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. 5. REFERENCES ALI, B.H. & HAROUN, E.M. (1984). Acute toxicity of Samorin (Isometamidium chloride) in rabbits. Comp. Biochem. Physiol., 78C: 419-423. BOSC, F., HUET, A.M. AUMONT, D., CAMPAGNA, J.F., ROLLAND, M.L. & WEIL, A. (1991a). Determination of isometamidium concentrations in rat plasma following intravenous administration of Trypanidium at a dose of 2 mg/kg. Description of animal phase carried out by Rhône Mérieux. Unpublished report No. MET 161 from Rhône Mérieux, Toulouse, France. Submitted to WHO by Rhône Mérieux, Toulouse, France. BOSC, F., HUET, A.M. AUMONT, D., CAMPAGNA, J.F., ROLLAND, M.L. & WEIL, A. (1991b). Determination of Isometamidium concentrations in rat plasma following repeated oral administration of Trypanidium at different doses during 21 days. Description of animal phase carried out by Rhône Mérieux. Unpublished report No. MET 162 from Rhône Mérieux, Toulouse, France. Submitted to WHO by Rhône Mérieux, Toulouse, France. BRIDGES, B.A. (1991). Genetic toxicity assessment of isometamidium. Unpublished report from the University of Sussex, Brighton, England. Submitted to WHO by Rhône Mérieux, Toulouse, France. BROOKER, A.J. & MYERS, D.P. (1991). Isometamidium : a screening study in pregnant and lactating rats for developmental toxicity. Unpublished report No. MRX 4 & 5/91167 from Huntingdon Research Centre Ltd, Huntingdon, England. Submitted to WHO by Rhône Mérieux, Toulouse, France. CLARE, C.B. (1989). Study to determine the ability of isometamidium chloride to induce mutations to 6-thioguanine resistance in mouse lymphoma L5178Y cells using a fluctuation assay. Unpublished report No. MAB 25/ML from Microtest Research Ltd, Heslington, United Kingdom. Submitted to WHO by Rhône Mérieux, Toulouse, France. COPPING, G.P. & EAST, P.W. (1986). Isometamidium chloride. Teratogenicity study by the intravenous route in the rat. Unpublished report No. R. Tox. 594 from May & Baker. Submitted to WHO by RMB Animal Health Ltd, Essex, England. Cited in toxicological evaluation of certain veterinary drug residues in food. WHO Food Additive Series, No. 25, p.135, 1990. CRICHTON, C., McGREGOR, D.B. & SMITH, I. (1977). Testing for mutagenic activity in isometamidium chloride and dinitrophenylphenanthridine. Unpublished report No. 894 from Inveresk Research International, Edinburgh, Scotland. Submitted to WHO by Rhône Mérieux, Toulouse, France. IRVINE, L.H.F. (1991). Review of May and Baker Report Number : R. Tox. 495 in comparison with background data compiled from studies conducted at Toxicol Laboratories. Unpublished report from Toxicol Laboratories Ltd, Ledbury, England. Submitted to WHO by Rhône Mérieux, Toulouse, France. KINABO, L.D., BOGAN, J.A., McKELLAR, Q.A. & MURRAY, M. (1989). Relay Bioavailability and Toxicity of Isometamidium Residues : A Model for Human Risk Assessment. Veterinary and Human Toxicology, 31: 417-421. KIRKLAND, D.J. (1984). Study to evaluate the chromosome damaging potential of M & B 2360 and M & B 4180A by their effects on the bone marrow cells of treated rats. Unpublished report No. MAB 3 & 4/RBM/AR/KF8 from Microtest Research Ltd, Heslington, United Kingdom. Submitted to WHO by Rhône Mérieux, Toulouse, France. LIGGETT, M.P. (1989). Acute oral toxicity to rabbits of isometamidium chloride. Unpublished report No. 891016D/RAH5/AC from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by Rhône Mérieux, Toulouse, France. MARSHALL, R.R. (1990). Study to evaluate the chromosome damaging potential of isometamidium chloride by its effects on cultured human lumphocytes using an in vitro cytogenetics assay. Unpublished report No. MAB 25/HLC from Microtest Research Ltd, Heslington, United Kingdom. Submitted to WHO by Rhône Mérieux, Toulouse, France. MATHESON, D.W. (1978). Evaluation of isometamidium chloride Batch H508 in the in vitro transformation of BALB/3T3 cells assay. Unpublished report No. 20840 by Litton Biometrics Inc., Kensington, MD, USA. Submitted to WHO by Rhône Mérieux, Toulouse, France. MIGNOT, A. & LEFEBVRE, M.A. (1991). Determination of isometamidium concentration in plasma of rats after intravenous administration and repeated oral administrations during 21 days. Unpublished report No. CB273 from Centre d'Etudes et de Recherche en Pharmacie Clinique, Saint-Benoît, France. Submitted to WHO by Rhône Mérieux, Toulouse, France. PALMER, A.K. (1992). Isometamidium and vertebral abnormality. Unpublished report from Huntingdon Research Centre Ltd, Huntingdon, England. Submitted to WHO by Rhône Mérieux, Toulouse, France. PETERS, D.H. STUART, V., CROOK, D., GIBSON, W.A., READ, R. & GOPINATH, C. (1991). Samorin (a commercial form of isometamidium chloride) toxicity to rats by repeated oral administration for 13 weeks. Unpublished report No. RAH 7/901283 from Huntingdon Research Centre Ltd, Huntingdon, England. Submitted to WHO by Rhône Mérieux, Toulouse, France. PROUDLOCK, R.J. (1991a). Isometamidium. Assessment of nuclear anomalies in rat stomach following 13-week oral exposure. Unpublished report No. MRX 6A/91822 from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by Rhône Mérieux, Toulouse, France. PROUDLOCK, R.J. (1991b). Isometamidium. Assessment of nuclear anomalies in various organs of weanling rats after acute oral exposure. Unpublished report No. MRX 6B/91823 from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by Rhône Mérieux, Toulouse, France. PROUDLOCK, R.J. & CAVALIERE, C. (1990). Isometanidium : analysis of rat bone marrow chromosomes following 13-week oral exposure. Unpublished report No. RAH 8/90932 from Huntingdon Research Centre Ltd, Huntingdon, England. Submitted to WHO by Rhône Mérieux, Toulouse, France.
See Also: Toxicological Abbreviations Isometamidium (WHO Food Additives Series 25) ISOMETAMIDIUM (JECFA Evaluation)