ISOMETAMIDIUM
First draft prepared by
Professor J.M. McLean
Swinburne University of Technology
Hawthorn, Victoria, Australia
1. EXPLANATION
Isometamidium had been previously evaluated at the
thirty-fourth meeting of the Joint FAO/WHO Expert Committee on Food
Additives (Annex 1, reference 85). The compound has a long history
of use as a trypanocide in animals. At the thirty-fourth meeting the
Committee had not been able to establish an ADI because the results
of adequate toxicity studies and information on absorption and the
nature of the metabolites were not available. Accordingly, in its
thirty-fourth report, the Committee suggested that certain
additional studies should be performed before it reviewed the
compound again, although it recognized that the data from these
studies might still not allow a full evaluation.
The commercial preparations of isometamidum (Samorinœ,
Trypanidium(R)) used in the studies reviewed by the Committee
contained four isomers and one bis-species; the isometamidium
content was 55-65% and the product contained less than 1% homidium.
Because of the nature of the synthetic process, the manufacturer has
not been able to prepare a product of greater purity, but the
composition is controlled within stated limits and this stable
mixture has been used in all studies.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion
A calf was given an intramuscular injection of 1 mg/kg bw of
isometamidium containing 14C-labelled compound. After 13 days the
animal was killed and the liver and kidneys removed, homogenized and
lyophilized. These two tissues were selected because they contained
the highest levels of radioactivity. The lyophilized material was
incorporated into a powdered commercial rat maintenance diet and
pelleted. One group of eight Sprague-Dawley rats was fed for 7 days
on the experimental diet and another group for 21 days. A control
group of six rats was fed for 21 days on a similar diet containing
lyophilized tissues from an untreated calf. A further group of 6
rats were given a single dose of 2.245 mg 14C-isometamidium/kg bw
by oral gavage as an aqueous solution and killed after 48 h. Tissues
and faeces were collected from all rats and analysed for
radioactivity.
The results showed that the drug accumulated in the tissues of
the calf and only 0.3% of the dose was excreted in the urine in
36 h, while 3.4% was eliminated in the faeces in 72 h. The
extractable radioactivity from the test rat diet was 24% and no
attempt was made to identify the nature of the residues. No
radioactivity was detected in either of the two groups of rats
receiving the test diet containing the radioactive lyophilized calf
tissues or in the tissues of the rats which received the
isometamidium by gavage. The cumulative excretion of radioactivity
in the faeces of all three treated groups accounted for 90 to 93% of
the total dose of radioactivity. The results of this experiment
showed that in the rat, radiolabelled isometamidium was not
bioavailable after oral dosing or administration in a relay study
using lyophilized bovine tissues. However, it was not possible to
accurately extrapolate these results directly to humans (Kinabo
et al., 1989).
Six groups of five female Sprague-Dawley rats received
intravenously a single dose of 2 mg isometamidium/kg bw and all
members of each group were sampled at either 10 min, 30 min, 2 h,
5 h, 10 h or 24 h after treatment and blood from all five animals
was then pooled for analysis. Isometamidium was detected in the
plasma for only 30 min after treatment. In a second experiment, rats
were gavaged daily for 21 days with isometamidium at 12.5, 50 or
200 mg/kg bw/day. On days 13 and 21 of treatment, five rats were
sampled 30 and 180 min after treatment and the plasma pooled for
analysis. Groups of five rats from a control group was similarly
treated. The method of analysis used HPLC and had a limit of
detection of 10 ng/ml plasma. Isometamidium was not detected in the
plasma from any of the rats that were dosed orally. The results of
this experiment showed that in the rat, isometamidium is not
absorbed when administered orally and is rapidly cleared from the
plasma when administered intravenously (Bosc et al., 1991a,b;
Mignot & Lefebvre, 1991).
2.2 Toxicological studies
2.2.1 Acute toxicity studies
The oral LD50 of a combined group of male and female New
Zeeland white rabbits was 455 mg/kg bw (Ligett, 1989), which was of
a similar order of magnitude as the values reported for the rat. In
a study reviewed at the thirty-fourth meeting, deaths were reported
in rabbits at single oral doses of 12.5 mg/kg bw and above (Ali &
Haroun, 1984). A re-examination of this study revealed significant
pre-existing lung and liver lesions in the rabbits used which
contributed to the abnormally high toxicity reported.
2.2.2 Short-term toxicity studies
2.2.2.1 Rats
Crl:CD(SD)BR rats (Charles River Laboratories) were divided
into groups of ten males and ten females and dosed by stomach tube
with either 50, 225 or 1000 mg/kg bw/day of isometamidium for 13
weeks. A control group received only the suspending vehicle (0.5%
aqueous methyl cellulose). Immediately after treatment, all animals
showed salivation, discoloured fur, hair loss and respiratory
distress, which was most severe in the two highest dose groups.
Three males and three females of the highest dose group died by week
three, all showing disturbed gait and posture, diarrhoea,
emaciation, distended abdomen and irregular breathing. The remaining
animals receiving 1000 mg/kg bw/day were sacrificed during the
fourth week and therefore were not reported further, as the effects
observed were clearly related to treatment.
There was some depression of body-weight in the group receiving
225 mg/kg bw/day, but this did not reach statistical significance at
any stage. Food and water consumption and efficiency of food
utilization were not affected in the two lowest dose groups.
Ophthalmoscopic examination of the group treated at 225 mg/kg bw/day
showed no abnormalities. Haematological investigations were
unremarkable. There were variations in serum aspartate and alanine
aminotransferases, total serum protein, serum albumin, and plasma
phosphorus in some groups and, although the changes achieved
statistical significance, they were minor and of no toxicological
importance. Similarly, there were minor but biologically
insignificant variations reported in the analysis of urine from some
of the treated groups. At autopsy there was a slight, but
significant, increase in relative spleen weight in the males treated
at 225 mg/kg bw/day. Distension of the ileum was recorded in both
sexes at 225 mg/kg bw/day while distension of the caecum was
reported in all treated groups. Histopathological examination of the
male rats treated at 225 mg/kg bw/day showed mild hyperplasia of the
caecal mucosa with varying degrees of inflammatory cell infiltration
and luminal dilation. Apart from the acute post-dosing clinical
signs, which were of pharmacological origin and caecal distension at
necropsy, there were no other treatment-related effects seen at
50 mg/kg bw/day, which was regarded as the non-toxic dose level in
this study (Peters et al., 1991).
2.2.3 Special studies on teratogenicity
Specific pathogen-free female rats [Crl:CD(SD)BR VAF/Plus
strain, Charles River Laboratories] were time-mated with males of
the same strain. Sixteen females in each group received either 60,
180 or 540 mg/kg bw/day isometamidium by gastric intubation on days
6 to 15 of pregnancy and to day 12 post-partum. A control group
received only the vehicle, which was 0.5% aqueous methyl cellulose.
Dosing at 540 mg/kg bw/day produced salivation, staining of
fur, dark and wet faeces, increased urine output and two deaths.
Doses of 180 mg/kg bw/day produced salivation, increased urine
output and dark faeces, while 60 mg/kg bw/day resulted in occasional
salivation and some dark faeces. Water consumption was increased at
540 and 180 mg/kg bw/day while food consumption and body-weight were
reduced at 540 mg/kg bw/day. In the 540 mg/kg bw/day group, one rat
was sacrificed because of a prolapsed uterus, two were not pregnant
and two lost their entire litters by day 12 post-partum. There were
three non-pregnant rats in each of the control and groups dosed at
60 and 180 mg/kg bw/day. There were no other treatment-related
effects on fertility or pregnancy. Pup weight and survival rates
were depressed at doses of 540 mg/kg bw/day and on postmortem at day
12, the stomachs of the pups from the high-dose group were distended
with ingesta with a firm consistency. There were no other
treatment-related effects observed and no developmental
abnormalities were reported. Discounting the clinical signs of
salivation, the NOEL for this study is 60 mg/kg bw/day (Brooker &
Myers, 1991).
Data from teratogenicity studies in the CD rat in which
isometamidium was administered intravenously were submitted to the
thirty-fourth JECFA (see Annex 1, reference 86). These data revealed
a very low incidence of abnormalities comprising absence of caudal
vertebrae, imperforate anus, rudimentary tails and malpositioned and
fused kidneys. These abnormalities were considered to be very rare
and had not been previously reported from the laboratory concerned
(Copping & East, 1986).
Historical control data covering the periods 1984 to 1986, 1976
to 1987 and 1990 to 1991 was reported from two other laboratories
using the same strain of rat in teratology and reproduction studies.
Evaluation of these two sets of data revealed a naturally occurring
syndrome which included all the abnormalities described above which
appeared sporadically in about 0.04% of pups. A reassessment of
these data including an examination of historical data and
reappraisal of statistics suggested that the effects were not
significant (Irvine, 1991; Palmer, 1992).
2.2.4 Special studies on genotoxicity
A summary of the results of the genotoxicity assays on
isometamidium is contained in Table 1.
Isometamidium induced frame-shift mutations in Salmonella spp
in the presence of metabolic activation, but there was no evidence
of genotoxicity in three different tests using cultured mammalian
cells or clastogenicity in three whole animal tests. It has been
postulated that isometamidium may require both bacterial and
mammalian metabolism to be transformed into a mutagen with effects
on the gut and liver, but an examination of the data from the acute
and 13-week studies does not support this hypothesis. The
intraperitoneal administration of isometamidium induced small but
statistically significant increases in numerical chromosomal
aberrations in the form of hyperdiploidy and endoreduplication in
the rat bone marrow cytogenetic assay. Such numerical aberrations
are a consequence of damage to the spindle and possibly other
non-DNA components of the nucleus and show a no-effect level, as
confirmed by the oral studies which revealed no evidence that the
numerical aberrations progressed to nicronuclei or other nuclear
abnormalities (Bridges, 1991). The Committee concluded that orally
administered isometamidium was not genotoxic.
Table 1. Results of genotoxicity assays on isometamidium
Test system Test object Concentration Results Reference
Ames test1 S. typhimurium 0.01-1 mg/plate2 Positive with Crichton
TA1535, TA100, metabolic et al.,
TA1537, TA1538, activation in 1977
TA98 TA1537, TA1538,
TA98
In vitro Cultured human 5.4-175 µg/ml3 Negative Marshall,
cytogenetics lymphocytes 1990
assay
Gene mutation1 Mouse lymphoma 0.09-300 µg/ml4 Negative Clare, 1989
L5178Y cells
(HGRPT locus)
In vitro cell Mouse embryo 0.02-0.31 µg/ml5 Negative Matheson,
transformation fibroblast cells 1978
assay (BALB/3T3)
In vivo Rat bone Intraperitoneal Equivocal7 Kirkland,
cytogenetics marrow 25 mg/kg bw6 1984
assay
In vivo Rat bone 50 or 225 Negative Proudlock &
cytogenetics marrow mg/kg/day orally Cavaliere,
for 13 weeks 1990
In vivo nuclear Rat stomach 50 or 225 Negative Proudlock,
anomaly assay epithelum mg/kg/day by 1991a
(glandular and gavage for 13 weeks
non-glandular)
In vivo nuclear Weanling rat Single gavage dose Negative Proudlock,
anomaly assay bone marrow, 1000 mg/kg8 1991b
liver, stomach,
small intestine,
colon
1 Both with and without rat liver post-mitochondrial (S9) fraction.
2 2-Aminoanthracene was used as a positive control.
3 Methyl methanesulphonate and cyclophosphamide were used as
positive controls.
Table 1 (continued)
4 4-Nitroquinoline-1-oxide (without S9) and benzo(a)pyrene (with S9)
were used as positive controls.
5 3-Methylcholanthrene was used as a positive control.
6 Cyclophosphamide was used as a positive control.
7 Increased numerical chromosomal aberrations (hyperdiploidy
and endoreduplication) without structural chromosomal damage.
8 1,2-Dimethylhydrazine and cyclophosphamide were used as positive
controls.
3. COMMENTS
The Committee considered data from studies of the uptake of
radiolabelled isometamidium from lyophilized bovine tissues,
short-term and teratogenicity studies in rats, and a range of
genotoxicity assays.
Isometamidium was not bioavailable in rats either after oral
dosing or after feeding animals from bovine tissues containing
residues of the drug (see companion residue monograph on
isometamidium, FAO Food and Nutrition Paper, No. 41/5).
Rats were given isometamidium by gavage at doses up to 200 mg
per kg of body-weight per day for 21 days or intravenously at a
single dose of 2 mg/kg bw. The results showed that isometamidium was
not absorbed after oral administration and was rapidly cleared from
the plasma when given intravenously.
The oral LD50 of a combined group of male and female New
Zeeland white rabbits was found to be 455 mg/kg bw, which was of the
same order of magnitude as the values previously reported for the
rat. In a study reviewed at the thirty-fourth meeting deaths were
reported in rabbits at oral doses of 12.5 mg/kg bw/day and above. A
re-examination of this study revealed significant pre-existing lung
and liver lesions in the rabbits used, which contributed to the
abnormally high toxicity reported.
In a short-term study in rats, isometamidium was administered
at doses up to 1000 mg/kg bw/day by gavage for 13 weeks. Immediately
after treatment all animals showed salivation and respiratory
distress. There were deaths in the highest dose group, with
diarrhoea and emaciation, which resulted in discontinuation of this
dose. Apart from the acute post-dosing clinical signs, which were of
pharmacological origin, and caecal distension at necropsy, no other
treatment-related effects were seen at 50 mg/kg bw/day, which could
be regarded as a non-toxic dose level.
A teratogenicity study was carried out in rats in which
isometamidium was administered by gastric intubation at doses up to
540 mg/kg bw/day. The maternal toxicity and fetotoxicity seen at the
highest dose affected pup survival, but neither effects on fertility
nor developmental abnormalities were seen. The NOEL was 60 mg/kg bw
per day. Data from a rat teratogenicity study reviewed at the
thirty-fourth meeting indicated that intravenous administration of
2 mg/kg bw/day produced vertebral abnormalities. A reassessment of
these data, including an examination of historical control data and
a reappraisal of the statistics, suggested that these effects were
not significant.
The genotoxic potential of isometamidium was investigated in a
range of studies. It induced frame-shift mutations in Salmonella
typhimurium in the presence of metabolic activation, but there was
no evidence of genotoxicity in any in vitro mammalian test
systems. When given by intraperitoneal injection isometamidium
increased the frequency of numerical chromosomal aberrations in rat
bone marrow in a cytogenetic assay, but oral administration produced
no genotoxic effects on the cells of the rat gastrointestinal tract.
All other in vivo genotoxicity tests were negative. The Committee
concluded that isometamidium was not genotoxic when administered by
the oral route.
4. EVALUATION
An ADI of 0-100 µg per kg of body-weight was established for
isometamidium based on the non-toxic dose level of 50 mg/kg bw/day
in the 13-week rat study and a safety factor of 500. The Committee
chose this safety factor because of the marginal pharmacological
effects seen at the lowest dose in the rat study and the limited
extent of the data available, although it recognized that neither
the drug nor its metabolites were bioavailable when given by the
oral route.
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