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        INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

        WORLD HEALTH ORGANIZATION



        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




    DICLAZURIL (addendum)

    First draft prepared by
    Professor F.R. Ungemach
    Institute of Pharmacology, Pharmacy and Toxicology
    Veterinary Faculty, University of Leipzig, Leipzig, Germany

    1.   Explanation
    2.   Biological data
         2.1  Toxicological studies
         2.2  Toxicokinetics
         2.3  Developmental toxicity
    3.   Comments
    4.   Evaluation
    5.   References

    1.  EXPLANATION

         Diclazuril, an anticoccidial drug, was evaluated at the
    forty-fifth meeting of the Committee (Annex 1, reference 119), when a
    temporary ADI of 0-20 g/kg bw was established on the basis of a NOEL
    of 3 mg/kg bw per day in a two-year study of toxicity and
    carcinogenicity in mice and a safety factor of 200. The Committee
    noted a lack of toxicokinetic data showing enteric absorption of
    diclazuril in the studies of teratogenicity in rabbits and concluded
    that there was no evidence that the exposure of the dams to diclazuril
    had been sufficient to allow evaluation of the embryotoxicity and
    reproductive toxicity of the drug. The Committee requested a further
    study of teratogenicity in rabbits and evidence that the doses
    administered were sufficiently high for the teratogenic potential of
    diclazuril to be adequately explored.

         Since the previous evaluation, two pilot dose range-finding
    studies including the toxicokinetics of diclazuril and a full-length
    study of teratogenicity with repeated doses have been conducted in
    rabbits. The additional information is summarized and discussed in
    this addendum.

    2.  BIOLOGICAL DATA

    2.1.  Toxicological studies

          Rabbits

         In two separate studies, diclazuril was administered by oral
    gavage as an aqueous suspension to groups of seven rabbits for two
    weeks, in one study at doses of 0, 80, 160, or 320 mg/kg bw per day
    (Dom  et al., 1995) and in the other at 0, 320, 640, or 1280 mg/kg bw
    per day (Dom  et al., 1996). These studies were designed as dose
    range-finding studies for a subsequent investigation of teratogenicity
    and were not performed in compliance with GLP. The animals were
    checked periodically for body weight, food consumption, clinical signs

    of adverse reactions, haematological and serum parameters, organ
    weights, and gross and histopathological appearance (the last in the
    first study only). At day 10, blood samples were taken from three
    randomly selected animals of each group for toxicokinetic analysis.

         No deaths, abnormal clinical signs, or adverse effects on body
    weight or food consumption were reported. Slight changes seen in some
    haematological and serum parameters and in the weights of the heart
    and pancreas were not dose-dependent and can be considered not to be
    related to treatment. A dose-dependent decrease in the weight of the
    gonads, with no gross or histopathological changes, was observed in
    the first study but not at the higher doses in the second study. The
    results indicate that orally administered diclazuril is well tolerated
    by female rabbits at doses up to 1280 mg/kg bw per day.

    2.2  Toxicokinetics

         In order to demonstrate oral absorption and systemic exposure of
    the animals to diclazuril during tthe two-week studies described
    above, serial blood samples were collected 0, 2, 4, 12, and 24 h after
    administration of the dose from three randomly selected animals of
    each group at day 10 in the first study (Sterkens, 1997a) and at day
    13 in the second (Sterkens, 1996). Plasma levels of diclazuril were
    also determined at day 18 in the study of teratogenicity described
    below in two animals per group, 1, 2, 4, 8, and 24 h after treatment
    (Sterkens, 1997b). Diclazuril was analysed by a validated gas
    chromatography method with electrochemical detection (limit of
    quantification, 50 ng/ml). The plasma levels declined very slowly in
    all studies, suggesting saturation and/or poor elimination. The
    quality of the data was not such as to enable an adequate evaluation
    of the Cmax or of the area under the concentration-time curve.
    Table 1 shows the peak plasma levels derived by visual inspection of
    the plasma concentration curves, indicating enteric absorption and
    systemic exposure of female rabbits to orally administered diclazuril.
    Owing to variations between animals and assays and saturation of
    absorption at doses > 640 mg/kg bw, no linear dose-dependency of
    the plasma levels of diclazuril could be observed.

    Table 1. Peak plasma levels (Cmax, g/ml) of  orally administered 
    diclazuril in rabbits

                                                                         
    Reference           Dose (mg/kg bw)
                                                                         
                        80        160      320        640       1280
                                                                         

    Sterkens (1997a)    3.7       3.8      6.40
    Sterkens (1996)                        6.06       8.20      7.41
    Sterkens (1997b)    4.2/7.9            7.4/21.2             13.1/14.9
                                                                         

    Values are means of three determinations

    2.3  Developmental toxicity

          Rabbits

         In a study of developmental toxicity, groups of 18 randomly
    selected pregnant albino rabbits were given an aqueous solution of
    diclazuril by oral gavage at doses of 0, 80, 320, or 1280 mg/kg bw per
    day on days 6-18 of pregnancy. The doses were selected on the basis of
    the findings of the two-week studies and the toxicokinetic analysis
    (Dom  et al., 1995, 1996; Sterkens, 1996, 1997a). The does were
    observed for body-weight changes, food intake, behaviour, and
    mortality. At day 18 of treatment, serial blood samples were taken
    from two animals of each group for analysis of the plasma levels of
    diclazuril. The toxicokinetics were reported separately (Sterkens,
    1997b) and are described above. Animals that aborted were killed and
    necropsied; the remaining animals were killed at day 28 of pregnancy,
    and the uteri, ovaries, and litters were checked for signs of
    embryotoxicity and teratogenicity. The study was carried out in
    compliance with GLP and according to the rules governing medicinal
    products in the European Community and to the Guidelines on Detection
    of Toxicity to Reproduction for Medicinal Products published by the US
    Food and Drug Administration. Appropriate statistical analysis was
    performed with non-parametric tests.

         No compound-related mortality was observed, and diclazuril had no
    adverse effect on body weight or maternal weight gain. Food
    consumption was sightly reduced in all treated groups but increased
    after cessation of treatment. The only clinical signs reported in
    animals at the intermediate and high doses were reduced faeces
    production in three and abortions in four. Uterine weights were
    comparable in all groups. A slight, nonsignificant increase in the
    number of total resorptions was seen at doses up to 320 mg/kg bw per
    day, and the highest dose caused a significant increase in the
    frequency of early and late resorptions and a slight decrease in the
    number of live fetuses and in mean litter size. No substance-related
    effect on preimplantation loss was observed, whereas the frequency of
    post-implantation loss was increased at 1280 mg/kg bw per day. The sex
    ratio and fetal weights were comparable in all groups. Examination of
    fetuses for external, visceral, and skeletal anomalies revealed minor
    abnormalities and variations in those at the low and intermediate
    doses. At 80 mg/kg bw per day, no difference from the vehicle controls
    was observed, but at 320 mg/kg bw per day an increased number of
    fetuses with minor variations, such as plaques in cranial sutures,
    fused sternum, and rudimentary first pair of ribs, was reported.
    Malformations were detected in a few fetuses at doses < 320 mg/kg
    bw per day, but these changes were considered not to be related to
    treatment because of the lack of dependency on dose, their absence in
    previous studies with diclazuril in rabbits at doses up to 160 mg/kg
    bw per day (Annex 1, reference 119), and the appearance of similar
    malforma-tions in untreated historical controls. The group at the
    highest dose showed a pronounced increase in the incidence of
    abnormalities and of major malformations (palatoschisis,
    cheiloschisis, deformation or absence of cranial bones, meningoceles,

    protrusion of brown masses in the brain, hyperossification of
    vertebrae, and sternum deformations), which occurred in 48% of
    fetuses, affecting 92% of the litters. These malformations were
    considered to be related to treatment. Thus, high doses of diclazuril
    (1280 mg/kg bw per day) were teratogenic to rabbits, whereas a dose of
    320 mg/kg bw per day was not teratogenic but slightly fetotoxic. The
    NOEL for developmental toxicity was 80 mg/kg bw per day (Dom  et 
     al., 1997).

    3.  COMMENTS

         The Committee considered additional information on the toxicity
    of diclazuril in non-pregnant female rabbits and on its toxicokinetics
    in pregnant and non-pregnant animals. The teratogenicity of the
    compound was also investigated. The studies were carried out in
    accordance with appropriate standards for study protocol and conduct.

         Two separate two-week pilot studies of toxicity including
    toxicokinetics were performed in female rabbits in order to define
    doses for a definitive study of teratogenicity. Diclazuril was
    administered by gavage to groups of seven animals at doses of 0, 80,
    160, or 320 (first study) and 0, 320, 640, or 1280 mg/kg bw per day
    (second study). The compound was well tolerated at all doses.

         For toxicokinetic analysis, serial blood samples were collected
    over 24 h from three rabbits at each dose on day 10 (first study) or
    day 13 (second study). The plasma concentrations of diclazuril showed
    only minor fluctuations throughout the sampling period and showed no
    clear dependency on dose and no linear increase with increasing dose.
    The results indicate saturable intestinal absorption of orally
    administered diclazuril and demonstrate systemic exposure of the
    treated animals to the test compound.

         In a study of developmental toxicity, groups of 18 pregnant
    rabbits were given daily doses of 0, 80, 320, or 1280 mg/kg bw
    diclazuril by gavage on days 6-18 of pregnancy. All animals were
    killed on day 28. Studies of toxicokinetics performed on day 18
    confirmed the previous findings of saturable absorption and plasma
    levels of diclazuril ranging from 4 g/ml at the lowest dose to
    15 g/ml at the highest. Diclazuril was well tolerated by the dams at
    all doses, with no deaths. The only clinical signs observed were
    slightly reduced food intake during treatment, reduced faeces
    production, and a few abortions at doses of 320 and 1280 mg/kg bw per
    day. Examination of the litter parameters revealed no compound-related
    adverse effects at doses up to 320 mg/kg bw per day, whereas the
    incidences of resorption and postimplantation loss were significantly
    increased at the highest dose. Examination of the fetuses revealed a
    low incidence of minor compound-related abnormalities at 320 mg/kg bw
    per day; at 1280 mg/kg bw per day, treatment with diclazuril
    significantly increased the number of abnormalities and induced
    malformations in 48% of the fetuses. The Committee concluded that
    diclazuril is teratogenic in rabbits at high doses, whereas doses up

    to 320 mg/kg bw per day are slightly fetotoxic but not teratogenic.
    The NOEL for developmental toxicity was 80 mg/kg bw per day.

    4.  EVALUATION

         The additional studies complied with the request of the Committee
    by showing enteric absorption and exposure of pregnant rabbits to
    diclazuril which was sufficient to enable the teratogenicity of the
    drug to be assessed. On the basis of these findings, the Committee
    considered the NOEL for diclazuril of 3 mg/kg bw per day in the
    two-year study of toxicity and carcinogenicity in mice reviewed at the
    forty-fifth meeting, based on histopathological changes in the lungs,
    as the most appropriate toxicological end-point for establishing an
    ADI. An ADI of 0-30 g/kg bw was established by applying a 100-fold
    safety factor to the NOEL. This provides a margin of safety of at
    least 10 000 for the teratogenic effect of the compound in rabbits.

    5.  REFERENCES

    Dom, P., Benze, J., Coussemont, W., Fransen, J., Lampo, A. & van
    Cauteren, H. (1995) Two-week oral toxicity study in rabbits.
    Unpublished report No. R064433, Exp. No. 3682 from Janssen Research
    Foundation. Submitted to WHO by Janssen Pharmaceutica NV, Beerse,
    Belgium.

    Dom, P., Benze, J., Coussemont, W., Vandenberghe, J., Lampo, A. & van
    Cauteren, H. (1996) Two-week oral toxicity study in rabbits.
    Unpublished report No. R064433, Exp. No. 3762 from Janssen Research
    Foundation. Submitted to WHO by Janssen Pharmaceutica NV, Beerse,
    Belgium

    Dom, P., Benze, J., Coussemont, W., Fransen, J., Lampo, A. & van
    Cauteren, H. (1997) Oral (gavage) study for effects on embryo-foetal
    development (segment II) with R064433 (Exp. No. 3373). Unpublished
    report from Janssen Research Foundation. Submitted to WHO by Janssen
    Pharmaceutica NV, Beerse, Belgium

    Sterkens, P. (1996) Toxicokinetics of diclazuril (R064433) in the
    albino rabbit in a two-week oral toxicity study (Exp. No. 3762).
    Unpublished protocol No. R064433/FK 2119 from Janssen Research
    Foundation. Submitted to WHO by Janssen Pharmaceutica NV, Beerse,
    Belgium

    Sterkens, P. (1997a) Non-clinical pharmacokinetics report: Addendum to
    the toxicity report exp. No. 3682. Unpublished report No. R064433/FK
    2037 from Janssen Research Foundation. Submitted to WHO by Janssen
    Pharmaceutica NV, Beerse, Belgium

    Sterkens, P. (1997b) Non-clinical pharmacokinetics report: Addendum to
    the toxicity report exp. No. 3373. Unpublished report No. R064433/FK
    2432 from Janssen Research Foundation. Submitted to WHO by Janssen
    Pharmaceutica NV, Beerse, Belgium
    


    See Also:
       Toxicological Abbreviations
       Diclazuril (WHO Food Additives Series 36)
       DICLAZURIL (JECFA Evaluation)