year study in rats with
    dietary levels of 0, 8, 80, or 800 ppm, the predominant effects were
    hepatocytic hypertrophy, thyroid follicular-cell hypertrophy, and an
    increase in thyroid follicular-cell adenomas; in addition, thyroid
    carcinomas were seen at the high dose. The NOAEL was 80 ppm, equal to
    3.0 mg/kg bw per day.

         The etiology of the hepatic and thyroidal effects in rats was
    further investigated in a 4-13-week study which illustrated the
    biological feedback mechanism in rats: hepatomegaly leading to
    increased metabolism and excretion of thyroxine, increased levels of
    thyroid stimulating hormone, and thyroid hypertrophy/hyperplasia. The
    effects seen after four weeks in this study were reversible. In
    studies designed to investigate the hepatotoxicity of fenbuconazole,
    hepatic effects were seen in rats and mice that were similar to those
    induced by phenobarbital. Increased cytochrome P450 activity (CYP2B
    form) was observed, with hepatocellular hypertrophy and proliferation.
    The NOAEL in mice after treatment for 13 weeks was 60 ppm (equal to 14
    mg/kg bw per day).

         Fenbuconazole was adequately tested for genotoxicity  in vitro 
    and  in vivo. The Meeting concluded that it is not genotoxic.

         Fenbuconazole was not teratogenic in either rats (at doses of 0,
    30, 75, or 150 mg/kg bw per day) or rabbits (at doses of 0, 10, 30, or
    60 mg/kg bw per day), but fetotoxicity was seen in both species, with
    an NOAEL of 30 mg/kg bw per day. The NOAELs for maternal toxicity were
    30 mg/kg bw per day in rats and 10 mg/kg bw per day in rabbits. No
    effects on reproductive parameters were seen in a multigeneration
    study in rats at dietary levels of 0, 8, 80, or 800 ppm, but
    fetotoxicity was again seen at high doses, with maternal toxicity. The
    NOAEL was 80 ppm, equal to 5.8 mg/kg bw per day.

         An ADI of 0-0.03 mg/kg bw was allocated, on the basis of the
    NOAEL of 3 mg/kg bw per day in the two-year study in rats and a safety
    factor of 100. The Meeting noted that the NOAEL in the 13-week study
    in rats and in the 78-week study in mice was 1.3 mg/kg bw per day, but
    it concluded that this figure should not be used to derive the ADI.
    The NOAEL from the 13-week study in rats was not considered to be
    relevant in the light of the results of the larger, two-year study.
    The Meeting concluded that the overall NOAEL in mice was 14 mg/kg bw
    per day. This figure was taken from the 13-week study, which included
    detailed investigations of hepatotoxicity. Hepatotoxicity was the
    critical effect in the long-term study in mice, and the NOAEL in the
    13-week study was lower than the lowest dose that was hepatotoxic in
    the long-term study.

    Toxicological evaluation

     Levels that cause no toxic effect

         Mouse:    60 ppm, equal to 14 mg/kg bw per day (13-week study of
                   hepatotoxicity)
                   10 ppm, equal to 1.3 mg/kg bw per day (78-week study of
                   toxicity)

         Rat:      20 ppm, equal to 1.3 mg/kg bw per day (13-week study of
                   toxicity)
                   80 ppm, equal to 3.0 mg/kg bw per day (two-year study
                   of toxicity)

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

                                                                                                                               

    Human exposure      Relevant route, study type, species               Results, remarks
                                                                                                                               

    Short-term          Oral toxicity, rat                                LD50 > 2000 mg/kg bw
    (1-7 days)          Dermal toxicity, rat                              LD50 > 5000 mg/kg bw
                        Inhalation toxicity, rat                          LC50 > 2.1 mg/L
                        Dermal irritation, rabbit                         Not irritating
                        Ocular irritation, rabbit                         Not irritating
                        Dermal sensitization, guinea pig                  Not sensitizing in Buehler test, weakly 
                                                                          sensitizing in maximization test

    Medium-term         Repeated oral, 1-year, toxicity, dog              NOAEL = 5.2 mg/kg bw per day: hepatic effects
    (1-26 weeks)        Repeated dermal, 4 weeks, toxicity, rat           NOAEL = 1000 mg/kg bw per day (highest dose 
                                                                          tested)
                        Repeated oral, reproductive toxicity, rat         NOAEL = 5.8 mg/kg bw per day: maternal and 
                                                                          fetal toxicity
                        Repeated oral, developmental toxicity, rabbit     NOAEL = 10 mg/kg bw per day: maternal 
                                                                          toxicity

    Long term           Repeated oral, 2 years, toxicity and              NOAEL = 3 mg/kg bw per day: hepatic and
    (> 1 year)          carcinogenicity, rat                              thyroid effects
                                                                                                                               
    

                   80 ppm, equal to 5.8 mg/kg bw per day (two-generation
                   study of reproductive toxicity)
                   30 mg/kg bw per day (maternal toxicity in a study of
                   developmental toxicity)

         Rabbit:   10 mg/kg bw per day (maternal toxicity in a study of
                   developmental toxicity)

         Dog:      150 ppm, equal to 5.2 mg/kg bw per day (one-year study
                   of toxicity)

     Estimate of acceptable daily intake for humans

         0-0.03 mg/kg bw

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

         Observations in humans

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    See Also:
       Toxicological Abbreviations