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    RONIDAZOLE

    1.  EXPLANATION

         Ronidazole (1-methyl-5-nitroimadozole-2-methanol carbamate) is a
    5-nitroimadazole with antiparasitic activity useful in the treatment
    of enterohepatitis in turkeys and dysentery in swine.  The normal
    level of inclusion of ronidazole in a feed is between 60 and 120 ppm. 
    Ronidazole has not been previously evaluated by the Joint FAO/WHO
    Expert Committee on Food Additives.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

         Absorption:  A number of studies using 14C-ronidazole in
    several animal species revealed that the drug is readily absorbed from
    the gastrointestinal tract (Rosenblum  et al., 1972; Wolf  et al.,
    1983). Plasma concentrations of 0.09 and 0.5 µg/ml were attained in
    rats 24 hours following oral administration of 2 and 10 mg/kg bw of
    14C-ronidazole respectively (Wolf  et al., 1983).

         Distribution: Data obtained from studies with 14C-ronidazole
    indicate that the drug is widely distributed in the body of animals
    (Rosenblum et al. , 1972; Wolf  et al., 1983). Ronidazole-related
    radioactivity was shown to be present in brain, fat, heart, kidney,
    liver, lung, muscle, pancreas, skin and spleen.

         Excretion: Ronidazole is excreted mainly in the urine and feces
    of animals. Up to 3% of the administered dose is exhaled as carbon
    dioxide. Animals receiving a single oral dose of ronidazole have been
    shown to excrete 30-36% of the dose in urine and 16-40% in feces
    during a 24-hour period (Rosenblum  et al., 1972); Wolf  et al.,
    1984). Subsequent excretion is slow and incomplete. In rats combined
    urinary and fecal excretion have been shown to decrease from 36% and
    40% in the first day and to 2% and 6% in the second day, respectively
    (Wolf  et al., 1984).

    2.1.2  Biotransformation

         The biotransformation of ronidazole to protein-bound metabolites
    was examined in rat liver by West  et al., (1982). The authors
    demonstrated that rat liver microsomal fraction catalyzes both NADH
    and NADPH-dependent covalent binding of ronidazole metabolites to
    protein under either aerobic or anaerobic conditions. NADPH was more
    efficient than NADH and the binding was greater under anaerobic
    conditions, suggesting that the metabolism of ronidazole to
    protein-bound metabolites occurs via a reductive pathway.

         Purified rat liver microsomal NADPH-cytochrome P-450 reductase
    was able to catalyze the activation of ronidazole to metabolites which
    bind covalently to protein. Like the reaction catalyzed by rat liver
    microsomes, protein alkylation by the purified reductase required
    reducing equivalents, was sensitive to oxygen, was inhibited by
    sulfhydryl-containing compounds and was stimulated several fold by
    either flavin mononucleutide or methylviologen. It was also
    demonstrated that both the phenobarbital and
    3-methylcholanthrene-inducible forms of cytochrome P-450 purified from
    rat liver microsomes are not involved in bound metabolite formation,

    suggesting that other cytochrome P-450 isozymes present in rat liver
    microsomes may be responsible for ronidazole activation (West  et al.,
    1982).

         Evidence that persistent tissue residues of ronidazole could
    result from metabolic degradation of the imidazole nucleus to one or
    two carbon fragments which could form endogenous substances capable of
    entering cellular macromolecules via normal protein synthesis
    reactions has been obtained in rats dosed with ronidazole labelled
    with 14C at different positions (Wolf  et al., 1984).

    2.2  Toxicological studies

    2.2.1  Acute Toxicity

    
                                                                                  

    Species        Sex       Route          LD50         Reference
                                          (mg/kg bw)
                                                                                  

    Mouse          F         Oral         2330, 2440     Peck, 1974

                   F         i.p.            1250        Lankas  et al.,
                                                         1988

                   F         s.c.            1730        Lankas  et al.,
                                                         1988

    Rat            M         Oral            2850        Peck, 1974

                   F         Oral            3140        Lankas  et al.,
                                                         1988

                   M         i.p.            1140        Lankas  et al.,
                                                         1988

                   F         i.p.             969        Lankas  et al.,
                                                         1988

                   M         s.c.            3080        Lankas  et al.,
                                                         1988

                   F         s.c.            3350        Lankas  et al.,
                                                         1988

    Rabbit         M&F       Oral            1250        Peck, 1974
                                                                                  
    
    2.2.2  Short-term studies

    2.2.2.1  Rats

         Groups of 15 FDRL strain albino rats/sex/dose were administered
    0,50, 100 or 200 mg/kg bw/day ronidazole orally by gavage five days a
    week for 13 weeks. Excessive salivation was noted in all treated
    animals and the time of onset of salivation was dose related, the
    earliest appearance being the third week at the 200 mg/kg bw/day
    dosage level. Excessive micturition was noted also in several animals
    of the middle and high dosage groups. A decrease of body weight gain
    was observed in rats of the middle and high dosage levels. There were
    no ophthalmological or hematological changes that could be
    attributable to treatment.

         Necropsy revealed that the testes of all rats from the high
    dosage group and 11 rats from the middle dosage group were reduced to
    about one-half normal size. Microscopic examinations of the testes
    showed moderate to marked tubular atrophy among the high dosage rats
    and very slight to marked tubular atrophy among the middle dosage
    rats. No spermatoza or normal spermatids were present in the high
    dosage rats. There was no difference in testicular size between the
    rats from the low dosage and the control groups. The average weight of
    the liver of the male rats in the high dosage group was increased as
    was that of the spleen. Very slight hepatic cellular enlargement was
    noted in the rats of the high dosage group. The ceca of treated rats
    of all dosage groups were increased in size, but there were no
    significant microscopical changes (Lankas  et al., 1988).

    2.2.2.2  Dogs

         Groups of 2 male and 2 female purebred beagle dogs were given
    ronidazole orally at dosage levels of 25, 50, 100, and 200 mg/kg
    bw/day, five days a week for 17 weeks. A similar number of dogs were
    used as a control group. The dogs in the control and 25 mg/kg bw/day
    groups remained in good health throughout the study. After one week,
    all 4 dogs from the 200 mg/kg bw/day group were sacrificed because of
    poor physical condition. These dogs had tonic convulsions and
    exhibited opisthotonus, fine tremors, ataxia, hindquarter stiffness,
    dry mouth and gums, slight tachycardia, and slow and shallow
    respiration. After two weeks, the dogs from the 100 mg/kg bw/day group
    showed similar signs and were sacrificed because of poor physical
    condition. Three of the 4 dogs from the 50 mg/kg bw/day group were
    also sacrificed at 5 and 8 weeks for the same reason.

         Two of the 4 dogs from the 100 mg/kg bw/day group had a widening
    of the Q-T interval of the ECG tracing after one week of treatment and
    the only dog from the 200 mg/kg bw/day group for which an ECG was
    taken showed a similar pattern. The dogs from the 100 and 200 mg/kg
    bw/day groups showed hemoconcentration. Slight increases of serum
    glucose and serum glutamic oxalacetic transaminase were noted in some

    dogs from the 50, 100, and 200 mg/kg bw/day groups. Two dogs from the
    200 mg/kg bw/day group had moderate increases of blood urea and
    alkaline phosphatase. All dogs that received 200 mg/kg bw/day of
    ronidazole had albuminurea and erythrocytes in the urine.

         Testicular hypoplasia was a common finding in dogs from 50, 100,
    and 200 mg/kg bw/day groups. In addition, epicardial, myocardial, and
    valvular hemorrhages, increased liver and kidney weights, lymphoid
    atrophy, lipid infiltration of the liver and kidney, and elevated
    adrenal weights were noted among the dogs from the 200 mg/kg bw/day
    dose level (Lankas  et al., 1988).

         Groups of 5 young adult dogs/sex/dose were administered 0, 10,
    20, or 40 mg/kg bw/day ronidazole orally in gelatin capsules for two
    years. After 34 days of treatment, the high dosage level was reduced
    to 30 mg/kg bw/day because of intolerance. At the completion of one
    year of the study, 2 dogs/sex/group were sacrificed for gross and
    microscopic examinations. The remaining dogs were sacrificed after two
    years of treatment.

         The dogs from the 10 mg/kg bw/day group showed occasional fine
    tremors and slight dehydration. The dogs from the 20 mg/kg bw/day
    group developed a nervous behaviour and became hyperreactive. Three of
    the dogs at this dosage level failed to survive the duration of the
    study. The dogs from the 30 mg/kg bw/day group exhibited the same
    signs with more intensity and longer duration. In addition, the dogs
    showed anorexia, weight loss, ataxia, and clonic and tonic
    convulsions. At the end of one year, 7 of 10 dogs in the high dosage
    group had either died or been sacrificed in a moribund condition.

         Hematological changes including leucocytopenia, elevations in
    erythrocyte sedimentation rates, and reductions in hemoglobin and
    hematocrit values were observed in dogs from the 20 and 30 mg/kg
    bw/day groups.

         Gross pathological lesions involving the ventral internal capsule
    at the level of the optic chiasma and the globus pallidus were seen in
    the brain of 2 male dogs from the 30 mg/kg bw/day group. Slight
    hydrocephalus, subdural hemorrhage and pale yellowish coloration of
    the brain were noted among the dogs from the 20 mg/kg bw/day group.
    Histopathological changes involving focal hemorrhage of the
    cerebellum, leukomalacia, vascularization with endothelial
    proliferation, neurophagia, and phagocytosis were seen in brain tissue
    of dogs from the 20 and 30 mg/kg bw/day groups.

         Hemorrhages in various areas of the heart occurred among dogs
    from the 20 and 30 mg/kg bw/day groups. Absolute testes weights of
    ronidazole-treated dogs at all dosage levels were decreased when
    compared with those of controls. Microsocopic examinations of

    testicular tissues revealed aspermatogenesis and oligospermia. These
    testicular lesions were considered by the authors to be treatment
    related (Wazeter  et al., 1969c; Lankas  et al., 1988).

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Mice

         Three groups of 60 male and 60 female Alderly Park strain mice
    were maintained on diets containing 5, 10, or 20 mg/kg bw/day
    ronidazole for 81 weeks. Two groups of 60 male and 60 female mice of
    the same strain were fed unmedicated diet and served as controls. Body
    weight and food consumption were measured weekly for all animals. At
    the end of the study, animals were necropsied and subjected to a
    complete gross examination. Microscopic examination was conducted on
    the tissues and gross lesions from all animals.

         Data submitted in summary form suggested that during the course
    of study there were no significant effects on body weight gain or food
    consumption in any of the treated groups. No treatment-related effects
    on the survival and physical condition of animals were seen in any
    group throughout the study.

         At necropsy there were no gross lesions attributed to treatment
    in any group. However, a dose-dependent increase in combined benign
    and malignant pulmonary tumors was observed in both sexes in the mid
    and high dosage groups as summarized in Table 1 (Lankas  et al.,
    1988).

        Table 1:  Incidence of pulmonary neoplasms in control and treated male
              and female mice in 81-week feeding study with ronidazole
                                                                                  
                                            Males
                                                                                  
         Group (N=60)        Adenoma        Carcinoma      Total
    Control 1                 4              3              7
    Control 2                 3              1              4
    5 mg/kg bw/day            8              2             10
    10 mg/kg bw/day           9              3             12
    20 mg/kg bw/day          191             81            271

                                            Females
                                                                                 
                             Adenoma        Cardinoma      Total
         Group (N=60)
    Control 1                 1              0              1
    Control 2                 5              1              6
    5 mg/kg bw/day            3              1              4
    10 mg/kg bw/day           8              2             10
    20 mg/kg bw/day          141             61            201
                                                                                  

    1.   P<0.05
    
    2.2.3.2  Rats

         In a 95 week study, ronidazole was administered in the feed at 0,
    10, 20, or 40 mg/kg bw/day to groups of 42 Manor Farm Albino rats of
    each sex. An increase in benign mammary tumors was seen in males in
    the 40 mg/kg bw/day group and in females in all ronidazole treated
    groups after 52 weeks. In addition, 5/41 malignant tumors occurred in
    the high dose females compared to 0/39 in the controls (Table 2). The
    biological significance of the malignant tumors was not clear. There
    was no dose-related effect, as 2 malignant tumors also occurred in the
    low dose group but none were observed in the mid-dose group. No
    information on the historical incidence of mammary tumors in this
    strain of rats was submitted for evaluation.

        Table 2: Incidence of mammary neoplasms in control and treated male
             and female rats in 95-week feeding study with ronidazole
                                                                                  
                                              Males
                                                                                  

    Group (N=42)           Adenoma/Fibroma    Adenocarcinoma    Total

    Control                    0/341             0/341          0/341
    10 mg/kg bw/day            0/40              0/40           0/40
    20 mg/kg bw/day            0/40              0/40           0/40
    40 mg/kg bw/day            5/32              1/32           6/32

                                                 Females
                                                                                  

    Group (N=42)           Adenoma/Fibroma    Adenocarcinoma    Total2

    Control                    7/391             0/391          7/391
    10 mg/kg bw/day            13/41             2/41           14/41
    20 mg/kg bw/day            21/41             0/41           21/41
    40 mg/kg bw/day            19/41             5/41           20/4l
                                                                                  

    1.   Denominator is number of rats surviving after 52 weeks. No
         tumors were seen in rats dying prior to 52 weeks.
    2.   Some rats had both benign and malignant tumors, hence this
         column is not a sum of the first two.
    
         During the first 52 weeks of the study no ronidazole-related
    pharmacodynamic or toxic signs were observed at any dosage level.
    During the later part of the study, rats from the 40 mg/kg bw/day
    groups did not gain weight as well as those from the control groups.
    There were no meaningful alterations in group mean food consumption,
    hematology, clinical biochemistry and urinalysis at any dosage level
    in either sex of rats. Testicular atrophy was observed in rats from
    the high dosage group and was considered by the authors to be a
    ronidazole-related effect (Wazeter  et al., 1969a).

         Ronidazole was administered in the diet for at least 104 weeks to
    three groups of Charles River CD rats with each group consisting of 60
    males and 60 females. The dietary concentration of ronidazole was
    adjusted to achieve dosage levels of approximately 5, 10, and 20 mg/kg
    bw/day. Satellite groups of 15 animals/sex/group were also treated
    with the drug at these same dosage levels and were used for blood
    sampling and urinalysis in weeks 6, 13, 25, 52 and 78. Two groups of
    60 animals/sex/group were fed unmedicated diet and served as controls.
    Ronidazole treatment continued for a minimum of 104 weeks with
    necropsies completed by week 108. Microscopic examination was
    conducted on tissues from all main groups.

         Data submitted in summary form suggested that there were no
    treatment-related physical signs during the study. A significant
    decrease in survival was noted in the 20 mg/kg bw/day groups during
    the last few months of the study. Survival in all other treated groups
    was comparable to controls. Body weight gain in the 10 and 20 mg/kg
    bw/day groups of both sexes decreased slightly compared to controls
    during the second year of the study.

         The only non-neoplastic lesion due to ronidazole treatment was an
    increased incidence of testicular atrophy at the 20 mg/kg bw/day
    dosage level. The only significant neoplastic effect was an increased
    incidence of benign fibro-epithelial mammary tumors in the 20 mg/kg
    bw/day males and in females at the 10 and 20 mg/kg bw/day levels. The
    incidence of these tumors is summarized in Table 3 (Lankas  et al.,
    1988).

    
    Table 3: Incidence of mammary neoplasms in control and treated
             male and female rats in 108-week feeding study with
             ronidazole
                                                                                  

                        No. of Rats with One or More Benign Mammary Tumors
    Group (N=60)                            Males               Females
    Control 1                               3                   45
    Control 1                               2                   42
    5 mg/kg bw/day                          3                   49
    10 mg/kg bw/day                         6                   531
    20 mg/kg bw/day                         81                  541
                                                                                  

    1.  P<0.05
    
    2.2.4  Reproduction studies

    2.2.4.1  Rats

         Groups of 10 male and 20 female Charles River CD strain albino
    rats, 35 days old at the beginning of the study, were maintained on
    diets containing 0, 0.02, 0.04 or 0.089% ronidazole, seven days a week
    70 days prior to the first mating and throughout the production of
    three generations. These dietary concentrations resulted in
    approximate dosage levels of 25, 30 and 60 mg/kg bw/day. Two litters
    were produced with each generation of parents. The second litter was
    used to produce the succeeding generation and the first litter was
    examined and then discarded at weaning.

         There were no alterations in behaviour, appearance, body weights
    or mean food consumption values of dams. No treatment-related
    abnormalities were noted in the pups at any dietary level of
    ronidazole. During each of the six whelping phases, the fertility,
    gestation period, viability and lactation indices were comparable for
    the control and treated group. There were no adverse effects on the
    average body weight of pups at birth. At 60 mg/kg bw/day, ronidazole
    significantly reduced the numbers of pups per litter as compared to
    the control or lower dosage levels. Although there were slightly fewer
    pups per litter at the 30 mg/kg bw/day level, the difference from the
    control group was not significant. Because of the smaller number of
    pups, the average pup weight at weaning was greater for the pups from
    the 40 and 60 mg/kg bw/day groups. There were no treatment-related
    gross or microscopic changes in any of treated pups of the second
    litter of the third generation (Wazeter  et al., 1969b).

    2.2.5  Special studies on embryotoxicity (and/or teratogenicity)

    2.2.5.1  Mice

         Ronidazole was administered by gavage to 3 groups of 20 pregnant
    mice (strain not specified) from days 6 through 15 of gestation at
    dosage levels of 50, 100, and 200 mg/kg bw/day. Two additional groups
    of 20 pregnant mice each served as controls. At 200 mg/kg bw/day,
    there was a significant decrease in average maternal weight gain as
    compared to the control group. The average number of implants,
    resorptions, and live fetuses per litter, and average fetal weight per
    litter from the low and mid dosage level groups were comparable to the
    control groups. The average number of implants and the average number
    of live fetuses per litter were slightly decreased at 200 mg/kg
    bw/day.

         External examination of all fetuses from the control and treated
    groups revealed no evidence of a teratogenic effect attributable to
    ronidazole. Visceral and skeletal examination of the fetuses from the
    control and the high dose level groups revealed no evidence of
    drug-related teratogenicity. The four visceral malformations observed
    at 200 mg/kg bw/day were from the same fetus and were considered to be
    spontaneous in origin (Zwickey  et al., 1975).

    2.2.5.2  Rats

         Ronidazole was administered by gavage to 3 groups of 20 pregnant
    Charles River-CD rats from days 6 through 15 of gestation at dosage
    levels of 50, 100, and 200 mg/kg bw/days in Study 1 and at dosage
    levels of 100, 150, and 200 mg/kg bw/day in Study 2, respectively. Two
    additional groups of 20 pregnant rats served as controls in both Study
    1 and 2. No drug-related embryotoxicity occurred at 50, l00, or 150
    mg/kg bw/day of ronidazole. A slight but significant increase in
    resorptions at 200 mg/kg bw/day was observed in Study 1 but not in
    Study 2.

         At 100 mg/kg bw/day and above, a decrease in average fetal weight
    per litter was seen in both studies. At 100 mg/kg bw/day average
    maternal weight gain was nonsignificantly and significantly retarded
    in Study 1 and Study 2, respectively. At dosage levels of 150 or 200
    mg/kg bw/day, average maternal weight gain was significantly retarded.

         External examination of all fetuses from the control and
    drug-treated group in Study 1 revealed no malformations at 50 mg/kg
    bw/day. At 100 mg/kg bw/day, microphthalmia occurred in one stunted
    fetus which also had severe hydrocephalus. At 200 mg/kg bw/day, 4
    fetuses exhibited malformations involving the head (2 microphthalmia,
    1 displaced eyes, and 1 micrognathia and cleft palate). Visceral and
    skeletal examination of all fetuses from the control and 200 mg/kg
    bw/day groups and of the one externally malformed fetus from the 100
    mg/kg bw/day group revealed no additional evidence of drug-related
    teratogenicity. However, an increased incidence of skeletal variations
    including unossified sternebrae, incompletely ossified interparietals,
    supraoccipitals and zygomatics were seen in the 200 mg/kg bw/day
    group. External examination of all fetuses from the control and
    treated groups in Study 2 revealed no evidence of treatment-related
    teratogenicity. One fetus from one of the control groups had
    microphthalmia of the left eye, and l fetus from the 200mg/kg bw/day
    group had edema of the neck, but not micrognathia. Visceral
    examination of approximately one-third of the fetuses from the two
    control groups and from the 200 mg/kg bw/day group revealed no
    evidence of drug-related teratogenicity. The 11 malformations observed
    in the 200 mg/kg bw/day group were considered to be spontaneous in
    origin, because they all occurred in the same fetus which was also
    severely stunted and externally malformed. An increased incidence of
    skeletal variations and unossified sternebrae were also seen in the
    200 mg/kg bw/day group (Zwickey  et al., 1976).

    2.2.5.3  Rabbits

         Two separate studies have been reported by Zwickey  et al.,
    (1975). In the first study, ronidazole was administered by gavage to
    3 groups of 15 pregnant New Zealand rabbits from days 7 through 15 of
    gestation at dosage levels of 3, 10, and 30 mg/kg bw/day. Two
    additional groups of 15 pregnant rabbits served as controls. The
    protocol of the second study was similar to the first study, except
    that the 3 mg/kg bw/day ronidazole group was omitted.

         No teratogenic, embryotoxic, or fetotoxic effects attributable to
    treatment with ronidazole were observed at dosage levels of 3 and 10
    mg/kg bw/day. A significant decrease in maternal weight gain and
    average fetal weight occurred with ronidazole at 30 mg/kg bw/day. 
    Fetuses from the 30 mg/kg bw/day group exhibited malformations
    involving the heart and great vessels. Heart and great vessel
    malformations have been observed to occur spontaneously in rabbits. In
    seven studies, the incidence of control fetuses with such
    malformations varied from 0.4 to 2.4% compared with an incidence of
    2.7 to 2.8% in fetuses from the 30 mg/kg bw/day dose levels in the two
    studies of ronidazole. The authors concluded that the cardiovascular
    malformations seen among the fetuses from dams given ronidazole were
    not related to treatment.

    2.2.6  Special studies on genotoxicity

        Table 4:  Results of genotoxicity assays on ronidazole
                                                                                  
                                         Concentration
                                            of
    Test System       Test Object        ronidazole      Results     Reference
                                                                                  

    Ames test (1)     S.typhimurim       0.03 mM         Positive    Voogd  et
                      TA1530, TA1532                                  al., 1974
                      TA1534,
                      Lt2his-G46

    Ames test (2)     S.typhimurim       10-50µg/plate   Positive    Hite  et
                      TA1530, TA1531                                  al., 1976
                      TA1532, TA1534
                      TA2535, TA1536
                      TA1537, TA1538

    Ames test (2)     S.typhimurim       0.1µg/ml        Positive    Mourot, 1988
                      TA97a, TA98
                      TA100, TA102

    Luria and         K.pneumoniae       0.01 mM         Positive    Voogd  et
    Delbruck's        E.Coli K12HfrH                                  al., 1974
    fluctuation test  C.freundii 425

    Sex-linked        D.melanogaster     10 mM           Positive    Kramers, 1982
    recessive
    lethal test

    Bone marrow       DF2S mice          100-200         Positive    Hite  et
    cytogenic assay                      mg/kg bw/day                 al., 1976

    Dominant lethal   CF2S mice          200             Negative    Hite  et
    assay                                mg/kg bw/day                 al., 1976

    Micronucleus      CF2 mice           200             Negative    Hite  et
    test                                 mg/kg bw/day                 al., 1976

    Micronucleus      Swiss/RIV mice     280             Negative    Oud  et
    test                                 mg/kg bw/day                 al., 1979
                                                                                  
    (1) Without rat liver S-9 fraction
    (2) Both with and without rat liver S-9 fraction.
    
    2.3  Observations in man

         No information is available.

    3.  COMMENTS

         Ronidazole is absorbed from the gastrointestinal tract in both
    laboratory and target species. In studies using radio-labelled
    ronidazole, the radioactivity has been found to be widely distributed
    in tissues and eliminated in the urine, feces and expired air of the
    animals. The parent compound accounts for part of the urinary
    excretion but is essentially absent from feces. Ronidazole is rapidly
    degraded in pig urine and feces. The ultimate fate of ronidazole
    metabolites has not been fully determined.

         The mutagenic potential of ronidazole was investigated in a range
    of studies. Positive findings were recorded in bacterial assays with
    and without metabolic activation, and in the sex-linked recessive
    lethal test in  Drosophila melanogaster. The bone-marrow cytogenetic
    assay in CF1S mice yielded weakly positive or negative results,
    while micronucleus tests and a dominant lethal assay were negative.

         In addition, a range of postulated and/or identified metabolites
    of ronidazole and extract of muscle from ronidazole-treated pigs gave
    negative results in Ames tests. The Committee found that these studies
    contributed to the safety assessment of ronidazole.

         The carcinogenicity studies most appropriate for evaluation were
    an 81-week feeding study in Alderly Park mice and a 104-week feeding
    study in Charles River CD rats. The data on individual animals were
    not available to the Committee. The mice received ronidazole in the
    diet at 0, 5, 10 or 20 mg/kg bw/day. The increased occurrence of lung
    adenoma/carcinoma was statistically significant at 20 mg/kg bw/day in
    males and females. The rats received ronidazole in the diet at 0, 5,
    10 or 20 mg/kg bw/day and the increased occur-rence of benign mammary
    tumors was statistically significant in females at 10 and 20 mg/kg
    bw/day and in males at 20 mg/kg bw/day. The NOEL in these studies was
    5 mg/kg bw/day.

         The mechanism by which ronidazole exerts its dose-dependent
    carcinogenic effects had not been elucidated.

         In the rat carcinogenicity study, it was also noted that
    testicular atrophy was present in males receiving 20 mg/kg bw/day
    which died between 52 weeks of treatment and the planned end of the
    trial. In a two-year study in which ronidazole was administered at 20
    or 30 mg/kg bw/day, the same phenomenon was observed, in terms both of
    a decrease in absolute testicular weight and of the presence of
    histopathological change. Clinical signs of toxic effects in the
    central nervous system were observed at all dose levels in this study.
    Compound-related histopathological changes were found only in brain
    tissues from five of seven dogs dosed at 30 mg/kg bw/day and killed in
    the first year of the study. A subsequent two-year study in dogs
    established a NOEL of 5 mg/kg bw/day for both these effects.

         In a three-generation reproduction study in Charles River CD
    rats, ronidazole was included in the diet at 200, 400 and 800 mg/kg.
    There were no adverse effects on reproduction nor were
    compound-related teratogenic effects seen, but the average number of
    pups per litter was significantly reduced at 800 mg/kg. In two other
    studies of Charles River CD rats, no drug-related teratogenic effects
    were evident at doses up to 200 mg/kg bw/day. Maternal weight gain was
    depressed at 200 mg/kg bw/day, while fetal weight was reduced at doses
    of 100 mg/kg bw/day and above. In other studies, CF1S mice received
    ronidazole at 50, 100 or 200 mg/kg bw/day by gavage and New Zealand
    rabbits received doses of 3, 10, 30 mg/kg bw/day. Despite evidence of
    maternal toxicity at the highest doses, there was no statistically
    significant evidence of teratogenicity.

         A NOEL of 5 mg/kg bw/day or higher was observed in these
    long-term and reproductive studies. On the basis of a NOEL of 5 mg/kg
    bw/day, therefore, and a safety factor of 200, the Committee
    established a temporary ADI of 0-0.025 mg/kg bw/day. The safety factor
    was selected by the Commiteee in the light of the results of
    genotoxicity studies on ronidazole in mammalian systems and of the two
    recent carcinogenicity studies in which no-observed-effect levels for
    carcinogenicity and for other toxicological effects of concern were
    identified. It was also influenced by the lack of mutagenicity of
    several metabolites of ronidazole.

    4.  EVALUATION

         Level causing no effect

         Rat: 5 mg/kg bw/day
         Dog: 5 mg/kg bw/day.

         Estimate of temporary acceptable daily intake

         0-0.025 mg/kg bw

         Further work or information

         Required (by 1993):

         (a)  A complete submission, including data on individual animals
              of the carcinogenicity studies.

         (b)  The results of studies aimed at investigating the mechanism
              of tumorigenesis.

    5.  REFERENCES

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    HITE, M., SKEGGS, H., NOVEROSKE, J. & PECK, H. (1976). Mutagenic
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    KRAMERS, P.G.N. (1982).  Studies on the induction of sex-linked
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    LANKAS, G.R., BOKELMAN, D.L.& SCOLNICK, E.M. (1988). Ronidazole
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    MATTIS, P.A., PECK, H.M., BAGDON, W.J., BOKELMAN, D.L., JENSEN, R.J.
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    MOUROT, D. (1988). Ronidazole, Test d'Ames avec et sans activations
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    Laboratories, Merck & Co., Inc., NJ, USA.


    See Also:
       Toxicological Abbreviations
       RONIDAZOLE (JECFA Evaluation)