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.
5. REFERENCES
ALI, B.H. & HAROUN, E.M. (1984). Acute toxicity of Samorin
(Isometamidium chloride) in rabbits. Comp.Biochem.Physiol., 78C,
419-423.
ALI, B.H. & HASSAN, T. (1984). Preliminary pharmacokinetic study of
isometamidium in camels. Res.Vet.Sci., 37, 376-377.
ALI, B.H. & HASSAN, T. (1986). Some observations on the toxicosis of
isometamidium chloride (Samorin) in camels. Vet. Human Toxicol., 28,
424-426.
BRAIDE, V.B. & EGHIANRUWA, K.I. (1980). Isometamidium residues in goat
tissues after parenteral administration. Res.Vet.Sci., 29, 111-113.
BRIDGE, C.M., SMITH, G.E., SMITH, J., TEMPLETON, R. & YOXALL, A.T.
(1982). Samorin: Study in the lactating cow, of the distribution and
excretion of radiolabelled drug derived products, following the
administration of a single intramuscular dose of [6-14C]-Samorin.
May & Baker Research Report, R.Ph.176, submitted to WHO by RMB Animal
Health Ltd., Essex, England.
CAMERON, B.D., CURRIE, M.R., DRAFFAN, G.H. & JOHNSTON, A.M. (1981).
[14C] Homidium chloride in the rat: Absorption tissue distribution
and excretion following oral and intratracheal administration.
Inveresk Research International Report No. 1947, submitted to WHO by
RMB Animal Health Ltd., Essex, England.
COPPING, G.P. & EAST, P.W. (1986a). Isometamidium chloride.
Teratogenicity study by the intravenous route in the rat. May & Baker
Research Report, R.Tox. 594, submitted to WHO by RMB Animal Health
Ltd., Essex, England.
COPPING, G.P., & EAST, P.W. (1986b). Isometamidium chloride.
Teratogenicity study by the intravenous route in the rabbit. May &
Baker Research Report, R.Tox. 589, submitted to WHO by RMB Animal
Health Ltd., Essex, England.
COPPING, G.P. & EAST, P.W. (1986c). Homidium chloride. Teratogenicity
study by the intravenous route in the rat. May & Baker Research
Report, R.Tox. 602, submitted to WHO by RMB Animal Health Ltd., Essex,
England.
COPPING, G.P. & EAST, K.S.M. (1986d). Homidium chloride.
Teratogenicity study by the intravenous route in the rabbit. May &
Baker Research Report, R.Tox. 600, submitted to WHO by RMB Animal
Health Ltd., Essex, England.
CRICHTON, C., MacGREGOR, D.B. & SMITH, I. (1977). Testing for
mutagenic activity in isometamidium chloride and
initrophenylphenanthridine. Inveresk Research International Report No.
894, submitted to WHO by RMB Animal Health Ltd., Essex, England.
FAYEULLE, J.P. (1985). Effects of gas-phase cigarette smoke in the
induction of the mitochondrial "petite" mutation by ethidium bromide
in yeast. Mutat.Res., 158, 69-75.
FELDMAN, M. (1986). Health surveillance of phenanthridine workers. May
& Baker Report, MF/HS, submitted to WHO by RMB Animal Health Ltd.,
Essex, England.
FUKUNAGA, M., MIZUGUCHI, Y., YIELDING, L.W. & YIELDING, K.L. (1984).
Petite induction in Saccharomyces cerevisiae by ethidium
analogs. Action on mitochondrial genome. Mutat.Res., 127, 15-21.
HOMEIDA, A.M., EL AMIN, E.A., ADAM, E.I. & MAHMOUD, M.M. (1980).
The effect of samorin (isometamidium chloride) on Trypanosoma
evansi infection in mice. Brit.J.Exp.Pathol., 61, 380-389.
INGHAM, B. (1978). Isometamidium chloride: Evaluation in the in vitro
transformation test of Balb/3T3 cells assay; Litton Bionetics Inc.,
Project No. 20840, submitted to WHO by RMB Animal Health Ltd., Essex,
England.
INGHAM, B. (1985). Isometamidium chloride and homidium chloride. Study
to evaluate the chromosome damaging potential of M & B 2360 and M & B
4190A by their effects on the bone marrow cells of treated rats.
Microtest Research Ltd., Report No. MAB3 AND 4/RMB/AFR/KF8, submitted
to WHO by RMB Animal Health Ltd., Essex, England.
KINABO, L.D.B., & BOGAN, J.A. (1987). Binding of imsometamidium to
calf thymus DNA and lipids: pharmacological implications.
J.Vet.Pharmacol.Therap., 10, 357-362.
KINABO, L.D.B. & BOGAN, J.A. (1988). Pharmacokinetic and
histopathological investigations of isometamidium in cattle.
Res.Vet.Sci., 44, 267-269.
KINABO, L.D.B., BOGAN, J.A., McKELLAR, Q.A. & MURRAY, M. (1989). Relay
bioavailability and toxicity of isometamidium residues: A model for
human risk assessment. Submitted to Vet. Human Toxicol.
LECOINTE, P., BICHET, N., FRAIRE, C. & PAOLETTI, C. (1981). The
hepatic metabolism of ethidium bromide to reactive mutagenic species:
Biochemical and structural requirements. Biochem.Pharmacol., 31,
601-609.
LE PECQ, J.B. & PAOLETTI, C. (1967). A fluorescent complex between
ethidium bromide and nucleic acids. J.Mol.Biol., 27, 87-106.
LINDAU, M. & SPIELBERGER, U. (1973). Effect of Samorin injections on
growth and milk yield of West African Zebu of the Azaouak breed.
Berliner Münch Tierarzt Wöchenschrift, 86, 129-130.
MacGREGOR, J.T. & CLARKSON, T.W. (1971). Metabolism and biliary
excretion of phenanthridium salts - I. Nature of biliary metabolites.
Biochem.Pharmacol., 20,2833-2846.
MacGREGOR, J.T. & JOHNSON, I.J. (1977). In vitro metabolic
activation of ethidium bromide and other phenanthridium compounds.
Mutagenic activity in Salmonella typhimurium. Mutat.Res., 48,
103-108.
PHILIPS, F.S., STERNBERG, S.S., CRONIN, A.P., SODERGREN, J.E. & VIDAL,
P.M. (1967). Physiologic disposition and intracellular localization of
Isometamidium. Cancer Res., 27, 333-349.
PRUTZ, W.A. (1984). Inhibition of DNA - ethidium bromide intercalation
due to free radical attack upon DNA. I. Comparison of the effects of
various radicals. Radiat.Environ.Biophys., 23, 1-6.
SCHILLINGER, D., MALOO, S.H. & ROTTCHER, D. (1985). The toxic effect
of intravenous application of the trypanocide isometamidium (Samorin).
Zbl.Vet.Med.A., 32, 234-239.
SLONIMSKI, P., PERRODIN, G. & CROFT, J. (1968). Ethidium bromide
induced mutation of yeast mitrochondria: Complete transformation of
cells into respiratory nonchromosomal petites.
Biochem.Biophys.Res.Commun., 30, 232-239.
SMITH, J., BRIDGE, C.M., & SMITH, G.E. (1981). "Samorin": Study in the
female rat, of the distribution and excretion of radiolabelled
drug-derived products after intramuscular or oral administration of a
single dose of [6-14C]-Samorin. May & Baker Research Report,
RES/3851, submitted to WHO by RMB Animal Health Ltd., Essex, England.
SORA, S. & CARBONE, M. (1987). Chloral hydrate, methyl mercury
hydroxide and ethidium bromide affect chromosomal segregation during
meiosis of Saccharomyces cerevisiae. Mutat.Res., 190, 13-17.
TOURATIER, L. (1981). The advantage of using isometamidium for the
control of animal trypanosomiasis. International Scientific Council
for Trypanosomiasis Research and Control, 16th Meeting, Yaoundé,
Cameroon, 1979. OAU/STRC. 308-316.
WALLACE, V.A., WARD, R.J. & GRIMMETT, J.E. (1984). Trypanocides:
Novidium: Acute toxicity and local tolerance studies in various
species. May & Baker Research Report, R.Tox. 319, submitted to WHO by
RMB Animal Health Ltd., Essex, England.
WARD, R.J. & WALLACE, V.A. (1983). Trypanocides: Samorin: Acute oral,
dermal and intravenous toxicity studies in rats. May & Baker Research
Report, R.Tox. 257, submitted to WHO by RMB Animal Health Ltd., Essex,
England.
See Also:
Toxicological Abbreviations
Isometamidium (WHO Food Additives Series 31)
ISOMETAMIDIUM (JECFA Evaluation)