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    FEBANTEL, FENBENDAZOLE AND OXFENDAZOLE

    First draft prepared by
    Dr R. Fuchs
    Department of Experimental Toxicology and Ecotoxicology
    Institute for Medical Research and Occupational Health
    Zagreb, Croatia

    Explanation

    Biological data

    Toxicological studies
         Febantel
         Fenbendazole
         Oxfendazole

    Comments

    Evaluation

    References

    1.  EXPLANATION

         Febantel, fenbendazole and oxfendazole were previously evaluated
    at the thirty-eighth meeting of the Committee (Annex 1, reference 97).

         A temporary ADI of 0-25 µg/kg bw was established for fenbendazole
    at that time, based on a NOEL of 5 mg/kg bw/day in a long-term
    toxicity/ carcinogenicity study in rats and a safety factor of 200.
    Additional information on the mechanism of the observed increased
    incidence of liver tumours in female rats at high doses, including the
    results of an  in vivo DNA binding study, was requested.

         A temporary ADI of 0-10 µg/kg bw was established for febantel at
    the thirty-eighth meeting, based on a NOEL of 2 mg/kg bw/day in a
    2-generation reproductive toxicity study in rats and a safety factor
    of 200. Additional information on the genotoxic and carcinogenic
    potential of febantel, including the results of an  in vivo DNA
    binding assay in rats, was requested.

         A temporary ADI of 0-4 µg/kg bw was established for oxfendazole
    at the thirty-eighth meeting, based on a NOEL of 0.7 mg/kg bw/day in a
    carcinogenicity study in rats and a safety factor of 200. Additional
    data from genotoxicity and teratogenicity studies were requested,
    including the results of an  in vivo DNA binding assay in rats.

         At its present meeting, the Committee considered the data
    available at the thirty-eighth Committee meeting (Annex 1,
    reference 97), together with the results of additional genotoxicity
    study on fenbendazole, which are summarized in this monograph
    addendum.

    2.  BIOLOGICAL DATA

    2.1  Toxicological studies

    2.1.1  Febantel

         Combined long-term toxicity/carcinogenicity studies on febantel
    in rats and mice did not reveal increased incidences of any tumour
    types (Bomhard & Kaliner, 1985; Bombard & Mager, 1987; Annex 1,
    reference 98).

         No evidence of a distinct mutagenic potential of febantel could
    be derived from a variety of mutagenicity tests. Febantel was positive
    in the dominant lethal test in male mice only at the extremely high
    dose of 2000 mg/kg bw/day given twice daily at 24 h intervals, but was
    negative at the lower test dose of 500 mg/kg bw/day. This level was
    far in excess of the therapeutic dose (Machemer & Dycka, 1976; Annex
    1, reference 98). As febantel had no point mutagenic effect, a DNA
    binding assay was not deemed necessary.

    2.1.2  Fenbendazole

    2.1.2.1  Long-term toxicity/carcinogenicity studies

         There were no increase in tumour incidence in a 2-year
    carcinogenicity study in mice with doses of fenbendazole up to
    405 mg/kg bw/day (Goldenthal, 1980; Annex 1, reference 98).

         A lifetime toxicity/carcinogenicity study (including an  in utero
    phase) of fenbendazole in Charles River CD rats used the F1
    generation of pups that had been exposed to the same dose levels
     in utero. Fenbendazole was fed in the diet at dose levels of 0, 5,
    15, 45 or 135 mg/kg bw/day.  In utero exposure to 45 mg/kg bw/day and
    135 mg/kg bw/day caused severe toxicity in the pups characterized by
    decreased body weights, diarrhoea, bloated stomachs, icterus and
    alopecia. F1 pups exposed to 135 mg/kg bw/day had 33% decreased body
    weights as compared to controls, while pups exposed to 45 mg/kg bw/day
    were 15% below controls. There was also increased mortality, 18% of
    pups exposed to 135 mg/kg bw/day and 14% of the pups exposed to
    45 mg/kg bw/day died by day 21 of age as compared to 6% in the
    controls. These data suggest that pups at 45 and 135 mg/kg bw/day had
    been dosed above the MTD prior to the start of the lifetime study.
    This toxicity and increased neonatal mortality severely limit the
    conclusions that can be drawn from this study.

         The histopathological changes observed in the liver of treated
    animals were reported as hepatocellular hypertrophy, vacuolation and
    bile duct proliferation in the 15, 45 and 135 mg/kg bw/day groups,
    hepatocellular hyperplasia and biliary cysts in the 45 and 135 mg/kg
    bw/day groups, and hepatocellular adenomas and carcinomas in the

    135 mg/kg bw/day group. A group of independent pathologists reviewed
    the histopathologic findings and concluded that fenbendazole treatment
    did not result in an increased incidence of hepatocellular neoplasms
    (Goldenthal, 1981; Lewis, 1982; Brown, 1982; Annex 1, reference 98).

    2.1.2.2  Special studies on genotoxicity

         The results of additional genotoxicity studies on fenbendazole
    are given in Table 1.
        Table 1.  Results of genotoxicity studies on fenbendazole
                                                                                              

    Test system         Test object    Concentration      Results     Reference
                                                                                              

    Forward mutation    Mouse          up to 100 µg/ml    Negative    Den Boer &
    assay1              lymphoma                                      Horn, 1986
                                                                                              

    1    Both with and without rat liver S9.
             Fenbendazole was assayed in L5178Y TK+/- mouse lymphoma cells
    using doses ranging from 2.5 to 10 µg/ml without activation and from
    4.0 to 10 µg/ml with metabolic activation. The observed toxicity
    ranged from low to moderate. The substance showed steep toxicity so
    that treatment in the range of 10 to 20% relative growth was not
    possible. All of the treatment, both with and without metabolic
    activation, induced mutation frequencies that were below the minimum
    criterion of mutagenesis. The authors concluded that fenbendazole was
    not mutagenic in the mouse lymphoma assay (Den Boer & Horn, 1986).
    However, the test concentrations used in this study were considerably
    lower than those used in the previous study when concentrations up to
    62 µg/ml were tested (Cifone & Myhr, 1983).

         In the opinion of independent experts, a DNA binding study would
    not result in further clarification of the clastogenic effects of
    fenbendazole  in vitro on V79 cells, but would merely show whether
    there might be an additional potential for point mutations (letter
    from professor C. Schlatter, Institute for Toxicology, Zurich,
    Switzerland, to Dr Müller, Hoechst AG, 1991; submitted to WHO by
    Hoechst AG, Frankfurt, Germany).

         Genotoxicity studies on all essential genetic endpoints gave
    negative results. Clastogenic effects of benzimidazoles  in vitro are
    related to the inhibition of tubulin formation.  In vivo assays for
    binding to DNA in liver following the oral administration of
    fenbendazole have not been performed.

         In an expert review on the toxicology of fenbendazole, the
    authors concluded that it showed no mutagenic, genotoxic or
    carcinogenic potential and that sufficient data were available to
    establish an ADI for fenbendazole residues in animal-derived food
    (Bolt & Gansevendt, 1991).

    2.1.3  Oxfendazole

         Oxfendazole has been tested only in a bacterial mutation assay
    that gave negative results (Mourot, 1990; Annex 1, reference 98).

         The doses used in the oral rat carcinogenicity study were
    selected on the basis of results of a 3-month oral dosing study in
    rats. Based on increased mortality at 600 mg/kg (equal to 48 mg/kg
    bw/day for males and 50 mg/kg bw/day for females), and elevated ALP
    levels at 200 mg/kg (equal to 17 mg/kg bw/day for males and 18 mg/kg
    bw/day for females), a level of 100 mg/kg (equal to 7.4 mg/kg bw/day
    for males and 7.8 mg/kg bw/day for females) was determined to be the
    maximum tolerated dose that could be administered over a 2-year
    lifespan of rats without compromising survival (these doses have been
    corrected and do not correspond exactly to those given in Food
    Additives Series 29 (Annex 1, reference 98). In addition, in the oral
    carcinogenicity study in rats, histopathologic findings of
    hepatocellular vacuolation in the liver of rats fed 30 mg/kg of feed
    (equal to 2.0 mg/kg bw/day for males and 2.4 mg/kg bw/day for
    females), and 100 mg/kg of feed (equal to 6.6 mg/kg bw/day for males
    and 7.8 mg/kg bw/day for females) further suggested that there was
    sufficient systemic exposure to oxfendazole to show that it had been
    tested adequately. There was no carcinogenic effect of oxfendazole at
    any dose level (De Pass & Bidlack, 1987; Annex 1, reference 98).

         The thirty-eighth Committee (Annex 1, reference 97) requested a
    teratogenicity study in rabbits using oxfendazole at sufficiently high
    doses to explore its teratogenic potential. This study was not
    performed.

    3.  COMMENTS

         Febantel and fenbendazole have been tested in a range of
    genotoxicity assays, while oxfendazole has been tested only in an Ames
    test in which four strains of  Salmonella typhimurium were used. The
    compounds consistently gave negative results in the Ames test, a test
    for DNA repair and  in vivo cytogenetic assays. Febantel,
    fenbendazole and a metabolite (2-amino-5-phenylsulfinyl-2-
    benzimidazole) were weakly positive in the mouse lymphoma forward
    mutation assay in the presence of a metabolic activation system.
    Febantel, when tested at a sufficiently high dose to reduce fertility,
    was found to induce dominant lethal mutations in mice.

         The present Committee noted that available data on the mode of
    action of benzimidazoles as inhibitors of the polymerization of
    tubulin to microtubulin, with consequent disruption of mitosis,
    together with the results of genotoxicity tests, do not provide any
    evidence of a direct interaction of these compounds with DNA. It
    therefore concluded that the positive results in some genotoxicity
    assays are likely to have been caused by an indirect mechanism, and
    that further studies on the binding of febantel, fenbendazole and
    oxfendazole to DNA are not required.

         The present Committee re-evaluated the long-term toxicity/
    carcinogenicity studies on all three compounds that were reviewed
    at the thirty-eighth meeting, and reached the conclusions given
    below.

          Febantel. Combined long-term toxicity/carcinogenicity studies
    on febantel in rats and mice did not reveal increased incidences of
    any types of tumour. At its thirty-eighth meeting (Annex 1, reference
    97), the Committee was of the opinion that higher doses could have
    been used in the carcinogenicity study in rats. However, in the group
    of rats given febantel at 500 mg/kg in the diet (equal to 40 mg/kg
    bw/day), decreases in body-weight gain, slight anaemia (in females
    only), increased liver weight, hepatocellular enlargement and
    vacuolization, and significant increases in serum ALP activity were
    observed. These changes were not seen at the lower doses or in the
    control groups. The present Committee considered that these changes
    indicated that the dose levels used were sufficient for the
    carcinogenic potential of febantel to be assessed, and concluded that
    there was no evidence that it was carcinogenic in mice or rats.

          Fenbendazole. In a lifetime study with an initial  in utero
    phase, rats were dosed with fenbendazole at doses of 0, 5, 15, 45 or
    135 mg of fenbendazole/kg bw/day. There were no increases in tumour
    incidence at 5, 15 or 45 mg/kg bw/day. At 135 mg/kg bw/day only a
    marginal increase in the incidence of liver tumours was observed. At
    the beginning of the lifetime study in rats, the mean body weights of

    the highest-dose group (84 g for males and 79 g for females) were more
    than 40% lower than those of the controls (142 g for males and 123 g
    for females). Furthermore, the rats in the highest-dose group showed
    signs of toxicity at that time, including alopecia, icterus,
    diarrhoea, and lethargy. The Committee considered that the results for
    this group could not be used for the assessment of the carcinogenicity
    of fenbendazole because the maximum tolerated dose had been exceeded
     in utero. However, proliferative liver lesions in the group
    receiving 45 mg/kg bw/day, described as hepatocellular hyperplasia and
    biliary cysts, which were not observed at lower doses, indicated that
    this dose level was high enough to permit an assessment of the
    carcinogenic potential of fenbendazole. On the basis of this study in
    rats and the negative results in the carcinogenicity study in mice
    reviewed at the thirty-eighth meeting (Annex 1, reference 97), the
    Committee concluded that there was no evidence that fenbendazole
    possessed carcinogenic potential.

          Oxfendazole. The highest dose used in the rat carcinogenicity
    study was selected in the light of the results of a 3-month oral
    toxicity study in rats. It was based on increased mortality at a dose
    level of 600 mg/kg in the diet (equal to 48 mg/kg bw/day for males and
    50 mg/kg bw/day for females) and elevated ALP levels at a dose level
    of 200 mg/kg in the diet (equal to 17 mg/kg bw/day for males and
    18 mg/kg bw/day for females). A dose level of 100 mg/kg in the diet
    (equal to 7.4 mg/kg bw/day for males and 7.8 mg/kg bw/day for females)
    was selected as the highest dose in the carcinogenicity study. No
    carcinogenic effects were seen at any dose level in this study. The
    present Committee considered that histopathological findings of
    hepatocellular vacuolation in the liver of rats fed more than 2 mg/kg
    bw/day provided evidence of sufficient systemic exposure to
    oxfendazole, and concluded that the study was adequate for assessing
    the carcinogenic potential of the compound. On the basis of this study
    and the negative results in the carcinogenicity study in mice reviewed
    at the thirty-eighth meeting (Annex 1, reference 97), the present
    Committee concluded that there was no evidence that oxfendazole
    possessed carcinogenic potential.

         At its thirty-eighth meeting (Annex 1, reference 97), the
    Committee had requested an additional teratogenicity study in rabbits
    using oxfendazole. This was not provided at the present meeting.
    Instead, the sponsor suggested that the available reproductive data in
    sheep were sufficient. The Committee concluded, however, that the
    study in sheep did not adequately explore the teratogenic potential of
    oxfendazole, because it was designed to assess the safety of the drug
    in the target species, not to study its teratogenicity.

    4.  EVALUATION

         The available data from reproductive and teratogenicity studies
    with each of the three drugs suggest that oxfendazole is the most
    potent in producing embryotoxic effects. The data on fenbendazole
    suggest that the rabbit is the species most sensitive to the
    embryotoxic and teratogenic effects of these three compounds. The
    Committee therefore decided that an adequate teratogenicity study in
    rabbits with oxfendazole was still required.

         The Committee noted that, on the basis of the available
    toxicological data, the NOELs identified at the thirty-eighth meeting
    and a safety factor of 100, ADIs of 0-20 and 0-50 µg/kg bw could have
    been established for febantel and fenbendazole, respectively. However,
    it also noted that febantel, fenbendazole and oxfendazole have common
    metabolic routes, and that the residue data demonstrate that
    oxfendazole is the major residue in food-producing animals following
    administration of these three compounds. Therefore, the Committee
    assigned a group temporary ADI of 0-4 µg/kg bw to febantel,
    fenbendazole and oxfendazole based on the NOEL of 0.7 mg/kg bw/day for
    oxfendazole identified at the thirty-eighth meeting and a safety
    factor of 200 (Annex 1, reference 97). The results of a teratogenicity
    study in rabbits, in which oxfendazole is administered at sufficiently
    high doses for its teratogenic potential to be adequately explored,
    are required by the Committee for evaluation in 1998.

    5.  REFERENCES

    Bolt HM & Gansevendt B (1991). Evaluation of the toxicology of
    fenbendazole. Unpublished report from the Institut fur
    Arbeitsphysiologie, Universitat Dortmund, Dortmund, Germany. Submitted
    to WHO, by Hoechst AC, Frankfurt, Germany.

    Bomhard, E & Kaliner G (1985). BAY Vh 5757 chronic toxicity and
    carcinogenicity studies in mice. 21-month feeding study. Unpublished
    report No. 13961 from Bayer AG. Submitted to WHO by Bayer AG,
    Leverkusen, Germany.

    Bomhard E & Mager H (1987). BAY Vh 5757 combined chronic toxicity and
    carcinogenicity studies in Wistar rats. Unpublished report No. 15844
    from Bayer AG. Submitted to WHO by Bayer AG, Leverkusen, Germany.

    Brown WR (1982). Lifetime oral toxicity study of fenbendazole in rats,
    histopathology - liver. Research pathology Serices Inc.,
    New Britain, PA, USA. Unpublished report. Submitted to WHO by Hoechst
    AG, Frankfurt am Main, Germany.

    Cifone MA & Myhr BC (1983). Mutagenicity evaluation of BAY L5156 in
    the mouse lymphoma forward mutation assay. Unpublished project
    No. 10989 from Litton Bionetics Inc. Kensington, MD, USA. Submitted to
    WHO by Bayer AG, Leverkusen, Germany

    De Pass LR & Bidlack DE (1987) Carcinogenicity study in rats with
    RS-8858 (oxfendazole) mixed in the feed. Unpublished report
    No. 53-R-83-8858-PO-CA from Syntex Inc. Palo Alto, CA, USA. Submitted
    to WHO by Syntex Inc. Palo Alto, CA, USA.

    Den Boer WC & Horn AJW (986) Mutagenic evaluation of S711881 in the
    L 5178 Y TK +/- mouse lymphoma forward mutation assay. Unpublished
    report from Hazleton Biotechnologies, Veenendaal, Netherlands
    No. 1003E. Submitted to WHO by Hoechst AG, Frankfurt, Germany.

    Goldenthal EJ (1980) 24-month oral carcinogenicity study in mice.
    Unpublished report, International Research and Development
    Corporation, Mattawan, NJ, USA. Submitted to WHO by Hoechst AG,
    Frankfurt am Main, Germany.

    Goldenthal EI (1981) Lifetime oral toxicity study in rats. Unpublished
    report. International Research and Development Corporation, Mattawan,
    MI, USA. Submitted to WHO by Hoechst AG, Frankfurt am Main, Germany.

    Lewis JC (1982) Review and conclusions of the histopathology data of
    "Lifetime oral toxicity study of fenbendazole in rats". Unpublished
    report No. 102E from Hoechst AG, Frankfurt, Germany. Submitted to WHO
    by Hoechst AG.

    Machemer L & Dycka G (1976) BAY Vh 5757 dominant lethal test on male
    mice to test for mutagenic effects. Unpublished report No. 6031 from
    Bayer A.G. Submitted to WHO by Bayer AG Leverkusen, Germany.

    Mourot D (1990) Oxfendazole Ames test. Unpublished report from
    Laboratoire des médicaments vétérinaires, La Haute-Manche-Jarene,
    Fougčres. Submitted to WHO by Centre National d'études vétérinaires et
    alimentaires, Javene, Fougčres, France.
    


    See Also:
       Toxicological Abbreviations