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        INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

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        TOXICOLOGICAL EVALUATION OF CERTAIN
        VETERINARY DRUG RESIDUES IN FOOD



        WHO FOOD ADDITIVES SERIES 41





        Prepared by:
          The 50th meeting of the Joint FAO/WHO Expert
          Committee on Food Additives (JECFA)



        World Health Organization, Geneva 1998




    FEBANTEL, FENBENDAZOLE, AND OXFENDAZOLE (addendum)

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

    1.   Explanation
    2.   Biological data
         2.1  Developmental toxicity
         2.2  Tumour promotion
    3.   Comments
    4.   Evaluation
    5.   References

    1.  EXPLANATION

         Febantel, fenbendazole, and oxfendazole were evaluated at the
    thirty-eighth and forty-fifth meetings of the Committee (Annex 1,
    references 97 and 119).

         Fenbendazole and oxfendazole are benzimidazoles and are
    metabolically interconvertible  in vivo. Febantel is a prodrug that
    can be converted  in vivo by cyclization to fenbendazole or after
    oxidation at the sulfur atom and subsequent cyclization to
    oxfendazole.

         At the forty-fifth meeting, the Committee established a temporary
    group ADI of 0-4 g/kg bw for febantel, fenbendazole, and oxfendazole,
    on the basis of a NOEL for oxfendazole of 0.7 mg/kg bw per day in a
    two-year study in rats, applying a safety factor of 200 because the
    ADI was temporary. The Committee requested the results of a study of
    teratogenicity in rabbits in which oxfendazole was administrated at
    sufficiently high doses for its teratogenic potential to be adequately
    explored.

    2.  BIOLOGICAL DATA

    2.1  Developmental toxicity

         In a pilot study of the developmental toxicity of oxfendazole
    (99.3% pure) in 0.5% carboxymethylcellulose, groups of eight pregnant
    New Zealand white rabbits were given doses of 0, 1, 5, 12, or 25 mg/kg
    bw per day by gavage. None of the doses (including the maximal dose)
    affected body weights, and no adverse clinical signs of toxicity were
    seen. Feed consumption and absolute and relative liver weights were
    not changed in comparison with controls. In the second phase of the
    study, groups of five does were given oxfendazole at 0 or 25 mg/kg bw
    per day by gavage on days 7-19 day of gestation. No maternal toxicity
    and no changes in body weight, body-weight gain, or feed consumption
    were seen. In phase III, the maximal tolerated dose for pregnant

    rabbits was determined. Groups of eight does were given 0, 100, or 200
    mg/kg bw per day on days 7-19 of gestation, and groups of four animals
    were given 500 or 1000 mg/kg bw per day on days 8-19 days of
    gestation. Treatment-related early resorptions, decreased litter size
    and fetal weight, fetal malformations, and changes in ossification
    were reported at doses of 100 and 200 mg/kg bw per day. The fetal
    malformations included biologically relevant differences in the
    interrelated average numbers of ossification sites per fetus in the
    thoracic vertebrae (increased), lumbar vertebrae (decreased), and ribs
    (increased) as compared with controls. The doses of 200, 500, and      
    1000 mg/kg bw per day resulted in mortality and/or abortion of
    litters. None of the fetuses at 500 or 1000 mg/kg bw per day survived
    to day 29 of gestation. On the basis of these results, doses of 0, 10,
    30, and 45 mg/kg bw per day of oxfendazole were used in the definitive
    study of developmental toxicity in rabbits. The highest dose used was
    ninefold higher than the therapeutic dose of oxfendazole used in
    cattle, sheep, and horses: a single dose of 5 mg/kg bw, which can be
    repeated every four to six weeks (Hoberman, 1997).

         In the definitive study, oxfendazole was given to groups of 20
    pregnant rabbits on days 7-19 of presumed gestation, at doses of 0,
    10, 30, or 45 mg/kg bw per day. All animals survived the treatment,
    and no abortions or premature deliveries occurred during the study. No
    effects were seen on the average body weights of does, body-weight
    gain, gravid uterine weight, or absolute (g/day) or relative (g/kg per
    day) feed consumption. No maternal or developmental toxicity occurred.
    It did not affect the number of fetuses, their viability, or the sex
    ratios and did not cause malformations or variations (Hoberman, 1996).

    2.2  Tumour promotion

         Induction of the cytochrome P450 isoenzymes CYP1A1/2, 2B1/2, and
    4A1 is associated with promotion of liver tumours, and rat liver
    tumour promoters inhibit gap-junction intracellular communication, as
    evidenced by a decrease in the amount of the protein connexin 32. The
    two-stage liver carcinogenesis model in rats was used to examine
    whether oxfendazole promotes liver tumours, and the activity of P450
    isozymes, gap-junction intracellular communication, and the presence
    of foci of the placental form of glutathione  S-transferase
    (GST-P+) were measured. Five groups of 10-15 four-week-old Fischer
    344 rats were initiated with a single intraperitoneal injection of 100
    mg/kg bw  N-nitrosodiethylamine (NDEA), and one week later they
    received a diet containing oxfendazole at 0, 10, 100, 250, or 500 ppm
    for eight weeks. Five animals from the NDEA-initiated groups treated
    with 0 or 250 ppm oxfendazole were killed after one week of receiving
    the diet, and the remaining animals were killed at the end of the
    treatment period.

         No significant difference in body-weight gain was seen between
    treated and control groups. After one week of treatment with  250 ppm
    oxfendazole, initiated animals had significantly increased relative
    liver weights. At the end of treatment with oxfendazole, liver weights
    were increased in animals treated with NDEA and 250 or 500 ppm

    oxfendazole or with 100, 250, or 500 ppm oxfendazole alone. Treatment
    with NDEA and 250 ppm oxfendazole for one week did not cause
    histological changes, but treatment with 100 ppm or more of
    oxfendazole for eight weeks caused hepatocellular hypertrophy and
    fatty degeneration, irrespective of pretreatment with NDEA. In animals
    given NDEA plus 500 ppm oxfendazole, single hepatocytes sometimes
    showed necrosis, and a marked increase in smooth endoplasmic
    reticulum, with the appearance of lipid droplets in the cytoplasm, was
    seen by elecron microscopy. The liver P450 isozymes CYP1A1/2, 2B1/2,
    and 4A1 were markedly induced in animals initiated with NDEA and
    treated with 250 or 500 ppm oxfendazole for one or eight weeks;
    induction of CYP2E1 and 3A2 was not remarkable in these groups.
    Significant induction of CYP1A1/2 was also seen in NDEA-initiated
    animals treated with 10 or 100 ppm oxfendazole for eight weeks, and
    the CYP2B1/2 and 4A1 isozymes were induced in those given NDEA plus
    100 ppm oxfendazole.

         The number of connexin 32-positive spots per hepatocyte was
    significantly decreased in the animals receiving NDEA plus 250 ppm
    oxfendazole after one week as compared with controls, and the numbers
    and areas of spots were significantly reduced in a dose-dependent
    manner in the rats treated with oxfendazole. In all NDEA-initiated
    animals, GST+ focal lesions, consisting of single hepatocytes or
    mini-foci of hepatocytes strongly positive for the GST-P marker
    enzyme, were observed. The number of GST-P+ single cells was
    significantly increased in the NDEA-initiated animals treated with 250
    or 500 ppm oxfendazole as compared with those given NDEA alone;
    however, NDEA-initiated animals treated with 100 ppm oxfendazole had
    fewer GST-P+ single cells than those that received only NDEA. No
    positive foci were found in uninitiated groups with no oxfendazole
    administration (Mitsumori  et al., 1997). The results strongly
    suggest that oxfendazole promotes liver tumours.

    3.  COMMENTS

         The Committee considered the results of studies of the
    developmental toxicity of oxfendazole in rabbits, which were conducted
    to appropriate standards for study protocol and conduct. In a
    preliminary study in three phases, doses up to 1000 mg/kg bw per day
    were given by gavage to pregnant rabbits during the critical days of
    gestation. Doses of 100 mg/kg bw per day and higher produced fetal
    malformations. On the basis of these results, the compound was
    administered by gavage to pregnant rabbits on days 7-19 of gestation
    at doses of 0, 10, 30, or 45 mg/kg bw per day.  No maternal toxicity,
    embryotoxicity, or effects on fetal morphology were observed.

         The Committee considered a published study of
    initiation-promotion with oxfendazole in rats, in which the activity
    of P450 enzymes, gap-junctional intercellular communication, and
    expression of the placental form of glutathione  S-transferase were
    examined. Although some enzymatic and immunohistochemical changes were
    seen that suggested that oxfendazole might have tumour-promoting
    potential in rats, the results of the studies of genotoxicity and

    carcinogenicity evaluated at the thirty-eighth and forty-fifth
    meetings presented sufficient evidence that oxfendazole is not
    carcinogenic.

    4.  EVALUATION

         The Committee established a group ADI of 0-7 g/kg bw for
    febantel, fenbendazole, and oxfendazole on the basis of a NOEL of 0.7
    mg/kg bw per day in a two-year study on oxfendazole in rats (evaluated
    at the thirty-eighth and forty-fifth meetings of the Committee) and
    applying a safety factor of 100. The Committee noted that this ADI
    provides a safety factor of 1000 for teratogenic effects in sheep,
    which was considered at the thirty-eighth meeting to be the most
    sensitive species for the teratogenic effects of oxfendazole.

    5.  REFERENCES

    Hoberman, A.M. (1996) An oral developmental toxicity (embryo-fetal
    toxicity/teratogenicity) definitive study with oxfendazole in rabbits.
    Unpublished study No. 101-026 from Argus Research Laboratories, Inc.,
    Horsham, Pennsylvania, USA. Submitted to WHO by Hoechst Roussel Vet,
    Wiesbaden, Germany.

    Hoberman, A.M. (1997) An oral development toxicity (embryo-fetal
    toxicity/teratogenicity) pilot study with oxfendazole in rabbits.
    Unpublished study  No. 101-026 from Argus Research Laboratories, Inc.,
    Horsham, Pennsylvania, USA. Submitted to WHO by Hoechst Roussel Vet,
    Wiesbaden, Germany.

    Mitsumori, K., Onodera, H., Shoda, T., Uneyama, C., Imazawa, T.,
    Takegawa, K., Yasuhara, K., Watanabe, T. & Takahashi, M. (1997) Liver
    tumour-promoting effects of oxfendazole in rats.  Food Chem. 
     Toxicol., 35, 799-806.
    


    See Also:
       Toxicological Abbreviations