INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION TOXICOLOGICAL EVALUATION OF CERTAIN VETERINARY DRUG RESIDUES IN FOOD WHO FOOD ADDITIVES SERIES 41 Prepared by: The 50th meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) World Health Organization, Geneva 1998 NICARBAZIN First draft prepared by Dr G. Roberts Chemical Products Assessment Section Commonwealth Department of Health and Family Services Canberra, Australia 1. Explanation 2. Biological data 2.1 Biochemical aspects 2.1.1 Absorption, distribution and excretion 2.2 Toxicological studies 2.2.1 Acute toxicity 2.2.2 Short-term toxicity 2.2.3 Long-term toxicity and carcinogenicity 2.2.4 Genotoxicity 2.2.5 Reproductive toxicity 3. Comments 4. Evaluation 5. References 1. EXPLANATION Nicarbazin has been used in starter rations for several decades as an aid in the prevention of faecal and intestinal coccidiosis in broiler chickens. It may be used in combination with ionophore coccidiostatics. Chemically, it is an equimolar complex of 1,3- N,N'-bis(4-nitrophenyl)urea and 4,6-dimethyl-2(1 H)-pyrimidone. These compounds are also known as 4,4 '-dinitrocarbanilide and 2-hydroxy-4,6-dimethylpyrimidine, respectively (see Figure 1). Nicarbazin is described as an electron donor-acceptor molecular complex; the sites of the interaction are the electron-poor NH amide groups of the acceptor phenylurea and the electron-rich lone pairs of the nitrogen in the pyrimidone donor ring. Nicarbazin has not previously been evaluated by the Committee.2. BIOLOGICAL DATA 2.1 Biochemical aspects Very little information was available on the absorption, distribution, biotransformation, and excretion of nicarbazin in laboratory animals. The available data were presented with few or no methodological details, and the results were given in summary form only, providing no opportunity for independent confirmation of the conclusions. 2.1.1 Absorption, distribution, and excretion Rats received single oral doses of 1, 5, or 10 mg/kg bw nicarbazin; one animal at each dose was killed 6 or 18 h after treatment, and the blood concentrations of the phenylurea and pyrimidone components were determined. Low concentrations of the phenylurea component were detected at 6 h, but it was not detected at 18 h. The pyrimidone component was found at considerably higher concentrations, which increased between 6 and 18 h. Qualitatively similar findings were obtained in rats given oral doses of 0.1, 1, or 5 mg/kg bw per day for eight days and killed 4 or 24 h after the last dose. The blood concentrations of the pyrimidone component were dose-related, while those of the phenylurea component showed a flat response. In the latter experiment, urine collected for 5 h after the last dose contained dose-related concentrations of each component of nicarbazin, although the concentrations of the pyrimidone were an order of magnitude higher than those of the phenylurea component (Kuna, 1955). 2.2 Toxicological studies 2.2.1 Acute toxicity The results of studies on the short-term toxicity of nicarbazin and its components are shown in Table 1. 2.2.2 Short-term toxicity A number of short-term studies of nicarbazin were available, but the reports were inadequate for detailed evaluation as they contained minimal details of the protocols used, limited data on toxicological findings, and were often in the form of progress reports. The summaries reported kidney damage in the form of crystalline deposits in the collecting tubules in rats at oral doses of 500 mg/kg bw per day and more. In dogs, bile-duct proliferation was the principal finding after an oral dose of 1600 mg/kg bw per day (Kuna, 1955). Table 1. Acute toxicity of nicarbazin, 4,4'-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) after oral administration Species Sex Substance LD50 (mg/kg bw) Mouse Unspecified Nicarbazin > 25 000 HDP approx. 4 000 DNC > 18 000 Rat Unspecified Nicarbazin > 10 000 From Kuna (1955) Dogs Groups of five male and five female beagle dogs were fed diets containing the phenylurea and the pyrimidone components (purity unspecified) in a ratio of 3:1 on six days per week for two years. The actual intakes were 0, 60, 180, or 600 mg/kg bw per day of the phenylurea component and 0, 20, 60, or 200 mg/kg bw per day of the pyrimidone component. Two animals of each sex per group were killed after one year. Clinical observations were made daily, and body weight, food consumption, and reflexes were determined weekly; water intake and urinary output were measured monthly. Haematological, clinical chemical, and urinary parameters were determined before treatment and in months 3, 6, 12, 18, and 24. A wide range of tissues from all dogs was examined grossly and microscopically. The study was conducted before the development of guidelines for the conduct of toxicological studies. No abnormal behaviour or physical signs were seen; however, one male at the intermediate dose died of unknown causes during week 44. A green-to-yellowish hue was seen in the excreta of all treated dogs. Body-weight gain, food intake, and haematological and urinary parameters were unaffected by treatment. Serum alanine aminotransferase activity was increased in several dogs at the highest dose and in one dog at each of the lower doses, but in most cases the effects were transitory. The highest values were observed at about 12 months, and elevated activity persisted in only two animals at the high dose. Organ weights and gross pathological appearance revealed no treatment-related changes. The histopathological appearance was unremarkable, apart from slight bile-duct proliferation in one dog killed after one year of treatment with the high dose. This animal had been found to have elevated serum alanine aminotransferase activity. Although the relationship between the hepatic findings and treatment was unclear, the conservative NOEL in this study is 240 mg/kg bw day (Vogin, 1969a). 2.2.3 Long-term toxicity and carcinogenicity Rats Groups of FDRL rats were fed diets containing the phenylurea and the pyrimidone components (purity unspecified) for two years at concentrations calculated to give doses of 0, 50, 150, or 300 mg/kg bw per day of the phenylurea component and 0, 17, 50, or 100 mg/kg bw per day of the pyrimidone component. The groups consisted of 50 males and 50 females for control and the high doses and 40 males and 40 females for the low and intermediate doses. Five rats of each sex from the control and high-dose groups were killed during months 6 and 18, and 10 rats of each sex per group were killed in week 56. The animals were observed daily for behaviour, physical appearance, and survival. Food consumption and the efficiency of food use were assessed weekly for the first 12 weeks and then periodically on 15 animals of each sex per group. Body weight was recorded weekly for the first 12 weeks, then biweekly until week 26 and monthly thereafter. Water intake and urinary output were measured on 10 rats of each sex per group during one week per month for the first three months. Limited haematological, clinical chemical and urinary parameters were examined on 10 rats of each sex per group at 3, 6, 9, 12, 18, and 24 months. A wide range of tissues from all rats was examined grossly and microscopically. The study was conducted before the development of guidelines for the conduct of toxicological studies. No abnormal behaviour was noted, and mortality was unaffected by treatment. Food and water intake and body-weight gain were similar in all groups. There were no treatment-related effects on haemoglobin, haematocrit, leukocytes, blood urea nitrogen, serum alanine aminotransferase activity, blood glucose, urinary parameters, organ weights, or gross pathological appearance. Changes in the kidney, such as calcareous material in the tubules, calcification in the renal pelvis, tubules, glomeruli, or medulla, and calculi, were more frequent in treated groups at 56 weeks, but the overall incidence was similar in all groups at the end of the study. The incidence of testicular atrophy was slightly elevated in some treated males at 104 weeks, but significance was not attained. Tumour incidences were unaffected. The NOEL was 400 mg/kg bw per day of the 3:1 mixture (Vogin, 1969b). 2.2.4 Genotoxicity The results of assays for the genotoxicity of nicarbazine and its components are shown in Table 2. Table 2. Results of assays for genotoxicity with nicarbazine, 4,4'-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) End-point Test object Substance Concentration S9 Results Reference (µg/plate) Reverse S. typhimurium Nicarbazine 100-500a +/- Negative Bradley & mutation TA98, TA100, DNC 100-300a +/- Negative Cook (1980) TA1535, TA1537 HDP 200-2000 +/- Negative Reverse S. typhimurium Nicarbazine 0-10 000 +/- Weakly Ohta et al. mutation TA98, TA1538 positive (1980) TA100, TA1535, NR +/- Negative TA1537 E coli WP2 hcr trp NR +/- Negative DNA H17Rec+ Nicarbazine NR +/- Negative Ohta et al. damage M45Rec- (1980) S9, 9000 × g fraction of rat liver; NR, not reported a Higher concentrations precipitated. 2.2.5 Reproductive toxicity (i) Multigeneration reproductive toxicity Rats Groups of 12 male and 12 female FDRL rats were fed diets containing the phenylurea and the pyrimidone components (purity unspecified) at concentrations calculated to achieve doses of 0, 50, 150, or 300 mg/kg bw per day of the phenylurea and 0, 17, 50, or 100 mg/kg bw per day of the pyrimidone. Treatment was administered continuously during the production of two litters per generation for three successive generations. The initial groups of animals were paired 10 weeks after the start of treatment to produce the F1a litter and were paired again seven days after weaning of the first litter to produce the F1b litter. At four weeks of age, 12 male and 12 female F1b offspring were mated according to the above schedule to produce the F2a and F2b litters, and the F2b offspring were used to produce the F3a and F3b litters. The animals were examined daily for survival, behaviour, and appearance. Body weight and food consumption were measured weekly in adults. After birth, each litter was limited to eight pups, which were weighed at birth and on postnatal days 4, 12, and 21. After the offspring had been weaned, the adult rats were autopsied, and the testes of F2 males in the control and high-dose groups were examined histopathologically. The liver, kidneys, heart, urinary bladder, and gonads from five male and five female F3b weaned pups from each group were also examined microscopically. The study was performed before the development of guidelines for the conduct of toxicological studies. Adult rats of each generation showed no effects on survival, body-weight gain, or food intake, and the results of gross autopsy and testicular examinations were unremarkable. The pregnancy rates and duration of gestation were unaffected. The body-weight gain of F1b pups at the high dose was slightly depressed during lactation, but a similar effect was not found in any other litter. In subsequent generations, the F2a and F3a litters at the high dose had slightly fewer pups, but the effect was not reproduced in the F2b or F3b litters. Histopathological examination of limited organs from F3b pups revealed no abnormalities attributable to treatment, and it was considered that there were no significant effects on reproduction. The NOEL was the highest dose tested, 400 mg/kg bw per day of the 3:1 mixture (Kirschner & Vogin, 1970). (ii) Developmental toxicity Rats Groups of 24-25 CD/CRJ pregnant rats were given nicarbazin (equimolar complex; purity unspecified) as a suspension in 1% carboxymethyl cellulose by gavage at doses of 0, 70, 200, or 600 mg/kg bw per day on gestation days 7-17. Details of the method were not given in the report, but the tabulated results indicate that food and water intake and body weights were recorded daily on gestation days 7œ21. The times of sacrifice of dams and examination of fetuses were not indicated. Fetuses were subjected to external, visceral, and skeletal examinations. Since the protocol was not provided, it is not known whether a recognized test guideline was followed or if quality assurance was undertaken, and the adequacy of the study could not be determined. Seven rats at 600 mg/kg bw per day died, mostly during the treatment period. The food intake and body-weight gain of dams at 600 mg/kg bw per day were depressed from gestation day 8. Implantations and in-utero survival were similar in all groups, while fetal body weight was lower at 600 mg/kg bw per day. The fetuses of the dams at 600 mg/kg bw per day had delayed ossification, hyperplastic and bent ribs (four fetuses), sacralization of the 6th or 7th lumbar vertebrae (two fetuses), cleft palate (two in same litter), subcutaneous oedema (three in same litter), hydronephrosis (five fetuses), cryptorchismus (one fetus), and remained Merkel's diverticulum (one fetus). The NOEL for maternal and fetal toxicity was 200 mg/kg bw per day (Tajima, 1979). 3. COMMENTS The Committee considered the results of studies on the toxicokinetics, acute, short-term, and long-term toxicity, genotoxicity, reproductive toxicity, and developmental toxicity of nicarbazin. The studies were performed before the establishment of guidelines for the conduct of toxicological studies. Some of the reports were presented in sufficient detail for independent assessment and were considered of acceptable quality. Additionally, an expert report was available for consideration (Fitzpatrick, 1997). After oral administration of nicarbazin to rats, low concentrations of the phenylurea component and high concentrations of the pyrimidone were found in blood. The urinary excretion of the latter was considerably higher than that of the phenylurea. The pyrimidone portion is therefore probably absorbed to a greater extent, and most of the phenylurea was excreted in faeces without absorption. No data were available on the metabolism of nicarbazin. The acute oral toxicity of nicarbazin in rodents was low, the LD50 values being > 25 000 mg/kg bw in mice and > 10 000 mg/kg bw in rats. The individual components also had low acute toxicity, the oral LD50 values in mice being 4000 mg/kg bw for the pyrimidone and > 18 000 for the phenylurea component. A number of short-term studies of nicarbazin were available, but the reports were inadequate for detailed evaluation as they contained minimal details of the protocols used and limited data on toxicological findings and were often in the form of progress reports. The summaries reported kidney damage in the form of crystalline deposits in the collecting tubules in rats at oral doses of 500 mg/kg bw per day and more. In dogs, bile-duct proliferation was the principal finding after an oral dose of 1600 mg/kg bw per day. The highest dose chosen in the two-year study of toxicity and in the study of reproductive toxicity in rats was 400 mg/kg bw per day. This dose was selected because of the formation of acetylated phenylurea, with resulting precipitation of crystals in the kidneys, in rats given oral doses of 500 mg/kg bw per day. In some toxicological studies, the test species were given phenylurea and pyrimidone components in a ratio of 3:1, since it was claimed that phenylurea and pyrimidone are present in that ratio in the muscle of treated chickens. More recent data suggest a ratio as high as 8:1 for the phenylurea to the pyrimidone residue. Dogs were fed diets containing phenylurea and pyrimidone components in a ratio of 3:1 for two years. The actual drug intakes were 0, 80, 240, or 800 mg/kg bw per day. Serum alanine aminotransferase activity was increased in several dogs, and slight bile-duct proliferation was observed in one dog at 800 mg/kg bw per day. No other treatment-related effect was observed. The NOEL was 240 mg/kg bw per day of the 3:1 mixture of phenylurea and pyrimidone. In the two-year study in rats, doses of 0, 67, 200, or 400 mg/kg bw per day phenylurea and pyrimidone components in a ratio of 3:1 were given in the diet. There was no treatment-related toxicity, and tumour incidences were unaffected. The NOEL was the highest dose, 400 mg/kg bw per day. Nicarbazin slightly increased the mutation frequency in Salmonella strains TA98 and TA1538 in one study but not in another. Mutations were not detected in Salmonella strains TA100, TA1535, or TA1537 or in E. coli WP2, and DNA damage was not induced in the rec assay. No other end-points were investigated. The examination of genotoxic potential was considered to be inadequate, since studies were carried out only in bacteria. A three-generation study of reproductive toxicity was conducted in rats given a 3:1 ratio of phenylurea and pyrimidone components in the diet at doses were 0, 67, 200, or 400 mg/kg bw per day. There were isolated occurrences of slightly reduced litter size at birth or depressed body-weight gain during lactation at the highest dose, but these findings were not replicated in most litters and showed no progression over the duration of the study. Therefore, the Committee concluded that nicarbazin did not have significant effects on reproduction. The NOEL was the highest dose tested, 400 mg/kg bw per day. Developmental toxicity was studied in rats given 0, 70, 200, or 600 mg/kg bw per day of nicarbazin, at an equimolar ratio of phenylurea: pyrimidone, by gavage on gestation days 7-17. At 600 mg/kg bw per day, maternal food intake and body weight were depressed, and seven of 25 animals died. At this dose, the finding of lowered fetal body weight and reduced ossification suggested retarded fetal development, and a number of abnormalities were observed, in particular hydronephrosis and hyperplastic and bent ribs. The NOEL was 200 mg/kg bw per day on the basis of maternal and fetal toxicity; no teratogenic effects were observed. 4. EVALUATION The Committee noted the absence of certain toxicological studies in support of nicarbazin; however, the other data available provided sufficient information to overcome most of these deficiencies. It was noted that nicarbazin has been used in veterinary medicine in many countries for over 40 years. On the basis of this long history of use and the fact that use is restricted to starter rations in broiler chickens, the Committee considered that an ADI could be supported. The Committee established an ADI of 0-400 µg/kg bw on the basis of the NOEL of 200 mg/kg bw per day in the study of developmental toxicity in rats and using a safety factor of 500, chosen to account for the limitations in the database. 5. REFERENCES Bradley, M.O. & Cook, M.M. (1980) Nicarbazin: Microbial mutagen tests. Unpublished report. Submitted to WHO by Koffolk, Rancho Santa Fe, California, USA. Fitzpatrick, S.C. (1997) Nicarbazin: Evaluation of available toxicology data on nicarbazin: Rewrite format as outlined in WHO Technical Report 832, Veterinary Drugs with a Long History of Use. Unpublished report. Submitted to WHO by Koffolk, Rancho Santa Fe, California, USA. Kirschner, S.L. & Vogin, E.E. (1970) Multigeneration reproduction and lactation studies with 4,4'-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine dihydrate (HDP). Unpublished report from Food and Drug Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by Koffolk, Rancho Santa Fe, California, USA. Kuna, S. (1955) Tolerance studies in mammals. Unpublished report. Submitted to WHO by Koffolk, Rancho Santa Fe, California, USA. Ohta, T., Moriya, M., Kaneda, Y., Watanabe, K., Miyazawa, T., Sugiyama, F. & Shirasu, Y. (1980) Mutagenicity screening of feed additives in the microbial system. Mutat. Res., 77, 21-30. Tajima, M. (1979) Teratogenicity test of nicarbazin with rats by oral administration. Unpublished report from Nisseiken (NIBS). Submitted to WHO by Koffolk, Rancho Santa Fe, California, USA. Vogin, E.E. (1969a) Two-year chronic toxicity studies with components of nicarbazin in dogs. Unpublished report from Food and Drug Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by Koffolk, Rancho Santa Fe, California, USA. Vogin, E.E. (1969b) Chronic toxicity studies with nicarbazin formulation in rats. Unpublished Report from Food and Drug Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by Koffolk, Rancho Santa Fe, CA, USA.
See Also: Toxicological Abbreviations NICARBAZIN (JECFA Evaluation)