DIMINAZENE First draft prepared by Dr M. Miller Center for Veterinary Medicine Food and Drug Administration Rockville, Maryland, USA 1. EXPLANATION Diminazene is a veterinary drug with a long history of use for the treatment of trypanosomiasis and babesiosis. Diminazene was evaluated at the thirty-fourth meeting of the Committee (Annex 1, reference 85). At that time the Committee was unable to establish an ADI and suggested that additional genotoxicity and teratogenicity studies should be performed before bringing the compound to the Committee for re-evaluation. This monograph addendum, which supplements the information provided in the original toxicological monograph (Annex 1, reference 86), summarizes the data that have become available since the previous evalution. 2. BIOLOGICAL DATA 2.1 Biochemical aspects The excretion of diminazene into milk was examined in four lactating cows, each treated with one i.m. dose of diminazine (3.56 mg diminazene/kg bw). The diminazene content of milk collected at 7.5, 24, 31.5, 48, 55.5, 72, 240, 360, and 480 hours after application was quantitated by an HPLC method with a detection limit of 50 ng/ml. The results showed that the diminazene concentration in milk from all 4 cows was below the detection limit of the assay at all time points (Klatt & Schmidt, 1991a). Residue levels of diminazene were determined in plasma and tissues of 14 young cattle following one i.m. administration of 3.56 mg diminazene/kg bw. Blood plasma levels were measured for 35 days after application and tissues residue analyses were conducted in liver, kidney, muscle, and injection site muscle at 21, 28, and 35 days post-treatment. The study reported that a plasma level of 1250 ng/ml was acheived one day after drug administration. This value decreased to 350 ng/ml after 7 days. After 25-28 days, plasma levels of diminazene were at or below the detection limit of the assay (50 ng/ml). The highest tissue concentration of diminazene occurred at the injection site followed by liver and kidney. The lowest values were found in the muscle (Klatt & Schmidt, 1991b). 2.2 Toxicological studies 2.2.1 Special studies on embryotoxicity 2.2.1.1 Rats The embryotoxicity of diminazene dissolved in distilled water was studied in mated female Wistar rats. Prior to the start of the study, the females were mated with fertile males. Pregnant animals (determined by detection of spermatozoa in the vaginal smear) were randomly assigned to 4 groups of 22-24 animals each. The test groups were gavaged daily on the seventh to sixteenth days of pregnancy with 5 ml of distilled water (control), or 200, 400, or 800 mg of diminazene/kg bw/day. Behaviour and general physical condition were assessed daily. Feed consumption was monitored continuously and body weight was checked once weekly. On day 21 of pregnancy, the dams were killed and delivered by caesarean section. The uteri were opened and live and dead fetuses, concepta under resorption, placentas, and corpora lutea were counted and examined microscopically. Increased salivation was observed sporadically in several treated animals at all doses. This effect was attributed to local irritation resulting from the compound's administration. Doses of 200 and 400 mg/kg bw/day did not affect general physical condition, feed consumption, or body weight development. The 800 mg/kg bw/day dose was maternotoxic, causing a decrease in food consumption, an increase in spleen weight, and the death of five dams. No effects on intrauterine development of the conceptuses were observed or the low and middle doses. Fetal development in dams treated with 800 mg diminazene/kg bw/day was retarded, as indicated by a slight increase in the number of fetuses with body weights under 3 g, poor skeletal ossification and low placental weights. Morphological examination of fetuses from the treated dams revealed no malformations. The incidence of minor anomalies and variations in fetal development in the compound-treated groups were no higher than incidences observed in the contemporary controls and were also within the range of historical controls. Because materno- and embryotoxicity were observed at 800 mg/kg bw/day, the NOEL was 400 mg/kg bw/day under the study conditions (Baeder et al., 1991). Diminazene dissolved in deionized water was administered to pregnant rats daily by gavage on days 8-15 of pregnancy at dose levels of 0 (vehicle only), 100, 250, 500, or 1000 mg/kg bw/day. On day 21 of pregnancy, the dams were killed and the number of implants, resorptions and live fetuses counted. All fetuses were examined by routine teratological methods. A significant increase in fetal resorptions and a decrease in fetal body weights were observed at 1000 mg/kg bw/day dose. No significant increase in the incidence of fetal anomalies was observed in external, skeletal, and internal examinations even at the maternally toxic dose of 1000 mg/kg. These data suggest that diminazene is not teratogenic. The NOEL was 500 mg/kg bw/day (Yoshimura, 1990). 2.2.2 Special studies on genotoxicity Table 1 summarizes the results of genotoxicity assays on diminazene. Table 1. Results of genotoxicity assays with diminazene Test system Test object Concentration Results Reference Ames test1 S.typhimurium 0-500 µg/ml Negative Muller, 1989 TA100, 1535, 1537, 1538, 98 E. coli WP2uvrA Micronucleus Mouse bone test2 marrow 0 and 1500 mg Negative Muller, 1988a Forward Fetal hamster 10-100 µg/ml Negative Muller, mutation assay lung V79 (-S9 mix)3 1988b (HGPRT locus) 10-150 µg/ml Negative (+S9 mix)4 1 Both with and without rat liver S9 fraction. 2 Reviewed at the thirty-fourth Meeting of the Committee. 3 Ethylmethansulfonate was used as the positive control. 4 9,10-dimethyl-1,2-benzanthracene was used as the positive control. 3. COMMENTS Diminazene was negative in the micronucleus test and in bacterial and mammalian genotoxicity assays. These data together with the data from the subchronic toxicology studies, which showed no lesions predictive of a carcinogenic response, suggest that there is no concern for the carcinogenic potential of the compound. The embryotoxicity of diminazene was examined in female Wistar rats dosed by gavage with 0, 200, 400, or 800 mg diminazene/kg bw/day on days 7 to 16 after mating. A dose of 800 mg/kg bw/day was maternotoxic, causing a decrease in food consumption and increased spleen weight and death. This dose also caused retarded fetal development. The NOEL for this study was 400 mg/kg bw/day. In another embryotoxicity study, diminazene was administered to rats by gavage on days 8 to 15 of pregnancy at dose levels of 0, 100, 250, 500, or 1000 mg/kg bw/day. The 1000 mg/kg bw/day dose produced maternotoxicity and a significant increase in the number of fetal resorptions and a decrease in fetal body weights. The NOEL for this study was 500 mg/kg bw/day. No fetal malformations were detected in either study. The previous monograph on diminazene (Annex 1, reference 86) summarized the toxicity studies in rats, dogs, and various food-producing animals. At low doses, i.m. administration of diminazene produced central nervous system lesions in several species including cows, donkeys, camels, and dogs. In dogs fed diminazene at 20 or 60 mg/kg bw/day for nine months, the higher dose caused brain damage, testicular atrophy and death. Rats dosed by gavage with 63 or 160 mg/kg bw/day for 3 months showed no signs of toxicity. This indicates that the rat is not a sensitive model for evaluating the toxicity of diminazene, including reproductive toxicity. The NOEL in this study was 20 mg/kg bw/day. 4. EVALUATION In keeping with general principles for the evaluation of veterinary drugs with a long history of use described at the fortieth Meeting of the Committee (Annex 1, reference 104), an ADI of 0-100 µg/kg bw/day was established for diminazene based on the NOEL of 20 mg/kg bw/day in the nine-month study in dogs and using a 200-fold safety factor. The Committee chose this safety factor to compensate for the inadequacies in the design of the study. 5. REFERENCES BAEDER, C., MAYER, D. & LANGER, K.H. (1991). Oral embryotoxicity study of Berenil in Wistar rats (Segment II). Unpublished report No.91.1190 from Pharma Development Toxicology, Hoechst Aktiengesellschaft, Frankfurt, Germany. Submitted to WHO by Hoechst AG, Frankfurt, Germany. KLATT, P. & SCHMIDT, D. (1991a) Report on the excretion of diminazene aceturate in the milk of cows after one intramuscular administration of the commercial preparation berenil(R) for veterinary use. Unpublished report No. K1 90/1 from Abt. Forschung Tiergesundheit, Hoechst AG, Frankfurt, Germany. Submitted to WHO by Hoechst AG, Frankfurt, Germany. KLATT, P. & SCHMIDT, D. (1991b) Report on blood plasma level determinations and residue studies in organs of cattle after one intramuscular administration of the commercial preparation berenil(R) for veterinary use. Unpublished report No. K1 90/1 from Abt. Forschung Tiergesundheit, Hoechst AG, Frankfurt, Germany. Submitted to WHO by Hoechst AG, Frankfurt, Germany. MULLER, W. (1988a). Diminazendiaceturat (Berenil) micronucleus test in male and female NMRI mice after oral administration. Unpublished report No.88.0248 from Pharma Research Toxicology and Pathology, Hoechst Aktiengesellschaft, Frankfurt, Germany. Submitted to WHO by Hoechst AG, Frankfurt, Germany. MULLER, W. (1988b). Berenil: Detection of gene mutations in somatic cells in culture HGPRT-test with V79 cells. Unpublished report No.88.0990 from Pharma Research Toxicology and Pathology, Hoechst Aktiengesellschaft, Frankfurt, Germany. Submitted to WHO by Hoechst AG, Frankfurt, Germany. MULLER, W. (1989). Berenil: Study of the mutagenic potential in strains of Salmonella typhimurium (Ames test) and Escherichia coli. Unpublished report No.88.1425 from Pharma Research Toxicology and Pathology, Hoechst Aktiengesellschaft, Frankfurt, Germany. Submitted to WHO by Hoechst AG, Frankfurt, Germany. YOSHIMURA, H. (1990). Teratological assessment of the antiprotozoal, diminazene diaceturate, in rats. Toxicol Lett., 54: 55-59.
See Also: Toxicological Abbreviations Diminazene (WHO Food Additives Series 25) DIMINAZENE (JECFA Evaluation)