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        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.

    FIGURE 1

    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
                                                              (g/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
    721. 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 g/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)