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    DIMINAZENE

    1.  EXPLANATION

         Diminazene aceturate, usually referred to simply as diminazene
    (other names are 4,4'-(diazoamino) dibenzamidine diaceturate, berenil)
    is used in tropical countries for the treatment of animal
    trypanosomiasis and babesiosis, usually as an intramuscular injection
    of 3-5 mg/kg. It is said to be effective in canine, ovine and bovine
    babesiosis and, unlike some drugs, is less susceptible to relapse. It
    may also possess antibacterial properties. This compound has not
    previously been evaluated by the Joint FAO/WHO Expert Committee on
    Food Additives.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

         There are few data available on the distribution and excretion of
    diminazene in laboratory animals and none on biotransformation. In the
    rat, absorption appeared to be moderate after oral administration. For
    example, after a dosage of 100 mg/kg bw, blood levels were about
    0.25-2.25 g/ml at 0-2 hours, 1.85 g/ml at 7 hours and 0.5-0.6 g/ml
    at 28-31.5 hours, whereas after subcutaneous dosing levels were 26.35
    g/ml at 0-2 hours, 6 g/ml at 7 hours and zero at 28-48 hours
    (Raether  et al., 1972).

         In a relay study where rats were given liver from a calf dosed 7
    days previously with 3.67 mg/kg diminazene (to give an oral dose to
    the rats of 0.28-0.32 mg/kg), a large proportion of the dose was
    eliminated in the urine (21-33%) and in the feces (37-48%). Only small
    amounts were found in the bile (0.24-0.43%). It was estimated that
    25-35% of the available dose was absorbed but the fractions present as
    parent compound and/or metabolites were unknown (Kellner  et al.,
    1985).

         When 3.5 mg/kg bw was given to rabbits by intramuscular
    injection, biphasic pharmacokinetics with maximum blood levels at 15
    minutes (1.3 g/ml) and 3 hours (0.116 g/ml) were noted. The highest
    tissue levels at 7 days were in the liver (40 ppm), brain (2.5 ppm)
    and kidney (3 ppm). Levels were low in other organs (0.4-2.0 ppm)
    including muscle (2.1 ppm). By 7 days, 40-50% of the dose had been
    excreted in the urine with 8-20% in the feces, the latter indicative
    of biliary excretion (Gilbert & Newton, 1982; Gilbert, 1983).

         In the monkey  (Maccaca mulatta), peak plasma levels were
    attained 25 minutes after an intramuscular dose of 20 mg/kg bw
    diminazene and 6 hours after an oral dose of 40 mg/kg bw. The
    elimination half-life was 15 hours for the oral dose and showed
    monophasic kinetics. For the intramuscular dose, biphasic kinetics
    were noted with half-lives of 1-2 hours and 18-19 hours (Raether  et
     al., 1974).

         When 2 calves were dosed intramuscularly with 3.5 mg/kg bw
    diminazene , maximum blood levels were reached after 15 and 45
    minutes. Plasma clearance was biphasic with half-lives of 2 and 188
    hours for each phase. By day 7, 47% of the dose had been excreted in
    the urine and 7.1% in the feces. This was indicative of biliary
    excretion in this species. Residue levels were low in edible tissues
    when the calves were killed on days 7 or 20 after dosing, except for
    the kidney, liver and myocardium (54.7, 75.5 and 6.6 ppm at day 7 and
    12.1, 24.4 and 2.9 ppm, respectively, at day 20). Levels in skeletal
    muscle were low at both days (0.5 and 0.3 ppm, respectively) (Kellner
     et al., 1985). The results were similar to those noted in another
    study where 3.5 mg/kg bw intramuscular diminazene produced peak plasma

    levels at 0.5 hours and a maximum level of 4.5 g/ml (Klatt & Hajdu,
    1971; Klatt & Hajdu, 1976). Milk collected at 6 and 24 hours contained
    0.2-0.5 g/ml diminazene but the level fell to 0.1-0.2 g/ml by 30
    hours and was undetectable at 48 hours following dosing (limit of
    detection 0.07 g/ml). Two metabolites were detected in the urine of
    calves; p-aminobenzamidine (22%) and p-aminobenzamidine (4%). The
    remainder was the parent compound (74%) and approximately 80% of the
    intramuscular dose was collected in the urine (Klatt & Hajdu, 1971).
    In cattle, diminazene appeared to bind irreversibly to blood proteins
    including hemoglobin (Alvi  et al., 1985).

         In goats given 3.5 mg/kg bw diminazene intramuscularly, systemic
    availability was calculated to be 44-46% (Aliu  et al., 1984). After
    achieving a peak plasma level within one hour of administration,
    plasma decay was triexponential. The plasma half-life (14-30h) was
    longer than in sheep (10-13h) but shorter than in cattle (40-138h).
    Following administration of diminazene (3.5 mg/kg bw) to sheep by the
    intramuscular route, maximum plasma levels of 6.3-7.6 g/ml were
    attained in 20-45 minutes and plasma protein binding was high (65-85%)
    and concentration-dependent. Based on the pharmacokinetic data in
    sheep and goats, withdrawal periods of around 26 days were recommended
    for tissue depletion although these times did not appear to take
    account of retention by any specific issues.

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         There are few acute toxicity data available with diminazene in
    the usual laboratory species. In the mouse (NMRI), in a preliminary
    study for a micronucleus test (see section 2.2.5), an oral dose of
    1500 mg/kg bw to 3 males and 3 females resulted in a single death
    (female). Signs of toxicity included increased spontaneous activity,
    tactile hyperesthesia and uncoordinated gait (Muller, 1988). The
    LD50 by the subcutaneous route in the mouse was 258 mg/kg bw
    (Berenil-Toxicology, 1988). Mice tolerated doses of 75 mg/kg bw but
    unfortunately no other details including those of route and vehicle
    were specified (Harant, 1979).

         Brain damage has been reported in asses and dogs given the drug
    (Aburu  et al., 1984; Losos & Crockett, 1969). In the dog, spastic
    paralysis, opisthotonos and nystagmus with involuntary running
    movements were noted in dogs treated 24-72 hours previously with
    diminazene. Intramuscular hemorrhage and diffuse intramuscular edema
    were noted at the injection site (Losos & Crockett, 1969). Similar
    signs accompanied by vomiting and death have been reported in dogs
    given 30-35 mg/kg bw (Fussganger & Bauer, 1958) although the
    recommended therapeutic dose is 3.5-8.0 mg/kg bw. In a small
    experimental study, groups of 2 dogs of unspecified strain were given
    single intramuscular doses of 10, 15, 20 or 60 mg/kg bw diminazene.

    Animals given 20-60 mg/kg bw died 36-54 hours after administration;
    clinical signs were the same as those reported for dogs treated
    therapeutically with the drug. Extensive hemorrhagic malacia of the
    brain stem was noted involving the mesencephalon and diencephalon
    (Losos & Crockett, 1969).

         Intramuscular injections of 8 mg/kg bw were well tolerated except
    in the buffalo cow where "quivering" and restlessness followed dosing.
    The animal recovered after an intravenous dose of dextrose (Verma  et
     al., 1970). Buffalo calves tolerated intramuscular doses of 20 mg/kg
    bw diminazene. No acute effects occurred in cattle when given 6 times
    the recommended dose (21 mg/kg bw) (Fairclough, 1963). Camels also
    tolerated diminazene when given intramuscularly at the recommended
    therapeutic dose of 3.5 mg/kg bw (Schillinger & Rottcher, 1986).

         From a total of 154 donkeys usually given 0.5 mg/kg bw every 3
    months to protect against trypanosome infection, a group of 31 became
    infected with T. brucei and were treated with 7 mg/kg bw diminazene.
    Around 48 hours later, 4 animals became weak and displayed staggering
    and ataxia and by 96 hours, 29 donkeys had developed CNS effects; 6
    died. The survivors recovered after 14-30 days. Post-mortem
    examination revealed macroscopic and microscopic hemorrhages in the
    cerebellum (Boyt  et al., 1971).

         Hepatoxicity in the dog has also been reported after single doses
    of 3.5 mg/kg bw but pre-existing liver disease could not be excluded
    as a contributing factor (Opping, 1969). Diminazene was also
    hepatotoxic to camels; 10 or 40 mg/kg bw given to 3 dromedary camels
    by the intramuscular route resulted in hyperaesthesia, salivation,
    intermittent convulsions, frequent urination and defecation and
    sweating. At necropsy, the lungs were congested and edematous while
    the liver was congested and hemorrhagic with evidence of fatty change.
    Congestion of the brain and urinary bladder was noted along with
    hemorrhaging and congestive changes in the kidneys and heart (Homeida
     et al., 1981).

    2.2.2  Short-term studies

    2.2.2.1  Rats

         Groups of 20 male and 20 female Wistar rats were fed diets
    containing 630, 1600 or 4000 ppm diminazene for 5 weeks after which
    the levels were increased by 50% to 945, 2400 and 6000 ppm. After 3
    months, 10 rats from each group were sacrificed and the remainder fed
    the treated diets for a total of 9 months. Two other groups of 15 male
    and 15 female rats were given gavage doses of 63 and 160 mg/kg bw/day
    diminazene for 3 months when 5 rats of each sex were killed and the
    remainder given the drug for a further 3 months. No signs of toxicity

    were noted and no effects on food intake, body weights, hematology,
    blood glucose or urine were observed. There were no macro- or
    microscopic drug-related effects in any of the major organs (Baeder
     et al., 1975).

    2.2.2.2  Dogs

         Diminazene was given to dogs for periods of up to 10 days. It was
    reported that "ages varied from 6 months to 7 years and the breed from
    purebred Alsatian to nondescript mongrels". When given 3.5 mg/kg bw on
    two successive days by the intramuscular route, no signs of toxicity
    were observed. A group of 3 males and 3 females were given 3.5 mg/kg
    bw/day intramuscularly until signs of toxicity developed. Two dogs of
    each sex showed signs of CNS toxicity on days 6-9 and all 4 were
    killed on day 10; the remaining 2 dogs were unaffected. A further
    group of 3 males and 3 females were treated in a similar manner with
    10.5 mg/kg bw/day diminazene. All the dogs died on days 3-5. All the
    affected animals showed cerebellar lesions characterized by
    hemorrhages and areas of malacia. They were generally bilateral (Naude
     et al., 1970).

         In an oral study, groups of 3 male and 3 female beagle dogs were
    given capsules containing 0, 20 or 60 mg/kg bw/day diminazene, 7 days
    per week for 9 months. One dog of each sex died at the high dose. Body
    weights were also reduced and general condition was poor in high dose
    males. There were, however, no specific effects on hematology, urine,
    serum analysis and blood glucose levels. In animals given 60 mg/kg
    bw/day diminazene there were foci of softening in the brain stem and
    cerebellum. There was also testicular atrophy and prostatic
    dysfunction (Scholz & Brunk, 1969).

         In a study where 10 dogs and 2 long-tailed monkeys were given an
    unspecified dose of diminazene by an unspecified route for an
    unspecified period of time, 7 dogs and both monkeys died within the
    first week. Paralysis and lameness were noted prior to death.
    Hemorrhagic necrosis in the CNS, mainly in the brain, was evident at
    necropsy. The lesion was confined to the brain stem and cerebellum and
    was thought to be due to necrosis of the capillaries and arteries
    (Schmidt  et al., 1977).

    2.2.2.3  Oxen

         An ox given intramuscular diminazene, 7 mg/kg bw/day for 15 days,
    developed CNS-like signs of toxicity including ataxia and muscle
    tremors, and the animal died on day 18. Necropsy revealed mild
    swelling of the thalamic glia (Naude  et al., 1970).

    2.2.3  Long-term/carcinogenicity studies

         No data are available.

    2.2.4  Reproduction studies

         No data are available.

    2.2.5  Special studies on genotoxicity

         There are few conventional studies available to assess the
    genotoxic potential of diminazene, although there are a number of
    publications suggesting a direct effect on DNA. It has been
    demonstrated that diminazene is not a DNA intercalating agent (Waring,
    1970 a-d; Bernard & Riou, 1980).

         Treatment of  Trypanosoma cruzi kinetoplast DNA with diminazine
    resulted in an increased proportion of double-branched circular DNA
    molecules. Replication of the DNA appeared to be blocked at specific
    sites. Treatment resulted in a changed electron microscopic appearance
    of the DNA which displayed a "lampbrush-like" appearance, possibly due
    to the formation of replicating circular DNA molecules resulting in
    specific blocking of the kinetoplast DNA replication (Brack  et al.,
    1972a and b). Berenil appears to bind to the trypanosome DNA including
    DNA denatured by heat or other agents (Festy  et al., 1970a and b).
    The significance of these findings for the possible genotoxic effects
    of diminazine on mammalian DNA is unknown.

         Diminazene-induced respiration deficient petite mutations in
     Saccharomyces cerevisiae (Mahler & Perlman, 1973; Perlman & Mahler
    1973; Villa & Juliani, 1980). It is thought that diminazene binds to
    yeast mitochondrial DNA (Perlman & Mahler 1973; Mahler 1973). Again,
    whether these findings are relevant to mammals is unknown although
    they appear to be involved in the trypanocidal activity of the drug
    possibly in association with its inhibitory effects on diamine oxidase
    and S-adenosylmethionine decarboxylase (Balana-Fouce  et al., 1986).

         In a micronucleus test, groups of 5 male and 5 female NMRI mice
    were given gavage doses of 0 or 1500 mg/kg bw (highest tolerated doze)
    of diminazene in water. Cyclophosphamide (50 mg/kg bw) served as a
    positive control. Mice were killed 24, 48 and 72 hours after dosing.
    There was no increased incidence of micronucleated polychromatic
    erythrocytes in any of the test groups (1000 polychromatic erythrocyes
    per mouse examined) but there was a marked increase in incidence in
    animals given cyclophosphamide (Muller, 1988).

    2.3  Observations in humans

         Ninety-nine patients who had been treated 12-109 months earlier
    with diminazene for early stage African trypanosomiasis were traced
    and subjected to a medical examination. Each patient had received 3
    doses of 5 mg/kg bw diminazene at one or two day intervals. Although

    diminazene is known to produce a number of side effects in humans,
    including pain in the soles of the feet, pyrexia, nausea, vomiting and
    paralysis, no adverse effects were noted in the group (Abaru,  et al.,
    1984).

    3.  COMMENTS

         No carcinogenicity studies on diminazene and only two
    conventional mutagenicity studies were available. Trypanosome
    kinetoplast studies suggested an interaction of diminazene with DNA
    but the relevance to genotoxicity was unknown; the substance was not
    an intercalating agent. Diminazene induced respiration-deficient
    petite mutations in yeast but gave negative results in the mouse
    micronucleus test. No teratology or other studies were available for
    use in assessing the reproductive toxicity of the compound. Acute
    toxicity studies in mice showed some evidence of effects on the
    central nervous system at high doses. In dogs, cattle and donkeys,
    intramuscular doses of 7-35 mg/kg bw/day diminazene produced clinical
    effects on the nervous system, and necropsy revealed cerebellar
    hemorrhages and edema. Hepatotoxic effects were reported in the dog at
    a dose of 3.5 mg/kg bw/day although pre-existing liver disease could
    not be ruled out. Intramuscular doses of 10-40 mg/kg bw/day were
    hepatotoxic in camels.

         Daily doses of 300-500 mg/kg bw diminazene for up to nine months
    in the diet of rats produced no signs of toxicity. In dogs, repeated
    intramuscular or oral administration produced signs of toxic effects
    in the central nervous system, which were confirmed at necropsy by the
    presence of cerebellar lesions. Feeding diminazene to dogs at 60 mg/kg
    bw/day for nine months produced testicular atrophy; in this study, the
    no-effect level was 20 mg/kg bw/day. There were no acceptable data in
    humans on which to base any assessment.

         The Committee was unable to establish an ADI because the results
    of adequate studies of toxicity, including studies of carcinogenicity
    (or genotoxicity), reproduction and teratogenicity, were not
    available.

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    See Also:
       Toxicological Abbreviations
       Diminazene (WHO Food Additives Series 33)
       DIMINAZENE (JECFA Evaluation)