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    ISOMETAMIDIUM

    1.  EXPLANATION

         Isometamidium chloride (other names are Samorin, Trypanidium,
    3-amino-8[3-[3-aminoiminomethyl)
    phenyl]-triazenyl]-5-ethyl-6-phenylphenanthridi-nium chloride) is
    widely used in tropical countries for the control of animal
    trypanosomiasis. It is used principally in cattle but also in sheep,
    goats, buffalos, donkeys, horses, camels and dogs, usually at doses of
    0.5 or 1.0 mg/kg bw by deep intramuscular injection. Structurally,
    isometamidium is closely related to homidium, a compound more widely
    known as ethidium.

         The commercially available products (Samorin and Trypanidium)
    also contain two isomers, a purple compound plus pseudo-isometamidium.
    In addition, these contain a bis-species and homidium (Touratier,
    1981; Bridge  et al., 1982).

         This substance has not previously been evaluated by Joint FAO/WHO
    Expert Committee on Food Additives.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

         Isometamidium appears to be poorly absorbed from the
    gastrointestinal tract of the rat, the only species investigated by
    this route. Around 40% of the administered intragastric dose of 20 mg
    (approximately 60 mg/kg bw) was excreted in the feces during 48 hours,
    with a further 5-9% as homidium. No isometamidium or related compounds
    were detected in urine (Philips  et al., 1967).

         After a single intragastric dose of 1 mg/kg bw
    6-14C-isometamidium (see diagram for numbering), more than 99% was
    voided in feces over a 168 hour period with the majority (95%) being
    detected after 96 hours (Smith  et al., 1981); around 38% was
    excreted in the first 48 hours. Less than 1% was excreted in the urine
    over the 168 hour period. Similarly, the contaminant and gastric
    metabolite homidium was mainly (94%) detected in the feces of rats
    over a 4-day period following oral administration of the
    14C-labelled compound, with less than 1% of the dose being found in
    the urine (Cameron  et al., 1981).

         Following oral administration in rats of 1 mg/kg bw
    6-14C-isometamidium, most of the radioactivity was associated with
    the lumen of the stomach, small intestine, caeum and colon at the 4
    hour sacrifice. Low levels were noted in the skin and large intestine
    at the 24 hour sacrifice (Smith  et al., 1981). Similar results were
    obtained with homidium. Very low levels were associated with the liver
    and kidney 1 hour after administration of 2 mg/kg bw of radiolabelled
    material (0.2 and 0.08 microgram equivalents/g) but after 96 hours the
    only organ with detectable radioactivity was the intestine (including
    contents)  (Cameron et al., 1981).

         When given by the intramuscular route to rats, 14% of the
    administered dose of 1 mg/kg bw of isometamidium remained in the body
    after 59 days. Of this, 72% (9.9% of the dose) was located at the
    injection site. Most of the remainder of the radioactivity was noted
    in the liver, kidney and spleen (2.03, 6.45 and 2.14 microgram
    equivalents/g at 24 hours, and 2.71, 8.78 and 3.15 microgram
    equivalents/g at 72 hours, respectively, slowly decreasing until the
    next sampling period at 168 hours) (Smith  et al., 1981). Findings
    were similar when [14C]homidium was given by the intratracheal route
    to male rats (Cameron  et al., 1981).

         In a relay disposition study in the rat, a single calf was given
    an intramuscular dose of 45 mg 14C-isometamidium and 73 mg of the
    unlabelled drug to give a total dose of 1.0 mg/kg bw. After 13 days
    the calf was sacrificed and the liver and kidney minced and

    lyophilized. These tissues were then formulated with powdered rat feed
    in the proportion of 1:8 (w/w). The test feed was given to two groups
    of rats, one group for 7 days and the other for 21 days. In both cases
    8 rats were used; a control group was given untreated feed. The
    average feed intake by control and treated animals was 15 g/rat/day.
    The calf kidney and liver were found to contain 2.39 and 0.94
    microgram isometamidium/g wet weight tissue and the extractable
    radioactivity of the feed was 23% and the unextractable 76%.

         No radioactivity was found in the tissues of rats given the
    treated feed for 7 or 21 days followed by sacrifice 48 hours after
    cessation of treatment. Similarly, no radioactivity was found in the
    tissues of rats which were given the drug as an aqueous suspension by
    gavage (2.25 mg/kg bw) (Kinabo  et al., 1989).

         When given intramuscularly to the lactating cow at a dose of 1
    mg/kg bw, 14C-isometadimium levels in whole blood were variable (up
    to 0.06 ppm at day 40). However, by 90 days isometadimium was still
    detectable in the blood at levels not dissimilar to those seen at the
    beginning of the study (Bridge  et al., 1982). Radioactivity was
    distributed to all tissues with the exception of the skin. The highest
    levels in organs was noted at day 3 (7.05, 5.84 and 0.16 microgram
    equivalents/g in the kidney, liver and spleen respectively), falling
    steadily until day 90 (0.44, 1.08 and 0.15 microgram equivalents/g,
    respectively). However, the absolute highest levels were found at the
    injection site (73.5, 65.2 and 14.3 microgram equivalents/g at days 3,
    12 and 90, respectively).

         Very similar results were noted when calves were given 0.5 mg/kg
    bw isometadimium by intramuscular injection. The highest concentration
    was found at the injection site (1.27, 0.32 and 0.21 microgram/g at
    days 7, 14 and 21, respectively) with the highest concentrations being
    noted in the spleen (Kinabo and Bogan, 1988).

         After a calf was given 1 mg/kg bw isometamidium intramuscularly,
    isometamidium and the purple isomer were quantifiable in plasma for up
    to 24 hours at concentrations of 17 and 13 mg/ml. The pseudo isomer
    was detectable until 6 hours after injection, while the bisomer and
    homidium were undetected (Oliver  et al.). When given to male Sokoto
    Red goats (0.5 mg/kg bw intramuscularly), isometamidium was still
    detectable in the kidney and liver 4 weeks after administration (2.51
    and 5.52 microgram/g, respectively) but not at 12 weeks. Similarly,
    isometamidium was also found at the injection site (2.51 microgram/g)
    at 4 weeks but not at 12 weeks after injection (Braide & Eghianruwa,
    1980).

         Levels in the blood fell rapidly after intravenous administration
    of isometamidium to rats. Within 10 minutes, 62% of the dose was
    detected in the liver. Maximal levels occurred in the kidney 1 minute
    after dosing. No measurements were taken after 20 minutes (Philips  et
     al., 1967). Isometamidium was detectable in the liver

    (6.78 microgram/g) and kidney (3.26 microgram/g) of goats l2 weeks
    after administration, but not in the spleen, skeletal muscle, adipose
    tissues or at the injection site (Braide & Eghianruwa, 1980). Blood
    levels rapidly fell after camels were given intravenous doses of
    isometamidium (0.5 or 1.0 mg/kg bw); levels declined from around 9 ppm
    1 hour after dosing to 1.7 ppm (high dose) and 6.7 ppm (low dose) at
    24 hours. None was detectable in the blood at 48 hours (Ali & Hassan,
    1984).

         After intravenous doses of 217-313 g isometamidium to dogs, the
    majority of the dose (14-49%) was found in the liver at day 65, with
    0.4-1.9% in the renal cortex. Similar findings were made in two
    monkeys given 80 or 133 mg with sacrifice at day 14/15 (Philips  et
     al., 1967).

         As has already been described, the majority of an oral dose of
    isometamidium is excreted in the feces of rats, with only small
    amounts being found in the urine (Philips  et al., 1967; Smith  et
     al., 1981). Similar findings were observed for homidium (Cameron  et
     al., 1981). The majority is excreted in the first 96 hours. Because
    isometamidium was found in the bile of rats after intravenous
    administration, the bile of rats given the substance orally was
    examined, but none of the samples examined revealed any drug-related
    material (Philips  et al., 1967).

         Following intramuscular administration to rats, biliary excretion
    was evident, as around 26% of the administered dose was found in the
    59-day feces with only 3.6% in the corresponding urine. The majority
    of the fecal output (24%) was collected during the first 8 days (Smith
     et al., 1981). An almost identical situation was seen when 1 mg/kg
    bw isometamidium was given intramuscularly to lactating cows. The
    majority of the dose (11.6%) was excreted in the feces over the first
    7 days and by day 70, 20.8% of the dose had been recovered. Only 5%
    was noted in the urine over this period (Bridge  et al., 1982).

         No isometamidium was found in goat urine after intramuscular
    injections were followed by a 12-week observation period (Braide &
    Eghianruwa, 1980). There are no studies available on the excretion of
    homidium following oral, subcutaneous or intramuscular administration.
    After intratracheal instillation, about 79% of the dose had been
    collected in urine and feces within 4 days, with the majority (77%)
    found in the feces. Within 48 hours, 51% and 19% was collected in the
    feces and urine, respectively (Cameron et al., 1981).

         After intramuscular injection of 1 mg/kg bw of isometamidium to
    lactating cows, only a small amount of 14C-label was noted in the
    milk and cream samples analyzed. This was found in 4/7 cows on day 3
    (mean 0.012 ppm) and in one cow on day 70, another cow on day 40 and
    a third cow on day 5 with a range of 0.014 to 0.017 ppm (Bridge  et
     al., 1982).

    2.1.2  Biotransformation

         The biotransformation of isometamidium has not been extensively
    investigated. It may be converted to homidium in the gut (Philips  et
     al., 1967). Homidium, and other phenanthridium compounds are
    N-acetylated  in vivo in rats and/or by metabolizing preparations
    (Lecointe  et al., 1981., MacGregor & Clarkson, 1971; MacGregor &
    Johnson 1977). N-acetylation appears to depend on the presence of the
    aromatic amino group at the 3-position and isometamidium does possess
    such a moiety and indeed N-acetylated products have been noted in the
    bile of rats given isometamidium (Lecointe  et al., 1981; Philips  et
     al., 1967). Acetylation may be dependent in part on activation by
    cytochrome-P448 (Lecointe  et al., 1981).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

    2.2.1.1  Oral

         Isometamidium chloride appeared to be more toxic in the rabbit
    after oral administration than in the rat. Groups of 5 male and 5
    female rats were given 0, 800, 1250 or 2000 mg/kg bw isometamidium in
    water by stomach tube. Excessive salivation that disappeared after 15
    minutes was noted in low-dose rats. In those given 1250 mg/kg bw,
    excessive salivation and a brown discoloration of the fur by saliva
    was seen; the animals were normal within 125 minutes. At the highest
    dose, 1 out of 5 females died and again excessive salivation and wet,
    brown fur were observed. Surviving high-dose animals were normal
    within 22 hours (Ward & Wallace, 1983).

         When groups of 4 rabbits (unspecified strain) were given 6.25,
    12.5, 25 or 50 mg/kg bw isometamidium in water, 3/4 of those given
    12.5 mg/kg bw died within 4-5 hours and the other within 10 hours
    while all the rabbits given the two highest doses died (within 10-30
    minutes at 50 mg/kg bw). None of those given 6.25 mg/kg bw died,
    suggesting that the oral LD50 in the rabbit lies between 6 and 12
    mg/kg bw when given in water. Signs of toxicity included tremors,
    convulsions, tachycardia, laboured breathing, hyperaesthesia, and
    head-shaking. Gross examination revealed congestion and/or fatty
    change in the liver and kidneys and hemorrhages in the brain and
    gastrointestinal tract. At 6.25 mg/kg BW, histopathologic examination
    revealed mild congestion of the liver and edema in some portal tracts.
    Congestion and edema were also noted in the brain, lungs and kidneys.
    At higher doses, degenerative changes and necrosis were evident in the
    liver and focal hemosiderosis, petechial hemorrhages and patchy areas
    of edema and congestion were noted. The kidneys also showed congestion
    and edema. Hydropic degeneration was seen in the cortex and medulla
    and necrosis of glomeruli was present. Pneumonia with congestion,
    edema, focal hemorrhages, mononuclear infiltration and fibroblastic

    proliferation occurred. Vacuolar changes were observed in the brain
    again with congestion, edema, focal hemorrhages and perivascular
    cuffing. Necrosis and hemorrhagic enteritis occurred in the duodenum
    (Ali & Haroun, 1984).

    2.2.1.2  Intravenous

         Following intravenous administration of isometamidium in 0.85%
    aqueous sodium chloride at doses of 0.6, 1.3, 2.5 or 5 mg/kg bw to
    rats, none of those given 0.6 or 1.3 mg/kg bw died while death
    occurred in less than 2 minutes in those given the two highest doses.
    A brief convulsive episode preceded death (Philips  et al., 1967).
    When given in water at doses of 0, 5, 6.25 or 8 mg/kg bw, 100%
    mortality occurred in rats given the highest dose with 40% and 10% at
    the 6.25 or 5 mg/kg bw levels respectively. The LD50 was 6.6 mg/kg
    bw. Convulsions and tremors occurred prior to death. In those given 8
    mg/kg bw, death occurred within one minute (Ward & Wallace, 1983).

         The maximum tolerated intravenous doses of isometamidium in water
    to cattle, goats, dogs and camels were 1.5, 0.5, 2.0 and 1.0 mg/kg bw
    respectively. At lower doses tachycardia, salivation and lacrimation
    occurred while at higher doses, these signs plus recumbency,
    convulsions and diminished reflexes were observed. Death occurred in
    goats, the most sensitive species, given 1.0 mg/kg bw while dogs, the
    most refractive, survived doses of up to 5 mg/kg bw. Necropsy revealed
    hemorrhages in the intestines, heart and brain stem. The spleen and
    liver were congested and edematous (Schillinger  et al., 1985).
    Camels given 0.5 or 1.0 mg/kg bw isometamidium intravenously developed
    lacrimation, salivation and trembling. The frequencies of defecation
    and urination increased and animals given the higher dose rapidly
    became recumbent. Recovery appeared complete within 3 hours (Ali &
    Hassan, 1986).

    2.2.1.3  Intraperitoneal

         When groups of 5-8 mice were given single intraperitoneal doses
    of isometamidium in distilled water, animals dosed at 40 or 80 mg/kg
    bw died soon after dosing. Mice given 0.5-10 mg/kg bw were unaffected.
    Severe degeneration of the liver and congestion of the heart, liver
    and kidney were noted at necropsy (Homeida  et al., 1980).

         No deaths occurred in 6 rats given intraperitoneal doses of 12.5
    mg/kg bw isometamidium in 0.85% aqueous sodium chloride and only 1/9
    died in a group given 25 mg/kg bw. However, 60-78% of those given
    doses of 50-200 mg/kg bw died within 2-3 hours (high dose) or 1-4 days
    (50 and 100 mg/kg bw). All doses caused depression, ataxia and
    dyspnoea 5 minutes after injection (Philips  et al., 1967).

    2.2.1.4  Subcutaneous

         Dose range-finding experiments resulted in extensive necrosis of
    the skin and further studies by this route were abandoned (Ward &
    Wallace, 1983). In an earlier study, doses of 125-500 mg/kg bw in
    0.85% aqueous sodium chloride did not produce fatalities but again
    extensive necrosis at the injection site occurred (Philips  et al.,
    1967).

    2.2.1.5  Intramuscular

         After single intramuscular injections of 0.5 mg/kg bw
    isometamidium to cattle, severe and extensive damage occurred at the
    reaction site (Kinabo & Bogan, 1988). Injection site reactions can
    cause lameness in cattle (Lindau & Spielberger, 1973).

    2.2.1.6  Percutaneous

         Two groups of 5 male and 5 female CD rats were given dermal doses
    of 0 or 2000 mg/kg bw as an aqueous suspension applied to depilated
    skin and covered with a 24 hour occlusive dressing. No signs of
    toxicity occurred in a 14-day observation period and no deaths
    occurred (Ward & Wallace, 1983).

    2.2.1.7  Homidium

         Homidium is much less acutely toxic than isometamidium, the acute
    oral and dermal LD50 values in the rat being greater than 2000 mg/kg
    bw. The LD50 after intravenous administration was 27 mg/kg bw.
    Following subcutaneous injection, an LD50 of 80 mg/kg bw was
    reported (studies with isometamidium suggested mainly local effects by
    this route). The major signs of toxicity noted after intravenous and
    subcutaneous dosing included tremors, prostration and sedation.
    Respiratory impairment was seen after intravenous administration while
    necrosis at the injection site occurred following subcutaneous
    infection. Homidium was a moderate eye irritant but not a skin
    irritant in the rabbit (Wallace  et al., 1984).

    2.2.2  Short-term studies

    2.2.2.1  Dogs

         Isometamidium has been given to dogs intravenously for a total of
    10 rapid injections on successive days excluding weekends, to a total
    dose of 20 mg/kg bw. The dogs responded acutely after each dose with
    vomiting, ataxia, weakness, defecation, lacrimation and salivation
    within 1-5 minutes. Slow and shallow respiration occurred. Recovery
    occurred within 30 minutes. Apart from these acute effects the dogs
    appeared normal throughout treatment. No adverse effects on hematology

    and blood biochemistry occurred. At necropsy 8 and 40 days after the
    last injection, pigment deposition in kidney or liver was noted and a
    few foci of hemorrhage in the liver of one dog were observed (Philips
     et al., 1967).

    2.2.2.2  Monkeys

         One cynomolgus monkey was given 10 daily intravenous doses of 2
    mg/kg bw isometamidium while one rhesus and one cynomolgus monkey were
    given a single dose of 2 mg/kg bw followed by 9 daily injections of 4
    mg/kg bw (excepting weekends). Acute toxicity was seen after each
    injection evidenced by weakness, ptosis and dyspnoea. These effects
    lasted for approximately 20 minutes.

         The rhesus monkey lost 11% of its weight by day 16 when it was
    dysponeaic, depressed and unable to stand upright. Necropsy of this
    monkey at day 16 revealed isolated, necrotic hepatocytes with erosions
    of the gastric mucosa, esophagitis, venous thrombi in the adrenals,
    and a decrease in nucleated elements of the bone marrow with
    hemorrhage and congestion in affected areas. There was severe fatty
    change in the kidney and liver. Foci of edema in one of the cynomolgus
    monkeys and scattered hepatic necrotic foci in the other were observed
    but other major organs examined from all 3 monkeys were normal
    (Philips  et al., 1967).


         As part of an extension to the relay distribution study described
    in section 2.1.1, specimens of kidney, liver, stomach and small
    intestine from 3 control rats and from 4 rats given the diet
    containing tissues from the calf dosed with radiolabelled drug were
    examined microscopically. No abnormalities were noted after 21 days of
    feeding (Kinabo  et al., 1989).

    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

         Isometamidium is a member of the phenanthridium drug group, many
    members of which are mutagenic. Ethidium bromide, for example, is a
    frame-shift mutagen in  Salmonella typhimurium strains with metabolic
    activation and it is also mutagenic to yeast  (Saccharomyces
     cerevisiae) producing respiratory deficient colonies (petite) by its
    effects on mitochondrial DNA (Lecointe  et al., 1981; MacGregor &
    Johnson, 1977; Slonimski  et al., 1968; Fukunaga  et al., 1984;

    Fayeulle, 1985). It also appears to affect segregation in yeast during
    meiosis (Sora & Carbone, 1987). Ethidium bromide forms a highly
    fluorescent complex with native DNA by intercalation between
    base-pairs (Le Pecq & Paoletti, 1967; Prutz, 1984). Isometamidium
    binds strongly to calf thymus DNA  in vitro (Kinabo & Bogan, 1987).

         Isometamidium was tested for mutagenicity in  S. typhimurium
    strains TA 1535, TA 100, TA 1537, TA 1538 and TA 98 with and without
    a rat liver S-9 metabolic activation system. The compound caused
    frameshift mutations, giving positive results in strains TA 1537, TA
    1538 and TA 98, in the presence of the metabolizing system (Crichton
     et al., 1977).

         The compound was also tested in an  in vivo cytogenetic assay
    using the rat. Animals were given intraperitoneal injections of what
    was suggested in another study to be the maximum tolerated dose (40
    mg/kg bw) but these proved too toxic and several animals died. The
    experiment was repeated using 25 mg/kg bw isometamidium with sacrifice
    at 6, 24 and 48 hours followed by harvesting and examination of the
    bone marrow. There were significant increases in the number of
    chromosomal aberrations, both including and excluding gaps. In
    comparison with controls, the total number of endoreduplicated and
    hyperdiploid cells from all 3 kill times were significantly elevated.
    The total numerical aberrations at 24 hours and after combining 6, 12
    and 24 hour kill values, were also significantly increased. Rats
    treated with homidium in the same manner showed similar but weaker
    effects (Ingham, 1985). Thus, the two compounds induced numerical but
    not structural chromosome aberrations in the rat.

         Isometamidium chloride was tested for its ability to induce cell
    transformation in the Balb/3T3 cell assay  in vitro. The substance
    was tested in water up to and including a concentration of 0.312
    microgram/ml in the absence of a metabolic activation system. Higher
    concentrations were cytotoxic. There was no evidence of cell
    transformation (Ingham, 1978).

    2.2.6  Special studies on teratogenicity

         Isometamidium

    2.2.6.1  Rats

         In a preliminary study, groups of 12 pregnant CD rats were given
    intravenous doses of 0 or 2 mg/kg bw isometamidium chloride in
    distilled water (2 ml) by the tail vein, on days 5, 7, 9, 11, 13, 15
    and 17 after mating. Maternal toxicity, ataxia, sedation, tremors,
    reduced maternal body weights and reduced food consumption were noted
    in the pregnant animals. On day 22, the animals were killed and the
    uteri and ovaries removed. Fetuses from animals given isometamidium
    were reduced in weight compared with those from controls. One control
    fetus had unilateral microphthalmos. Two fetuses in different litters
    from the dams treated with isometamidium had major disruption and
    absence of various vertebrae. In addition, these two fetuses showed
    imperforate anus and rudimentary tails. One pup from the treated
    animals had malpositioned and fused kidneys. The vertebral
    abnormalities were considered to be very rare having not been observed
    previously at the laboratory (2286 control fetuses). Hence, the study
    was repeated using groups of 20 male and 20 female CD rats. Again, one
    control fetus had microphthalmos. Six abnormal fetuses were noted from
    treated dams. One showed major disruption and absence of vertebral
    bones as in the first study, three had hydrocephalus (one with
    bilateral microphthalmia), one exhibited situs inversus totalis and
    the remaining fetus had a slight spinal kink with lumbar scoliosis
    (Copping & East, 1986a).

    2.2.6.2  Rabbits

         Groups of 1-3 pregnant New Zealand white rabbits were given
    intravenous doses of 0, 0.25 or 0.5 mg/kg bw isometamidium into the
    lateral ear vein on days 6, 10, 13, 16 and 19 after mating. The
    pregnancy rate was poor in all the groups and the study was repeated
    using groups of 5-8 pregnant rabbits. Signs of toxicity included
    ataxia, sedation, cyanosis, and tonic and chronic convulsions after
    dosing. The number of fetuses per litter was lower in the group given
    0.5 mg/kg bw but there were no reductions in fetal weight and no other
    signs indicative of embryo- or fetotoxicity. There were no similar
    effects in the groups given 0.25 mg/kg bw, and no evidence of
    teratogenic effects in either group (Copping & East, 1986b).

             Homidium

    2.2.6.3  Rats

         Groups of 10 or 11 CD pregnant rats were given intravenous doses
    of 0 or 10 mg/kg bw homidium via the tail vein, on days 5, 7, 9, 11,
    13, 15 and 17 after mating. Ataxia, sedation, prostration and tremors
    were seen in the dosed groups but most animals recovered within
    minutes of dosing. Dams given homidium showed marked reductions in
    body weight gain and food intake. However, there was no evidence of
    fetotoxicity and no teratogenic effects were seen (Copping & East,
    1986c).

    2.2.6.4  Rabbits

         Groups of 1-3 New Zealand white rabbits were given intravenous
    doses of 0, 2 or 4 mg/kg bw homidium on days 6, 10, 13, 16 and 19
    after dosing, via the lateral ear vein. There was a poor pregnancy
    rate which, as in the study with isometamidium, may have been due to
    the use of poor quality rabbits (see section 2.2.6.2). A second study
    was conducted with pregnant rabbits (groups of 7/8) from another
    supplier and pregnancy rates were normal. An increase in
    post-implantation losses was seen in dams given 4 mg/kg bw. Delayed

    ossification of the skeleton was also noted in fetuses from dams given
    4 mg/kg bw but there were no other effects and no differences between
    treated animals and controls in the 2 mg/kg bw group (Copping & East,
    1986d).

    2.3  Observations in humans

         There are no published reports on effects in humans following
    exposure to isometamidium. In an unpublished report from a plant
    producing the material, 140 men and 21 women were identified as having
    been exposed to the substance although there were no details of length
    of exposure. These were subject to health screening using medical
    histories, a general medicalexamination and blood tests. There were no
    apparent effects on reproductive function, morbidity, hematology and
    biochemistry. The only finding considered significant was a single
    case of acute myeloid leukaemia, a condition said to be rare and to
    occur once in 1.7 for 105 population/years. The occurrence of this
    single case of acute myeloid leukaemia was not significant at the 5%
    level and the authors concluded that it was highly likely that it
    occurred by chance (Feldman, 1986).

    COMMENTS

    3.   Isometamidium appeared to be poorly absorbed from the
    gastrointestinal tract of the rat, with about 99% of an oral dose
    being excreted in the feces. Similar findings were obtained for
    homidium (ethidium), a contaminant of the commercial product. In the
    gastrointestinal tract, isometamidium may be converted into homidium
    but there are insufficient data on this and there is no information on
    the formation of any other metabolites. Small amounts of radio-active
    label were excreted in the milk of cows after intramuscular injection
    of radiolabelled isometamidium.

         No carcinogenicity data were available. Isometamidium was a
    frame-shift mutagen in  Salmonella typhimurium in the presence of
    metabolic activation, like the closely related contaminant and
    possible metabolite homidium, which is a known DNA intercalating
    agent. Isometamidium was also mutagenic in yeast. In an  in vivo
    cytogenetic assay in the rat, it produced numerical changes including
    hyperdiploidy and endoreduplication but not structural chromosome
    abnormalities. It gave negative results in a cell-transformation test.

         Both isometamidium and homidium were subjected to teratogenicity
    tests in the rat and rabbit by the intravenous route. For all studies,
    dosing occurred on only selected days of gestation. Isometamidium
    produced a weak teratogenic and fetotoxic response in the rat at 2
    mg/kg bw/day, the highest dose tested, but in the rabbit only a very
    weak fetotoxic response was observed. The NOEL was 0.25 mg/kg bw/day.
    There was no evidence of fetotoxic effects in the rat at 10 mg/kg
    bw/day and only a weak fetotoxic effect in the rabbit at 4 mg/kg
    bw/day when homidium was tested by the intravenous route. No studies
    were available on isometamidium given orally, but the poor absorption
    following administration in this way suggests that any NOEL would be
    much higher by this route.

         Isometamidium had a low order of acute toxicity when given in
    water to the rat. Rats given a single oral dose of 1250 mg/kg bw
    showed signs of toxicity characterized by excess salivation, while the
    majority of those given 2000 mg/kg bw died. Rabbits appeared more
    susceptible to single oral doses of aqueous isometamidum, deaths
    occurring at doses at 12.5 mg/kg bw and above. It was more toxic in
    rats by the intravenous and intraperitoneal routes. Homidium appeared
    less toxic in the rat. There were no adequate short-term toxicity
    studies, and no adequate studies of effects on humans.

         Poor absorption of orally administered isometamidium is found in
    rodents, but also there is no adequate evidence to suggest that either
    the compound itself or homidium was similarly absorbed in humans. The

    Committee was not able to establish an ADI because the results of
    adequate toxicity studies, including carcinogenicity (or genotoxicity)
    studies and teratogenicity and short-term studies with oral
    administration of the drug, were not available nor was there any
    information on the nature of the metabolites.

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    See Also:
       Toxicological Abbreviations
       Isometamidium (WHO Food Additives Series 31)
       ISOMETAMIDIUM (JECFA Evaluation)