IPRONIDAZOLE
1. EXPLANATION
Ipronidazole (2-isopropyl-1-methyl-5-nitroimidazole) is used for
the treatment of histomoniasis in turkeys and in swine dysentery. This
compound has not been previously reviewed by the Joint FAO/WHO Expert
Committee on Food Additives.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion
After an oral administration of 22.5 mg 2-14C-ipronidazole/kg
bw to five bile cannulated female Charles River rats for four
consecutive days, 31% of the total radioactivity was found in the
feces and in the contents of the gastrointestinal tract, 34% was found
in the bile, 27% in urine, and 1.4% in the carcass, liver and gut.
Total recovery of radioactivity was 92% (Weiss et al., 1981).
2.1.2 Biotransformation
Two major metabolites were isolated in the feces of rats and
turkeys after an oral administration of ipronidazole. In turkeys the
metabolite was identified as
1-methyl-2-(2-hydroxyisopropyl)-5-nitroimadazole, which together with
the unchanged drug accounted for about 40% of the excreted dose. In
the aqueous extract of the feces of rats the metabolite was identified
as 2,3-dihydro-2-(2-hydroxypropyl)-3-methyl-4-nitro-1H-imidazol-5-ol,
which represented 12.4% of the administered dose. Although
experimental details were not presented, it was stated that no
nitro-reduced metabolite was isolated (Weiss et al., 1981).
Metabolism studies with the isomer of ipronidazole in which the 2 and
5 positions are interchanged
(5-isopropyl-1-methyl-2-nitro-1H-imidazole) in female beagle dogs
revealed that after an oral administration of 50 mg 2-14C-labelled
drug/kg bw, 50% of the total radioactivity was found in the urine.
Together with a small amount of unchanged drug three main metabolites
still containing the nitro group were isolated. One metabolite was
identified as the isomer of one of the main metabolites of
ipronidazole (1-methyl-2-(2-hydroxyisopropyl)-5-nitroimidazole). The
formation of the two more polar metabolites also involved oxidative
steps in the isopropyl chain leading to a diol and a carboxylic acid
at the primary carbon (Assandri et al., 1978).
2.2 Toxicological studies
2.2.1 Acute toxicity
Species Sex Route LD50 Reference
(mg/kg bw)
Mouse M&F oral 970 Banzinger & Hane,
i.p. 600 1967.
s.c. >400
Rat M&F oral 920 ibid.
(adult)
Rat M&F oral 205 Pool & Steivis,
(neonate) 1968.
Rabbit M&F oral 960 Banzinger & Hane,
1967.
2.2.2. Short-term studies
2.2.2.1 Rats
Diets containing ipronidazole at levels of 0, 20, 80 or 320 mg/kg
bw/day were fed to groups of 5 Charles River rats of each sex for
thirteen weeks. The rats were observed daily for clinical signs of
toxicity and body weights were recorded weekly. Growth rate and food
consumption were similar in all groups. Hematology and urine analysis
values were not affected by ipronidazole treatment. Gross autopsy of
the tissues did not reveal pathological changes due to drug treatment.
Histopathological examinations of the tissues were not performed.
Absolute kidney and liver weight of male animals from all treated
groups were elevated; relative liver weights of all rats of the
highest dose groups were also elevated (Sadek & Banzinger, 1968).
A rat study using the same protocol as the previous study with 8
Charles River rats per sex per group was performed. The rats were
observed daily for signs of clinical toxicity and body weights were
recorded weekly. The body weights were significantly decreased in
males and females of the 320 mg/kg bw/day group. Mean food consumption
was similar in all groups with the exception of the females
administered 20 mg/kg bw/day. This group ate more food than the other
female rats. Opthalmologic examinations, hematology, clinical
chemistry and urine analysis values revealed no differences between
control and treated groups. There was a decrease in absolute and
relative ovarian weight, which was not accompanied by histological
changes. Hepatocellular hypertrophy occurred in the high dose male
group. The NOEL was 80 mg/kg bw/day. (Banarjee et al., 1968).
2.2.2.2 Dogs
Ipronidazole was administered orally by capsule at levels of 0,
20, 80 and 160 mg/kg bw/day 6 days a week for thirteen weeks to groups
of two beagle dogs of each sex. The dogs were observed daily for
clinical signs of toxicity and body weights were recorded weekly.
Hematologic studies and urine analysis were performed at 2 to 4 week
intervals. In the 160 mg/kg bw/day group two dogs died and another was
moribund and sacrificed; the fourth dog was taken off treatment 7´
weeks after the start of the study. All dogs of that group revealed
loss of body weight, dehydration, salivation, lacrimation, muscular
tremor, ataxia and dilatation of the pupils. Autopsy of the tissues
revealed congestion of internal organs and hepatomegaly. Dogs of the
80 mg/kg bw/day group showed mild lacrimation and salivation, slightly
decreased body weights and increased liver weights. Histology of the
liver revealed a mild granularity of the cytoplasm and an increase of
mesenchymal cells in the biliary tract. For this study the NOEL was 20
mg/kg bw/day (Sadek & Banzinger, 1968).
2.2.3 Long-term/carcinogenicity studies
2.2.3.1 Mice
Diets containing 0, 20, 200 or 1000 ppm ipronidazole were fed to
groups of 80 Charles River derived CD-1 mice of each sex (2 control
groups were used) for 89 weeks for males and for 100 weeks for females
(the time at which survival was 20%). The average consumption of
ipronidazole based on body weight was 0, 3, 30 or 150 mg/kg bw/day.
The mice were observed daily for clinical signs of toxicity and body
weights were recorded weekly for the first 13 weeks and every two
weeks thereafter. The group mean body weights and food consumption
were comparable in treated and control groups. There were no
significant differences in survival rates between treated and control
animals.
Gross autopsy did not show any treatment-related changes.
However, in males and females of the highest dose group there was a
significant increase (P<0.05) in hyperplasia and adenoma of the lung
(Table 1).
Although only adenomas and hyperplasia were significantly
increased at the highest dose group, a slight increase in tumor
multiplicity in both sexes and carcinoma of males was also seen at the
highest dose administered. Although adenoma of the lung is a common
tumor in this strain of mice, the high dose incidence exceeded the
historical control range from 8 studies in the same laboratory (2/50
to 10/59 for males and 2/59 to 11/59 for females). Historical control
data were not available for hyperplasia but the increase over
concurrent controls is significant (P<0.05). The NOEL in this study
was 200 ppm, equal to 30 mg/kg bw/day based on hyperplastic and
neoplastic lesions of the lung (Reno et al., 1981).
Table 1: Hyperplastic and neoplastic lesions of the lung of mice
treated with irponidazole
MALE FEMALE
drug in the diet (ppm)/number of animals
0 0 20 200 1000 0 0 20 200 2000
78 79 77 78 78 79 78 80 79 79
Hyperplasia 8 8 3 11 36* 8 8 7 13 25*
Adenoma 8 8 7 13 25* 6 5 3 4 20*
Adenoma 0 2 1 2 7 0 0 1 1 6
(multiple)
Carcinoma 3 2 3 1 7 0 3 0 3 2
Carcinoma 0 0 0 0 1 0 0 0 1 1
(multiple)
Adenoma and/or 11 9 10 13 27 6 8 3 6 21
carcinoma
* P<0.05
Additional ophthalmologic examinations were carried out in 13
weeks intervals and at the end of the study. There were no findings
that indicated any effect of ipronidazole treatment on the eye (Reno
et al., 1981).
2.2.3.2 Rats
A combined carcinogenicity and chronic toxicity study was
performed in rats. Diets containing 0, 20, 200 or 2000 ppm
ipronidazole were fed to groups of 50 Sprague Dawley derived CFY rats
of each sex per group for 109 weeks. The average consumption of
ipronidazole based on body weight was 0, 1, 10 or 110 mg/kg bw/day.
The rats were observed daily for clinical signs of toxicity and body
weights were recorded weekly for the first 15 weeks and every two
weeks thereafter. Comprehensive hematological examinations and
clinical chemical tests were performed on 15 rats of each sex of each
group; urine analysis was performed from pooled urine of 5 rats from
each sex per group. These examinations were done on pretest, and at 6,
13, 19 and 26 months. The body weights of male and female rats of the
2000 ppm group were significantly decreased at 51 weeks. Due to the
presence of large mammary tumors in some rats no statistical analysis
was carried out thereafter; however, during the second year of the
study group mean body weights of all treated groups were lower than
those of the controls. Food consumption was similar to controls in the
three dosed groups. Survival rates of male rats were similar in all
groups; survival rates of low and high dosed female rats were
decreased when compared to female controls.
An increase in the incidence of neoplasms of the mammary gland
was observed in high dosed females (incidence: 42/50, 32/50, 37/50,
and 48/50 at 0, 20, 200 and 2000 ppm, respectively). The time of
appearance of tumors was sooner in the 2000 ppm group; after 75 weeks
the incidences were 7, 12, 10, and 18 at 0, 20, 200, and 2000 ppm
respectively. In addition, the number of mammary tumors per tumor
bearing rat was increased in the high dosed group compared to controls
(mean number of mammary tumors per rat: 2.76, 2.72, 3.08, and 3.68 at
0, 29, 200 and 2000 ppm, respectively). Historical control data
obtained from three previous studies revealed a high incidence of
benign mammary tumors in untreated females; the incidences were 22/43,
31/44, and 29/47 in these studies. The results of this study indicate
an effect of ipronidazole on mammary tumor formation in high dosed
female rats. However, the high incidence of tumors in the control
animals, which is common in this rat strain, precluded the
determination of a definite NOEL.
No clinical signs could be attributed to the administration of
ipronidazole. Macroscopic and histological examinations of the tissues
of treated animals did not reveal any additional differences that
could be associated with ipronidazole intake. Changes were observed on
certain hematological and clinical biochemistry parameters. These
changes were slight and in view of the fact, that no such alterations
were observed in a 13-week-study, using higher dosages, these findings
were not considered to be of major biological significance (Brentnall
et al., 1977).
2.2.3.3 Dogs
Diets containing 0, 20, 200 or 2000 ppm ipronidazole were fed to
groups of 8 beagle dogs of each sex per group. An interim sacrifice of
2 dogs of each sex per group at about one year was scheduled. The
other dogs remained on this diet for 104 weeks. Dosage based on body
weight, was equal to 0, 0.51, 5.4 or 62 mg/kg/ bw/day. The dogs were
observed daily for clinical signs of toxicity and body weights were
recorded weekly. Every three months physical and ophthalomological
examinations and hematology, clinical biochemistry and urine analysis
were carried out. The body weights of the male and female dogs of the
2000 ppm group were significantly lower than in the controls at the
end of the study. Although average food consumption was variable
throughout the study, treated dogs tended to eat less than the
controls. However, no significant dose-response relationship was
established. With the exception of one male dog which died during
study week 99, all animals survived the study. Clinical signs observed
more frequently in the treated groups included dermatitis, otitis and
salivation. Ophthalomologic examinations revealed no drug related
effects. In both male and female dogs of the 2000 ppm group, alkaline
phosphatase was significantly increased throughout the study; in
addition, potassium levels were significantly decreased in males (with
the exception of 24 months) in females, potassium levels revealed
significant decreases only after 6 and 9 months. At sacrifice, fat
depletion was noted in 10/11 animals of the high dose group, relative
liver weights were significantly increased in males and females and
relative lung weights were increased in the females in the high dose
group. Histological examination showed an increase of
intrahepatocellular granular pigment in the high dose group animals.
In the lower dose groups no drug related effects were observed. In
this study the NOEL was 200 ppm, equal to 5.4 mg/kg bw/day for dogs
(Tucek, 1980).
2.2.4 Reproduction studies
2.2.4.1 Rats
In a three generation study CFY rats were fed diets containing 0,
20, 200 or 2000 ppm ipronidazole. The average consumption of
ipronidazole was equivalent to 0, 1, 10 or 100 mg/kg bw/day,
respectively. Each group consisted of 20 female and 10 male rats which
were fed these diets for 80 days after which the rats were mated.
Mating performance, parturition, litter size and postnatal growth were
unaffected by ipronidazole treatment. However, reduced growth in rats
of both sexes and in pregnant dams was observed at a dietary
concentration of 2000 ppm. The fertility indices were not affected,
although degenerative changes in the testes (damage of the tubules,
loss of spermatogenesis) occurred in one animal in all treated groups.
The NOEL for this study was 200 pm, equivalent to 10 mg/kg bw/day
(Dale, 1976).
2.2.5 Special studies on cataractogenicity
See under long-term/carcinogenicity studies.
2.2.6 Special studies on embryotoxicity and teratogenicity
2.2.6.1 Rats
Pregnant Füllingsdorf albino rats were treated by stomach tube
with doses of 0, 10 or 100 mg ipronidazole/kg bw/day from day 6 to day
15 of gestation. Each group consisted of 39 animals. Control and
treated animals were divided into two subgroups. Females from subgroup
1 were sacrificed on day 20 of gestation and uteri and fetuses were
examined microscopically. Females from the second subgroups were
allowed to give birth and to keep their young through the lactation
period. The young rats were then sacrificed on day 22 of age. No
skeletal or visceral malformations were found. Conception rate and
litter size, litter weight and resorption rates were similar in all
groups. Survival and development of the offspring were not affected.
Though the doses should have been higher in order to induce some overt
maternal toxicity, such as weight loss, the NOEL in this study was 100
mg/kg bw/day (Backes, 1976).
2.2.6.2 Rabbits
Pregnant Yellowsilver rabbits were treated by gavage with doses
of 0, 1, 10 or 100 mg/kg bw/day from day 6 to day 18 of gestation.
Each group consisted of 20 animals. On day 29 of gestation all animals
were sacrificed and autopsied. No drug related skeletal and visceral
malformations were observed in the fetuses. Toxic effects were found
in the 100 mg group in the form of significantly delayed maternal
weight development, sedation and a significant increase in the number
of resorbed fetuses. The NOEL was 10 mg/kg bw/day. (Backes, 1977).
2.2.7 Special studies on genotoxicity
Results of genotoxicity studies on ipronidazole
Concentration
Test System Test Object of ipronidazole Results Reference
Ames Test (1) S.typhimurium 0.1-0.5 mmol/1 Positive Cantelli-Forti
TA 100 et al., 1983.
Ames Test (1) S.typhimurium 1-4 mg/ml Positive Schüpbach, 1976
TA 1530
TA 1532
TA 1964
Fluctuation Klebsiella 0.02-1.0 mmol/l Positive Voogd, 1981
Test pneumoniae
E.coli 0.02-1.0 mmol/l Positive
Citrobacter 0.1-1.0 mmol/l Positive
freundii
Host Mediated S.typhimurium 100 or 150 Positive Schüpbach, 1976.
Assay TA1530 mg/kg bw
(Mouse) p.o.
(1)without rat liver S9 fraction.
Three other tests (Micronucleus Test, Dominant Lethal Test and Human
Cytogenetics Assay) were conducted. The results of these tests were
negative. However, the experimental designs of these studies were
inadequate (Schüpbach, 1976).
3. COMMENTS
Pharmacokinetic data from rats and data from studies on
carcinogenicity in mice and rats, mutagenicity, embryotoxicity and
teratogenicity in rats and rabbits and reproduction in rats, and from
long-term and short-term studies in rats and dogs were considered.
In the rat, after oral administration of 14C-labelled
ipronidazole, 27% of the total radioactivity is excreted in urine, 34%
in bile and 31% in the feces. Two hydroxylated metabolites still
containing the 5-nitro group have been identified in the feces of
turkeys and rats after oral administration of ipronidazole.
Ipronidazole showed mutagenic properties in bacterial test
systems. Because of the inadequate design of studies in mammalian test
systems, the Committee could not properly evaluate the genotoxic
potential of this drug.
In a carcinogenicity study in Charles River CD1 mice in which
ipronidazole was administered in the diet, a significant increase in
the incidence of benign proliferative lesions (adenoma and
hyperplasia) in the lung was observed at 1000 ppm in both sexes.
Although this type of tumor is common in this strain of mice, the
number of tumors also exceeded the range reported for the laboratory
historical controls. In this study, the no-observed-effect level was
200 ppm in the diet, equal to 30 mg/kg bw/day for mice.
In a combined carcinogenicity and chronic toxicity study with
Sprague Dawley CFY rats, dietary concentrations of ipronidazole of 0,
20, 200 and 2000 ppm were used. Although a high incidence of mammary
tumors was seen in all treated female groups (74-96%) and control
female rats (84%), the incidence was higher in the high-dose group
than in the controls. In addition, mammary tumors appeared sooner and
the number of mammary tumors per tumor-bearing rat was higher in the
females receiving high doses. The Committee concluded that the results
of this study indicated an effect of ipronidazole on mammary tumor
formation in female rats in the high-dose group. However, the high
incidence of tumors in the control animals, which is common in this
rat strain, precluded the determination of a definite
no-observed-effect level. Although the results of this study showed
changes in some hematological and clinical biochemical parameters,
similar effects were not observed in a 90-day rat study in which
higher doses of ipronidazole were used.
In a chronic toxicity study in dogs, which received ipronidazole
at 0, 20, 200 or 2000 ppm in the diet, decreased body weight, changes
in clinical biochemical values, fat depletion and changes in liver and
lung weight were observed in the high-dose group. The
no-observed-effect-level was 200 ppm bw/day, equal to 5.4 mg/kg
bw/day.
Studies on embryotoxicity and teratogenicity in rats and rabbits
did not reveal any effects at levels of 100 mg/kg bw/day for rats or
10 mg/kg bw/day for rabbits, which represent the
no-observed-effect-level for these studies.
In a three-generation study in rats fed diets containing 0, 20,
200 or 2000 ppm of ipronidazole, reduced growth was noted in the
highest-dose group. The fertility indices were not affected, although
degenerative changes in the testes occurred in one animal in each of
the treated groups. The no-observed-effect-level for this study was
200 ppm, equal to 10 mg/kg bw/day.
In a 13-week study in which rats were given ipronidazole in the
diet, a no-observed-effect-level of 80 mg/kg bw/day was established on
the basis of hepatocellular hypertrophy.
In a 13 week study in dogs in which the compound was administered
in capsules, all of the dogs in the highest-dose group either died or
were taken off treatment. Loss of body weight, dehydration and ataxia
were the most prominent signs of toxicity. These clinical signs
occurrred to a lesser degree in the middle-dose group. The
no-observed-effect-level was 20 mg/kg bw/day in this study.
The Committee was not able to establish an ADI because the rat
carcinogenicity study was inadequate to determine a no-effect level
for ipronidazole.
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