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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




    AZAPERONE (addendum)

    First draft prepared by
    G. Kirby, L. Ritter
    Canadian Network of Toxicology Centres
    Department of Environmental Biology
    University of Guelph, Guelph, Ontario, Canada
    and
    Dr L.-E. Appelgren
    Department of Pharmacology and Toxicology
    Faculty of Veterinary Medicine
    The Swedish University of Agricultural Science, Biomedical Centre,
    Uppsala, Sweden

    1.   Explanation
    2.   Biological studies
         2.1  Biotransformation
         2.2  Structural similarity to known carcinogens
         2.3  Reproductive toxicity
    3.   Comments
    4.   Evaluation
    5.   References

    1.  EXPLANATION

         Azaperone, a butyrophenone neuroleptic tranquillizer, was
    reviewed at the thirty-eighth and forty-third meetings of the
    Committee (Annex 1, references 97 and 113). A temporary ADI of 0-3
    g/kg bw was established at the forty-third meeting, which requested
    the following information before subsequent review:

    1.   Results of studies to determine the genotoxic potential of the
         metabolites of azaperone, which have been reported to be
         mutagenic in  Salmonella spp.

    2.   Evidence to support the claim that azaperone or its degradation
         products are not structurally similar to known carcinogens

    3.   Results of a study to assess the effects of azaperone on
         reproduction and fertility in male laboratory animals.

    This monograph addendum summarizes the data that have become available
    since the previous evaluation.

    2.  BIOLOGICAL STUDIES

    2.1  Biotransformation

         No further studies of mutagenicity were carried out, as the
    results of tests in  S. typhimurim with metabolic activation provided
    by rat liver microsomes (Ames' test) were considered to reflect the
    mutagenic potential of azaperone and its metabolites. To support this

    statement, the biotransformation of azaperone was studied  in vitro 
    with microsomal fractions from the livers of Arochlor 1254-treated
    (500 mg/kg bw) male Wistar rats in order to identify the metabolites
    formed under the conditions of Ames' test. Biotransformation was
    assessed by incubating 14C-azaperone (0.01, 0.1, 0.5, or 2.0
    mmol/plate) with rat liver fractions in buffer containing histidine,
    biotin, nutrient broth and a NADPH generating system at 37C for 30
    and 120 min. Samples were analysed by radio-high-performance liquid
    chromatography, and the major metabolites were identified by
    comparison of the chromatograms with those for unlabelled reference
    compounds  and by liquid scintillation counter and mass spectrometry
    of representative samples. Five metabolites were characterized, which,
    with unmetabolized azaperone, corresponded to 91100% of the total
    radiolabel applied. The main metabolite was an oxidized pyridinyl
    derivative. The other metabolites, which occurred in small quantities,
    were identified as the nor-piperazine derivative, the  N-oxide, the
    alcohol metabolite (azaperol), and a second oxidized pyridinyl
    derivative (Vermeir, 1997). The Committee noted that almost all of the
    primary metabolites previously found  in vivo in rats and pigs were
    produced under the conditions of Ames' test and concluded that the
    mutagenic potential of azaperone and its metabolites in bacteria had
    been adequately evaluated in the previously conducted tests and
    particularly in the second, more throrough study described in the
    previous evaluation, in which the presence of metabolites was
    questioned. In view of this new information, the weight of evidence is
    that azaperone is unlikely to be genotoxic.

    2.2  Structural similarity to known carcinogens

         A brief discussion of the lack of similarity of azaperone and its
    metabolites with known carcinogens was included in the previous
    evaluation. It was noted that the only reactive subgroup on azaperone,
    a neuroleptic of the butyro-phenone class, and its metabolites is
    pyridine, a heterocyclic moiety (Sanderson & Earnshaw, 1991; see
    Figure 1). Moreover, the report noted that human neuroleptics of the
    same class had no structural similarity to known carcinogens. The
    argument was reinforced by the lack of genotoxicity for azaperone and
    other butyrophenones, including haloperidol, pimozide, bromperidol,
    and bromperidol decanoate. Finally, apart from unspecified
    prolactin-mediated effects on the pituitary and mammary gland, no
    evidence of carcinogenicity had been found for the neuroleptics
    haloperidol, pimozide, and bromperidol (references were not included).
    Mammary gland stimulation is an epigenetic change induced by dopamine
    antagonists at doses lower than those required for promotion in
    prolactin-releasing tumours. Azaperone, however, is a relatively weak
    prolactin-releasing agent in rats, stimulating the mammary gland at
    doses > 40 mg/kg bw. There is therefore no evidence that azaperone
    and its degradation products are similar to known carcinogens.

    FIGURE 1

    2.3  Reproductive toxicity

         Studies were conducted to evaluate the effect of azaperone on
    reproduction and fertility in male rats. Groups of 24 male Wistar rats
    were given doses of 5, 20, or 80 mg/kg bw daily by gavage for 74 days.
    Signs of toxicity (severe sedation and ptosis, decreased body weight
    and feed consumption) were observed at the highest dose throughout
    treatment, and slight to moderate sedation and ptosis were also seen
    at the intermediate dose. Mild, possibly drug-related haematological
    changes (decreased thrombocytes) were seen, with alterations in serum
    biochemistry (increased chloride and decreased calcium, total protein,
    albumin, cholesterol, triglycerides, and phopholipids). The weight of
    the thymus was slightly increased, and that of the adrenal glands was
    decreased. Copulation and fertility rates and pre-coital intervals
    were comparable in groups at different doses, and maternal and litter
    parameters were not affected. No adverse effects on body weight, food
    consumption, or number of corpora lutea were observed in untreated
    females bred to treated males. Furthermore, embryofetal development
    (i.e. numbers of live and dead fetuses, mean litter size, number of
    implantations, number of resorptions, and pre- and post-implantation
    loss) was not adversely affected. There were no dose-related
    teratogenic effects in the offspring of untreated females mated with
    male rats at the highest dose. It was concluded that azaperone had no
    adverse effects on fertility in males or on embryofetal development in
    the offspring of untreated females bred to treated males. The NOAEL
    was 5 mg/kg bw per day on the basis of paternal toxicity (Dom, 1997).

    3.  COMMENTS

         The Committee considered new studies to address the issues that
    had been raised at the forty-third meeting. The studies were carried
    out according to appropriate standards for study conduct and
    protocol. A study was performed to address the issue of the genotoxic
    potential of azaperone metabolites. Various concentrations of
    radiolabelled azaperone were incubated with 9000   g  fractions

    prepared from the livers of Arochlor-treated rats to determine the
    metabolites formed under the conditions used in Ames' test. The
    metabolites were isolated and found to be similar to those produced in
    the target species. The Committee concluded that the results of the
    Ames' tests with azaperone that were reviewed at the thirty-eighth and
    forty-third meetings were adequate to assess the genotoxic potential
    of azaperone and its metabolites in bacteria. Taking into account the
    results of all of the genotoxicity studies performed, the Committee
    concluded that azaperone is unlikely to be genotoxic.  The Committee
    reviewed a brief report on the lack of structural similarity of
    azaperone and its metabolites with known carcinogens. It was noted
    that azaperone, a neuroleptic of the butyrophenone class, and its
    metabolites contain no reactive subgroups. The argument was reinforced
    by the lack of evidence for genotoxicity in a battery of studies. The
    Committee concluded that azaperone is unlikely to be carcinogenic.    
    Studies were conducted to evaluate the effects of azaperone on
    reproduction and fertility in male rats. Azaperone was administered by
    gavage daily to male rats for 74 days at doses of 5, 20, or 80 mg/kg
    bw per day. Signs of toxicity, including severe sedation and ptosis,
    decreased body weight and feed consumption, were observed in animals
    at the highest dose throughout treatment. Slight to moderate sedation
    and ptosis were seen at the intermediate dose.  Copulation and
    fertility rates and pre-coital intervals were comparable in all
    groups. There were no adverse effects on male fertility or on
    embryofetal development in the offspring of untreated females mated to
    treated males. The NOEL was 5 mg/kg bw per day on the basis of
    paternal toxicity.

    4.  EVALUATION

         The Committee concluded that the pharmacological effects of
    azaperone are the most relevant for determining an ADI. Therefore, an
    ADI of 06 g/kg bw was established on the basis of the NOAEL of 630
    g/kg bw for neurobehavioural effects in dogs after oral
    administration (as recommended by the forty-third meeting of the
    Committee) and a safety factor of 100.

    5.  REFERENCES

    Dom, P. (1987) Study on male fertility and on effects on embryo-foetal
    development in SPF Wistar rats with R001929 by oral gavage (Report No.
    R001929). Unpublished report from Janssen Research Foundation.
    Submitted to WHO by Janssen Animal Health, Beerse, Belgium.

    Sanderson, D.M. & Earnshaw, C.G. (1991) Computer prediction of
    possible toxic action from chemical structure; the DEREK system.
     Hum. Exp. Toxicol., 10, 261-273.

    Vermeir, M. (1997) A study on the metabolism of azaperone in liver
    9000g supernatant fractions of Arochlor pretreated rats (Report No.
    R001929/FK2533). Unpublished report from Janssen Research Foundation.
    Submitted to WHO by Janssen Animal Health, Beerse, Belgium.
    


    See Also:
       Toxicological Abbreviations
       Azaperone (WHO Food Additives Series 29)
       Azaperone (WHO Food Additives Series 34)
       AZAPERONE (JECFA Evaluation)