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    DIMINAZENE

    First draft prepared by
    Dr M. Miller
    Center for Veterinary Medicine
    Food and Drug Administration
    Rockville, Maryland, USA

    1.  EXPLANATION

         Diminazene is a veterinary drug with a long history of use for
    the treatment of trypanosomiasis and babesiosis. Diminazene was
    evaluated at the thirty-fourth meeting of the Committee (Annex 1,
    reference 85). At that time the Committee was unable to establish an
    ADI and suggested that additional genotoxicity and teratogenicity
    studies should be performed before bringing the compound to the
    Committee for re-evaluation. This monograph addendum, which
    supplements the information provided in the original toxicological
    monograph (Annex 1, reference 86), summarizes the data that have
    become available since the previous evalution.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

         The excretion of diminazene into milk was examined in four
    lactating cows, each treated with one i.m. dose of diminazine (3.56 mg
    diminazene/kg bw). The diminazene content of milk collected at 7.5,
    24, 31.5, 48, 55.5, 72, 240, 360, and 480 hours after application was
    quantitated by an HPLC method with a detection limit of 50 ng/ml. The
    results showed that the diminazene concentration in milk from all 4
    cows was below the detection limit of the assay at all time points
    (Klatt & Schmidt, 1991a).

         Residue levels of diminazene were determined in plasma and
    tissues of 14 young cattle following one i.m. administration of 3.56
    mg diminazene/kg bw. Blood plasma levels were measured for 35 days
    after application and tissues residue analyses were conducted in
    liver, kidney, muscle, and injection site muscle at 21, 28, and 35
    days post-treatment. The study reported that a plasma level of 1250
    ng/ml was acheived one day after drug administration. This value
    decreased to 350 ng/ml after 7 days. After 25-28 days, plasma levels
    of diminazene were at or below the detection limit of the assay (50
    ng/ml). The highest tissue concentration of diminazene occurred at
    the injection site followed by liver and kidney. The lowest values
    were found in the muscle (Klatt & Schmidt, 1991b).

    2.2  Toxicological studies

    2.2.1  Special studies on embryotoxicity

    2.2.1.1  Rats

         The embryotoxicity of diminazene dissolved in distilled water was
    studied in mated female Wistar rats. Prior to the start of the study,
    the females were mated with fertile males. Pregnant animals
    (determined by detection of spermatozoa in the vaginal smear) were
    randomly assigned to 4 groups of 22-24 animals each. The test groups
    were gavaged daily on the seventh to sixteenth days of pregnancy with
    5 ml of distilled water (control), or 200, 400, or 800 mg of
    diminazene/kg bw/day. Behaviour and general physical condition were
    assessed daily. Feed consumption was monitored continuously and body
    weight was checked once weekly. On day 21 of pregnancy, the dams were
    killed and delivered by caesarean section. The uteri were opened and
    live and dead fetuses, concepta under resorption, placentas, and
    corpora lutea were counted and examined microscopically.

         Increased salivation was observed sporadically in several treated
    animals at all doses. This effect was attributed to local irritation
    resulting from the compound's administration. Doses of 200 and 400

    mg/kg bw/day did not affect general physical condition, feed
    consumption, or body weight development. The 800 mg/kg bw/day dose was
    maternotoxic, causing a decrease in food consumption, an increase in
    spleen weight, and the death of five dams.

         No effects on intrauterine development of the conceptuses were
    observed or the low and middle doses. Fetal development in dams
    treated with 800 mg diminazene/kg bw/day was retarded, as indicated by
    a slight increase in the number of fetuses with body weights under 3
    g, poor skeletal ossification and low placental weights. Morphological
    examination of fetuses from the treated dams revealed no
    malformations. The incidence of minor anomalies and variations in
    fetal development in the compound-treated groups were no higher than
    incidences observed in the contemporary controls and were also within
    the range of historical controls. Because materno- and embryotoxicity
    were observed at 800 mg/kg bw/day, the NOEL was 400 mg/kg bw/day
    under the study conditions (Baeder  et al., 1991).

         Diminazene dissolved in deionized water was administered to
    pregnant rats daily by gavage on days 8-15 of pregnancy at dose levels
    of 0 (vehicle only), 100, 250, 500, or 1000 mg/kg bw/day. On day 21 of
    pregnancy, the dams were killed and the number of implants,
    resorptions and live fetuses counted. All fetuses were examined by
    routine teratological methods. A significant increase in fetal
    resorptions and a decrease in fetal body weights were observed at 1000
    mg/kg bw/day dose. No significant increase in the incidence of fetal
    anomalies was observed in external, skeletal, and internal
    examinations even at the maternally toxic dose of 1000 mg/kg. These
    data suggest that diminazene is not teratogenic. The NOEL was 500
    mg/kg bw/day (Yoshimura, 1990).

    2.2.2  Special studies on genotoxicity

         Table 1 summarizes the results of genotoxicity assays on
    diminazene.

    
    Table 1.  Results of genotoxicity assays with diminazene
                                                                                  
    Test system    Test object         Concentration   Results    Reference
                                                                                  

    Ames test1      S.typhimurium      0-500 g/ml     Negative   Muller, 1989
                   TA100, 1535,
                   1537, 1538, 98
                    E. coli WP2uvrA

    Micronucleus   Mouse bone
    test2          marrow              0 and 1500 mg   Negative   Muller, 1988a

    Forward        Fetal hamster       10-100 g/ml    Negative   Muller,
    mutation assay lung V79            (-S9 mix)3                1988b
                   (HGPRT locus)       10-150 g/ml    Negative
                                       (+S9 mix)4
                                                                                  

    1    Both with and without rat liver S9 fraction.
    2    Reviewed at the thirty-fourth Meeting of the Committee.
    3    Ethylmethansulfonate was used as the positive control.
    4    9,10-dimethyl-1,2-benzanthracene was used as the positive
         control.
    
    3.  COMMENTS

         Diminazene was negative in the micronucleus test and in bacterial
    and mammalian genotoxicity assays. These data together with the data
    from the subchronic toxicology studies, which showed no lesions
    predictive of a carcinogenic response, suggest that there is no
    concern for the carcinogenic potential of the compound.

         The embryotoxicity of diminazene was examined in female Wistar
    rats dosed by gavage with 0, 200, 400, or 800 mg diminazene/kg bw/day
    on days 7 to 16 after mating. A dose of 800 mg/kg bw/day was
    maternotoxic, causing a decrease in food consumption and increased
    spleen weight and death. This dose also caused retarded fetal
    development. The NOEL for this study was 400 mg/kg bw/day. In another
    embryotoxicity study, diminazene was administered to rats by gavage on
    days 8 to 15 of pregnancy at dose levels of 0, 100, 250, 500, or 1000
    mg/kg bw/day. The 1000 mg/kg bw/day dose produced maternotoxicity and
    a significant increase in the number of fetal resorptions and a
    decrease in fetal body weights. The NOEL for this study was 500 mg/kg
    bw/day. No fetal malformations were detected in either study.

         The previous monograph on diminazene (Annex 1, reference 86)
    summarized the toxicity studies in rats, dogs, and various
    food-producing animals. At low doses, i.m. administration of
    diminazene produced central nervous system lesions in several species
    including cows, donkeys, camels, and dogs. In dogs fed diminazene at
    20 or 60 mg/kg bw/day for nine months, the higher dose caused brain
    damage, testicular atrophy and death. Rats dosed by gavage with 63 or
    160 mg/kg bw/day for 3 months showed no signs of toxicity. This
    indicates that the rat is not a sensitive model for evaluating the
    toxicity of diminazene, including reproductive toxicity. The NOEL in
    this study was 20 mg/kg bw/day.

    4.  EVALUATION

         In keeping with general principles for the evaluation of
    veterinary drugs with a long history of use described at the fortieth
    Meeting of the Committee (Annex 1, reference 104), an ADI of 0-100
    g/kg bw/day was established for diminazene based on the NOEL of 20
    mg/kg bw/day in the nine-month study in dogs and using a 200-fold
    safety factor. The Committee chose this safety factor to compensate
    for the inadequacies in the design of the study.

    5.  REFERENCES

    BAEDER, C., MAYER, D. & LANGER, K.H. (1991). Oral embryotoxicity study
    of Berenil in Wistar rats (Segment II). Unpublished report No.91.1190
    from Pharma Development Toxicology, Hoechst Aktiengesellschaft,
    Frankfurt, Germany. Submitted to WHO by Hoechst AG, Frankfurt,
    Germany.

    KLATT, P. & SCHMIDT, D. (1991a) Report on the excretion of diminazene
    aceturate in the milk of cows after one intramuscular administration
    of the commercial preparation berenil(R) for veterinary use.
    Unpublished report No. K1 90/1 from Abt. Forschung Tiergesundheit,
    Hoechst AG, Frankfurt, Germany. Submitted to WHO by Hoechst AG,
    Frankfurt, Germany.

    KLATT, P. & SCHMIDT, D. (1991b) Report on blood plasma level
    determinations and residue studies in organs of cattle after one
    intramuscular administration of the commercial preparation
    berenil(R) for veterinary use. Unpublished report No. K1 90/1 from
    Abt. Forschung Tiergesundheit, Hoechst AG, Frankfurt, Germany.
    Submitted to WHO by Hoechst AG, Frankfurt, Germany.

    MULLER, W. (1988a). Diminazendiaceturat (Berenil) micronucleus test in
    male and female NMRI mice after oral administration. Unpublished
    report No.88.0248 from Pharma Research Toxicology and Pathology,
    Hoechst Aktiengesellschaft, Frankfurt, Germany. Submitted to WHO by
    Hoechst AG, Frankfurt, Germany.

    MULLER, W. (1988b). Berenil: Detection of gene mutations in somatic
    cells in culture HGPRT-test with V79 cells. Unpublished report
    No.88.0990 from Pharma Research Toxicology and Pathology, Hoechst
    Aktiengesellschaft, Frankfurt, Germany. Submitted to WHO by Hoechst
    AG, Frankfurt, Germany.

    MULLER, W. (1989). Berenil: Study of the mutagenic potential in
    strains of  Salmonella typhimurium (Ames test) and  Escherichia coli.
    Unpublished report No.88.1425 from Pharma Research Toxicology and
    Pathology, Hoechst Aktiengesellschaft, Frankfurt, Germany. Submitted
    to WHO by Hoechst AG, Frankfurt, Germany.

    YOSHIMURA, H. (1990). Teratological assessment of the antiprotozoal,
    diminazene diaceturate, in rats.  Toxicol Lett., 54: 55-59.


    See Also:
       Toxicological Abbreviations
       Diminazene (WHO Food Additives Series 25)
       DIMINAZENE (JECFA Evaluation)