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. 5. REFERENCES ASSANDRI, A., PERAZZI, A., ZERILLI, C.F., FERRARI, O., & MARTINELLI, E. (1978). Metabolism of 5-isopropyl-1-methyl-2-nitro-1H-imadozole. Drug Metab.Dispos., 6, 109-113. BACKES, G. (1976). Embryotoxic studies in the rat by oral administration of Ro-7-1554 (Ipronidazole). Unpublished report from Hoffmann-La Roche, Inc. Nutley, N.J., USA. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. BACKES, G. (1977). Embryotoxic studies in rabbits by oral administration of Ro-7-1554 (Ipronidazole). Unpublished report from Hoffmann-La Roche, Inc., Nutley, N.J., USA. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. BANARJEE, B.N., HOWARD, D.J., WOODARD, M.W. & WOODARD, G. (1968). Ro-7-1554, safety evaluation by repeated oral administration to rats for 13 weeks. Unpublished report from Woodard Research Corporation, Herndon, Virginia, USA. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. BANZINGER, R. & HANE, D. (1967). Acute toxicity and dog tolerance testing of Ro-7-1554 (Histomonacide). Unpublished report from Hoffman-la Roche, Inc., Nutley, N.J., USA. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. BRENTNALL, D.W., DALE, E., INGHAM, B. & FARR, M.J. (1977). Ipronidazole: Toxicity and tumorgenicity study in rats. Unpublished report from May & Baker, Ltd., Dagenham, Essex, England. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. CANTELLI-FORTI, G., ALCARDI, G., GUERRA, M.C., BARBARO, A.M. & BIAGI, G.L. (1983). Mutagenecity of a series of 25 nitroimadazoles and two nitrothiazoles in Salmonella typhimurium. Terat.Carcinog.Mutag., 3, 51-63. DALE, M.J. (1976). Ipronidazole: study of effects on reproductive performance in rats for three generations. Unpublished report from May & Baker, Ltd., Dagenham, Essex, England. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. POOL, W. & STEIVIS, D. (1968). Acute neonatal toxicity of Ro-7-1554 (Ipronidazole). Unpublished report from Hoffmann-La Roche, Inc., Nutley, N.J., USA. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. RENO, F.E., DUDEK, L.E., KUNDZIUS, W., VOELKER, R.W. & MISTRETTA, L.M. (1981). Chronic toxicity study in mice, ipronidazole base (Ro-7-l554), final report. Unpublished report No. 131-128 from Hazleton Laboratories America Inc., Vienna, VA, USA. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. SADEK, S. & BANZINGER, R. (1968). Preclinical toxicity study of Ro-1554 in rats and dogs for thirteen weeks. Unpublished report from Hoffmann-La Roche, Inc., Nutley, N.J., USA. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. SCHUPBACH, M. (1976). Mutagen tests with Ro-7-1554 (Ipronidazole). Unpublished report from Hoffmann-La Roche, Inc., Nutley, N.J. USA. Submitted to WHO by the US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. TUCEK, P.C. (1980). Chronic feeding study in dogs. Unpublished report from International Research and Development Corporation, Mattawan, Michigan. Submitted to WHO by US Coordinator of the Codex Alimentarius, US Department of Agriculture, Washington, D.C. WEISS, G., ROSE, N., DUKE, P. & WILLIAMS, T.H. (1981). A major rat faecal metabolite of irponidazole. Xenobiotic, 11, 207-215. VOOGD, C.E. (1981). On the mutagenicity of nitroimiedazoles. Mut.Res., 3, 243-277.
See Also: Toxicological Abbreviations IPRONIDAZOLE (JECFA Evaluation)