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    CARBADOX

    1.  EXPLANATION

         Carbadox is a growth-promoting and antibacterial drug added to
    swine feed at the rate of 50 ppm.  In most areas of the world it is
    used in animals up to 4 months of age with a 4 week withdrawal period
    prior to slaughter for human consumption.  Carbadox has not been
    evaluated previously by the Joint FAO/WHO Expert Committee on Food
    Additives.

         The structure of carbadox is shown in Figure 1.

    FIGURE 1

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

         The elimination of carbadox was studied in rats, swine and
    monkeys.  Pigs received 3.5 mg/kg of 14C-carbadox after several of
    weeks of receiving feed containing 50 g/ton of unlabelled carbadox,
    while rats and monkeys received a single dose of 5 mg/kg 14C-carbadox. 
    Urine and faeces were collected and assayed for radioactivity.  The
    urinary metabolites were evaluated qualitatively using TLC.  Nearly
    all (13/15) of the metabolites present in swine urine are found in rat
    and monkey urine.  One of the other two was a glycine conjugate of
    quinoxaline-2-carboxylic acid.  All species excreted more than 50% of
    the dose in urine (swine: 74%, monkey: 61%, rat: 54%) during the 72
    hour collection period.  The following activities were reported for
    faeces during the 72 hour period: swine, 17%, monkey, 8-10%, and rat,
    29%.  Total excretion appeared to be in the 70-90% range over the 72
    hour period.  The author concluded that the distribution of
    radioactivity was similar in the three species (von Wittenau, 1969).

    2.1.2  Biotransformation

         Seven-week-old swine received feed containing 50 g/ton of
    unlabelled carbadox for several weeks, then received a single oral
    dose of either 3.5 mg/kg or 0.8 mg/kg of 14C-carbadox labelled in the
    phenyl ring.  Peak radioactivity was observed in plasma approximately
    3 hours after dosing.  The following were identified in plasma at 5-8
    hours post dose:  carbadox (13%), desoxycarbadox (9-19%),
    carbadoxaldehyde (13%), and quinoxaline-2-carboxylic acid (19%) (all
    expressed in terms of total plasma radioactivity).  The presence of
    carbadoxaldehyde in stomach contents was confirmed.  Carbadox was
    rapidly eliminated.  Approximately 2/3 of the dose was eliminated in
    the urine and the remainder in the faeces (total of approximately 90%)
    within 48-72 hours.  Radioactivity equivalent to approximately 0.1 ppm
    carbadox was found to be retained in the liver at 14 days post dose. 
    Attempts to identify this residual radioactivity were not successful. 
    The only metabolite identified in liver after 24 hours was the major
    urine-eliminated metabolite, quinoxaline-2-carboxylic acid (Figdor
    et al., 1969).

         In a second study seven-week-old pigs received unlabelled
    carbadox in feed at the rate of 50 g/ton for several weeks followed by
    a single oral dose of 14C-carbadox labelled in the carbonyl position
    via stomach tube.  The following evaluations were made:  expired air
    was evaluated for 14CO2, labelled material in liver and urine was
    evaluated to determine if it was methyl carbazate-related, and plasma
    and urine were evaluated for free hydrazine.  Maximum plasma
    concentrations of radiolabelled material occurred at approximately 3

    hours.  While early plasma concentrations were similar to those found
    for ring-labelled carbadox, concentrations at 24 hours remained
    somewhat higher.  Approximately 50% of the radiolabelled material in
    the plasma at 3 hours was identified as carbadox, while methyl
    carbazate was estimated to be 30%.  The major route of radiolabel
    excretion was urinary.  However, less than half the total amount
    recovered with ring-labelled carbadox was recovered from carbonyl-
    labelled carbadox (37% vs 88%).  The reason for this is the apparently
    high conversion of the radiolabel to CO2 (verified in the rat as up
    to 36%).  Radiolabelled material equivalent to 0.1 - 0.34 ppm carbadox
    was present in liver at 5 days.  The authors concluded that some of
    this material was incorporated CO2 (potentially 25%).  The pig
    receiving 7 mg/kg was found to have eliminated 7% of the dose as free
    hydrazine in the urine at 24 hours, while pigs receiving lesser doses
    were not found to have any identifiable hydrazine in their urine
    (Figdor, 1969).

         The reported studies support the metabolism scheme illustrated in
    Figure 2.

         Extensive studies have been performed not only on carbadox, but
    also on its metabolites.  Studies are summarized separately on each
    substance in this monograph.  

    2.1.3  Effects on enzymes and other biochemical parameters

         The persistence of carbadox-induced adrenal lesions was studied
    in swine.  Groups of 13 animals each received 0, 25, 50, 100, or 200
    ppm carbadox in feed for 10 weeks.  Five and 11 weeks after withdrawal
    2 pigs from each group were necropsied and adrenals evaluated
    histologically.  At five weeks the pigs in the 25 and 50 ppm groups
    showed recovery of adrenal lesions.  At 11 weeks pigs in the 100 and
    200 ppm groups had not completely recovered.  Plasma aldosterone
    levels were monitored at 1, 2, 4, 6, and 8 weeks post treatment.  At
    one week aldosterone levels were depressed in the 100 and 200 ppm
    groups vs controls.  All other aldosterone measurements of treated
    groups were similar to control values (van der Molen et al.,
    1989a).

         The effect of carbadox on the renin-angiotensin system was
    examined in swine.  Five groups of 13 five-week-old weaned pigs
    received feed containing 0, 50, 100, 150, and 200 ppm carbadox to
    provide target doses of 0, 2, 4, 6, and 8 mg/kg body weight for 10
    weeks.  By nine weeks the plasma renin levels of all treated groups
    were significantly higher than controls.  At 5 and 10 weeks, renal
    immunoreactive renin from two to three pigs was evaluated.  All pigs
    demonstrated an increase in immunoreactive renin.  It was concluded
    that carbadox induces activation of the renin-angiotensin system,
    secondary to suppression of mineralocorticoid secretion (van der Molen
    et al., 1989b).

    FIGURE 2

         The in vitro effect of carbadox on adrenal aldosterone production
    was evaluated in porcine adrenal slices obtained from 3-5 week old
    pigs.  The adrenal slices were maintained in Krebs solution and
    exposed to 1-40 g carbadox/ml.  Aldosterone was estimated using RIA
    at hourly intervals.  A dose-dependent decrease in aldosterone
    production occurred with a reduction of 25-35% at 40 g/ml
    (Spierenburg et al; 1988).

    2.2.  Toxicological Studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with carbadox are shown in
    Table 1.

    Table 1:  Carbadox acute toxicity data
                                                            
                             LD50
    Species   Sex   Route    (mg/kg b.w.)   Reference
                                                            

    Mouse     M     oral      2810          Pfizer, undated a 
              F     oral     >2810
                    i.p.      1050

    Rat       M     oral      850
                    i.p.      810
                                                            

         A study was done to examine the emetic effect of carbadox on
    monkeys and dogs.  Three animals/group received a single dose of
    carbadox orally via capsule.  Doses were 25, 10, 5, and 1 mg/kg
    b.w./day.  In the monkey 1/3 exhibited emesis in the 2 higher dose
    groups, while in dogs all dose groups had emesis.

    2.2.2  Short-term studies

    2.2.2.1  Rats

         Twenty Charles River C-D weanling rats were divided and dosed for
    30 days as follows:  2-3 males and females at 50 mg/kg b.w./day and
    100 mg/kg b.w./day, and 10 males as untreated controls.  Carbadox was
    administered in the diet.  Weight and food consumption were observed
    weekly and drug levels adjusted as necessary.  CBC at 30 days, urinary
    electrolytes at 1, 2, 3, 14, and 30 days, cholesterol at 30 days and
    urinalysis at 14 and 30 days were evaluated.  Rats received a gross
    necropsy at the end of the study and tissues were evaluated
    microscopically.  A dose-dependent decrease in weight gain and food-
    consumption were noted.  The authors concluded that all other
    parameters were normal (Pfizer, 1963).

    2.2.2.2  Dogs

         The short-term toxicity of carbadox was studied by dosing dogs 6
    days/week for three weeks.  Beagle dogs (1/sex/dose) were initially
    dosed with 25 or 50 mg/kg b.w./day carbadox via oral capsule.  These
    doses were later reduced to 10 and then 15 mg/kg b.w./day due to
    emesis.  A variety of parameters were evaluated including clinical
    chemistry, gross necropsy, and microscopic tissue examination at the
    termination of the study.  The dogs lost weight and had elevated
    SGPT's.  It was concluded that carbadox is emetic and possibly
    hepatotoxic in the dog (Stebbins, 1964).

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Rats

         The chronic toxicity and potential tumorigenicity of carbadox
    were studied in rats.  One hundred twenty Charles River C-D rats were
    divided into 6 groups (10/sex/dose) and received carbadox in the diet
    at rates providing doses of 100, 50, 25, 10, 5, and 0 mg carbadox/kg
    b.w./day for 26 months.  Feed concentration was adjusted weekly. 
    Clinical signs, body weights, and food consumption were recorded
    weekly.  Haematology and urinalysis were evaluated in 5 rats/sex/dose
    at 3, 6, 12, 18, and 25 months.  Rats were sacrificed at 14 and 112
    weeks and received gross necropsies.  Tissues were examined
    histopathologically.

         Survival was reduced for animals in the high-dose groups.  The
    following observations were reported for animals up to and including
    the interim sacrifice:

    100 mg/kg b.w./day (10 rats/sex necropsied) - Decreased weight gain
    and food consumption, reduced haemoglobin, RBC's and neutropenia. 
    Microscopic changes reported include pulmonary hemorrhage and edema,
    adrenalcortical hemorrhage and degeneration, splenic haemosiderin, and
    thymic atrophy.  No animals survived beyond the 14 week sacrifice.

    50 mg/kg b.w./day (10 rats/sex necropsied) - Decreased weight gain
    and food consumption.  Microscopic changes reported include pulmonary
    hemorrhage and edema, adrenalcortical hemorrhage, necrosis, and
    degeneration, splenic haemosiderin and renocorticomedullary fatty
    metamorphosis.  No animals survived beyond the 14 week sacrifice.

    25 mg/kg b.w./day (3 rats/sex necropsied) - Reduced weight gain,
    slight adrenal cortical atrophy, degeneration/necrosis, and renal
    tubular fatty change.

    5 and 10 mg/kg b.w./day (3 rats/sex necropsied) - No clinical, gross
    or microscopic changes reported at 3 months.

         The following observations were reported for rats from 3 months
    up to and including the 26 month sacrifice:

    25 mg/kg b.w./day - One female died at 51 weeks with no drug-related
    changes noted.  All 13 remaining rats were sacrificed by 73 weeks due
    to palpable abdominal masses.  At necropsy all rats had multiple
    hepatic nodules.  Ten of 13 rats were diagnosed microscopically to
    have benign nodular hyperplasia, while the remaining 3 rats were
    determined to have malignant transformation based on metastatic foci
    observed in other organs (lung, kidney and lymph nodes).

    10 mg/kg b.w./day - One rat died after 67 weeks with
    reticuloendothelial neoplasia, a common tumour of aged rats.  The
    remaining 13 rats were sacrificed between the 64th and 112th weeks. 
    Eleven of these were observed to have hepatic benign nodular
    hyperplasia.  

    5 mg/kg b.w./day - One male died at 20 weeks due to pulmonary
    abscesses.  The remaining 13 died or were sacrificed between the 61st
    and 112th weeks.  Five or these were observed to have hepatic benign
    nodular hyperplasia.

    Controls  One male was sacrificed at 33 weeks with a forestomach
    papilloma and pulmonary atelectasis and a second male died at 93 weeks
    with myocarditis, nephrosclerosis and peribronchitis.  The remaining
    animals were sacrificed between weeks 80 and 112 of the study.  No
    animals in the control group were observed to have hepatic nodular
    hyperplasia.  The authors suggested that the observed nodular
    hyperplasia in treated groups was associated with hepatic injury and
    concluded that the rats failed to tolerate carbadox at any of the
    tested dose levels.  A treatment-related increase in total tumours was
    also reported for carbadox-treated rats.  The tumour incidence was
    lower in the high-dose group, however mortality occurred earlier in
    this group than in other treatment groups (Stebbins & Coleman, 1967).

         A follow-up study was performed to determine a chronic carbadox
    level tolerated by rats.  Sixty male Charles River rats (124-173 gms)
    and 60 female rats (107-152 gms) of the same strain were divided into
    3 groups (20/sex/dose) and received 2.5, 1.0, or 0 mg/kg b.w./day of
    carbadox in the diet.  Signs, weights and food consumption were
    recorded weekly and drug levels were adjusted accordingly. 
    Haematology, complete urinalysis, and ophthalmoscopic examination were
    done at 3, 6, 12, 18, and 24 months on 5 rats/sex/dose.  An interim
    sacrifice of 5 rats/sex/dose was conducted at 54 weeks with the
    remaining rats sacrificed at 106 weeks.  Each rat received a gross
    necropsy and microscopic examination of standard tissues including: 
    brain, spinal cord, eye, sub-maxillary gland, thyroid gland, thymus,
    heart, aorta, lung, sternum, liver, spleen, pancreas, kidney, adrenal,
    stomach, mesenteric lymph node, reproductive tract, urinary bladder,
    femoral nerve, femoral bone marrow, and mammary gland with skin.

         All parameters evaluated, including histopathologic examination,
    were within normal limits at 54 weeks.  Survival at 2 years was as
    follows:  control - 38%, 1.0 mg/kg b.w./day - 45%, 2.5 mg/kg b.w./day
    - 43%.  At the 2.5 mg/kg b.w./day level 7/27 rats displayed hepatic
    benign nodular hyperplasia and 7/27 showed peliosis hepatis.  At the
    1.0 mg/kg b.w./day level 1/29 rats was found to have hepatic benign
    nodular hyperplasia and 3/29 displayed peliosis hepatis.  In the
    control group 3/29 rats had benign nodular hyperplasia and 2/29 had
    peliosis hepatis.  Additionally, an increase in total mammary tumours
    was reported in the 2.5 mg/kg b.w./day dose group.  The authors
    concluded that the 1.0 mg/kg b.w./day dose level was tolerated by the
    rats for 2 years with no adverse effects, based on the similar
    incidence of both hepatic lesions and tumours in other organs in
    treatment and control groups (Sigler, 1969a).

         Three groups of 28 rats (14/sex) were treated with a target dose
    of 25 mg/kg b.w./day of carbadox, or one of two lots of desoxycarbadox
    for 11 months to compare the oncogenic activity of carbadox and
    desoxycarbadox.  An equal sized group served as untreated controls. 
    The test material was given in the feed for 10 months.  Two to four
    rats were sacrificed at 30, 60, 90, 191, and 309 days.  Animals were
    observed daily for toxicity and mortality.  Weights and food
    consumption were recorded weekly until the 38th week and then every
    other week.  Rats received a gross necropsy at sacrifice and liver and
    adrenals were evaluated histopathologically.  A moderate decrease in
    weight gain occurred in both sexes receiving carbadox.  No other
    significant clinical observations were made.  At gross necropsy all
    groups treated for 10 months showed evidence of hepatic changes
    including necrosis and nodule formation.  Changes in the carbadox-
    treated group were less severe than in the desoxycarbadox-treated
    group.  Histopathologically all desoxycarbadox-treated rats showed
    evidence of hepatocellular carcinoma, while 2/18 rats receiving
    carbadox showed evidence of hepatocellular carcinoma.  There was a
    significant increase in adrenal cortical hemorrhage in both carbadox
    and desoxycarbadox-treated groups (King, 1976).

         The tumorigenicity of carbadox administered in various dosing
    regimens was studied more recently in rats.  A total of 119 Wistar
    rats of each sex were divided into groups of various size (2 - 18
    rats) following parturition and treated with various regimens of
    carbadox i.p. (prior to weaning for 8 to 20 days) and/or in the feed
    at 300 ppm until study termination at 1 year.  Rats were examined and
    weighed routinely during the study.  Tumours were evaluated grossly
    and histopathologically at the end of the study.  Weight gains were
    reduced.  Animals receiving 300 ppm carbadox in feed all developed
    hepatic tumours.  Rats which received carbadox i.p. developed a higher
    incidence of hepatic tumour than untreated controls.  The largest
    number of spontaneous and rare tumours occurred in groups receiving
    carbadox via both routes (Sykora & Vortel, 1986).

    2.2.3.2  Monkeys

         A study was performed to assess the long-term toxicity of
    carbadox in primates.  Twenty-eight monkeys were divided into 4 groups
    of 7 animals (3 or 4/sex) and were dosed with carbadox in gelatin
    capsules 5 days/week.  The doses were 20 (5 mg/kg QID), 10 (5 mg/kg
    BID) or 5 mg/kg b.w./day and controls.  A variety of parameters were
    evaluated at 1, 3, 6, 12, and 24 months including haemoglobin,
    haematocrit, RBC's, WBC's, clotting time, prothrombin time, complete
    urinalysis, blood glucose, BUN, alkaline phosphatase, SGOT and SGPT. 
    Ophthalmoscopic examinations were performed at the same intervals.  At
    3 months and 2 years animals were sacrificed and necropsied.  The diet
    contained 0.2% isoniazid as prophylaxis for tuberculosis.

         Elevated transaminase levels were detected at the 3 and 6 month
    evaluations in both treated and control animals.  The authors
    concluded that monkeys tolerated 20 mg/kg/day for two years with no
    adverse effect (Coleman, 1967).

    2.2.4  Reproduction studies

    2.2.4.1  Rats

         A 3-generation reproduction study with 2 litters per generation
    was conducted using feed levels adjusted to provide target doses of
    2.5, 1.0 and 10 mg/kg b.w./day.  Feed levels were identical to those
    used for a concurrent 2 year chronic study.  Sixty female and 30 male
    Charles River C-D rats were divided into groups of 20 females and 10
    males per level for the 1st generation.  Two females were placed with
    each male for mating.  First litter offspring were sacrificed
    following a gross examination on lactation day 21.  Following a 10 day 
    rest period the females were rebred to different males.  Twenty females 
    and 10 males from the 2nd litter of each dose level were selected to 
    become the 2nd generation.  These rats were mated at 100 days of age.  
    Dose levels were maintained and the 2nd and 3rd generation were 
    treated from weaning throughout the breeding period.  Reproductive 
    parameters evaluated included:  fertility, length of gestation, number 
    of livebirths, stillbirths, number alive at 4 days, number weaned, 
    litter weight at 24 hours, litter weight at weaning, sex at weaning, 
    viability index, lactation index, growth index, live birth index, and 
    gross abnormalities.  In addition 10 weanlings/dose level from the 2nd 
    litter of the 3rd generation received an ophthalmoscopic examination, 
    gross necropsy, and a standard array of tissues were evaluated 
    microscopically.  Although occasional differences from control group 
    parameters were noted, these were sporadic and not thought to be 
    treatment related.  A reduced pregnancy rate was reported for all 
    groups of the third generation, with only the first litter of the 
    1.0 mg/kg b.w./day treatment group above 50%.  Additionally a high 
    incidence of cannibalism (30 - 44%) occurred in treated groups of the 
    3rd generation as well as in the second litter of the untreated control 

    (19%).  This cannibalism produced a non dose-related reduction in 
    lactation index in the treated groups of the 3rd generation 
    (59 - 79% in treated group litters vs 93 - 98% in controls).  No 
    effects were reported in the first two generations.  The authors 
    concluded that carbadox had no effect on fertility, lactation or 
    the neonate at any dose level (Sigler, 1969b).

    2.2.5   Special studies on genotoxicity

         The results of genotoxicity studies with carbadox are shown in
    Table 2.

    2.2.6  Special studies on relay toxicity

    2.2.6.1  Rats

         A study was done to assess the possible risk of toxicity of pork
    from pigs fed carbadox.  To accomplish the study pigs weighing 60-80
    kg were fed carbadox at the rate of 0, 20, and 200 ppm in their feed
    for 30 days at which time they were slaughtered.  Meat from these pigs
    was divided into pieces of approximately 200 grams and frozen at -18
    C.  

         Weanling Wistar rats were divided into groups of 12/sex/dose
    (except the high dose which had 24 females).  Rats received a dietary
    supplement of 10% pork liver from swine fed the carbadox regimens
    outlined above.  This dietary regimen was continued for 23 - 25
    months.  All rats received gross necropsies and the following organs
    were evaluated microscopically: liver, kidney, adrenal, testes, ovary,
    and thyroid.  Weights and mortality were recorded.  There was no
    effect on mortality.  No treatment-related histologic changes were
    reported (Ferrando et al., 1975).

    2.2.6.2  Dogs

         In another study 12 beagle dogs weighing about 4.5 kg were fed
    for one month with 100 g of control pork as part of a diversified
    diet.  The dogs were then distributed into 3 treatment groups and
    received pork from hogs treated with the above regimens of carbadox at
    the rate of 100 gm/day for the first 5 months and then 200 gms for the
    remainder of the treatment period (85 months).  The dogs were weighed
    periodically.  Every fourth month haematology, clinical chemistry, and
    urinalysis parameters were evaluated.

         All dogs were in good health at sacrifice.  The dogs received a
    gross necropsy at study termination and a standard set of tissues were
    evaluated histopathologically.  No treatment-related effects were
    reported in this study.  The authors concluded that there is no danger
    to the public health when carbadox is used in pig feed as a growth
    promoter at 50 ppm (Ferrando, et al.,1978).


        Table 2:  Results of genotoxicity assays on carbadox
                                                                                                                          
    Test System              Test Object              Concentration                 Results         Reference
                                                                                                                          
    Ames test                S. typhimurium           0.0004 - 0.125                Positive        Pfizer, undated b
                             TA1535                   (mg/plate)                                    

                             TA1536, TA1537,                                        Equivocal       
                             TA1538, C340                                                           

    Ames test                TA100                    3.1 - 61.7                    Positive        Beutin et al, 
                             TA98 (+/- S9)            (nM)                                          1981

    Ames test                TA100                    2.5 - 15 g/plate             Positive        Yoshimura et al, 
                             TA98 (+/- S9)                                                          1981

    Ames test                TA100, TA98              .1 umole/plate                Positive        Negishi, et al, 
                                                                                                    1980

    Ames test                TA100                    0.001-.02                     Positive        Voogd et al., 
                                                                                                    1980

    Mutagenicity Ames        E. coli WP2hcr                                         Positive        Ohta et al, 1980
    test                     TA100+/-S9               5 - 50 g/plate               Positive        
                             TA98                                                   Positive        
                             TA1538                                                 Negative        
                             TA1537                                                 Negative        
                             TA1535                                                 Negative        

    Host mediated Assay      S. typhimurium           0,1,2, 2.5, 5, 10,            Negative        Pfizer, undated b
                             TA1534, TA1952           0,1,2.5 5,7.5,                Positive        
                                                      10,12.5, 20 mg/kg                             

    Repair test              B. subtilis (rec)        1 - 100 g/disc               Positive        Yoshimura et al., 
                             S. typhimurium (urv)                                                   1981

    Fluctuation test         K. pneumoniae            .00002 -                      Positive        Voogd et al, 1980
                             E. coli K12              .005 mmoles/l                                 
                                                                                                                          

    Table 2 (contd)
                                                                                                                          
    Test System              Test Object              Concentration                 Results         Reference
                                                                                                                          

    Mutagenicity test        S. cerevisiae D4         .02%                          Positive        Voogd et al, 1980

    Micronucleus test        rat bone marrow          5 - 240 mg/kg i.p.            Positive        Cihak and Srb, 
                                                                                                    1983
    Repair test              B. subtilis M45          .1 - 100                      Positive        Ohta et al, 
                                                      umole/disk                                    1980

    Chromosomal damage       mouse bone marrow        200 mg/kg acute,              Positive        Pfizer, undated b
                                                      10 mg/kg day seven                            
                                                      days                                          

    Chromosomal damage       in vitro human           0 - 250 g/ml                 Positive        Pfizer, undated b
                             lymphocytes                                                            

    Dominant lethal          CD-1 mice                10,50,150,30 mg/kg            Negative        Pfizer, undated b
    assay
                                                                                                                          
    

    QUINOXALINE-2-CARBOXYLIC ACID

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with
    quinoxaline-2-carboxylic acid are given in Table 3.

    Table 3:  Acute toxicity of quinoxaline-2-carboxylic acid
                                                                 
                             LD50
    Species        Sex       Route     (mg/kg)        Reference
                                                                 
    Rats           M         Oral      4450           Briggs, 1971
    Mice           M         Oral      1360
                                                                 

    2.2.3  Long-term/carcinogenicity Studies

    2.2.3.1  Mouse

         Groups of 50 Charles River CD mice/sex/dose (100 in controls)
    received quinoxaline-2-carboxylic acid in feed for 19 months in a
    study of its chronic oral toxicity and tumorigenic potential.  Fresh
    diets were prepared weekly.  Feed and water intake were monitored
    weekly for the initial three months and monthly thereafter.  Feed
    levels of quinoxaline-2-carboxylic acid were adjusted at these
    intervals to provide target doses of 0, 25, 50, and 100 mg/kg
    b.w./day.  Haematology and clinical chemistry were evaluated once,
    prior to sacrifice.  Necropsy was performed on all animals.  Organ
    weights were obtained for brain, heart, kidneys, liver, testes, and
    ovaries.  Histopathologic examination was performed on a standard
    array of tissues.

         No treatment-related effects were noted in any parameter examined
    during the study.  Cumulative mortalities were 20% and 15% for control
    females and males, respectively, while treatment group cumulative
    mortalities ranged from 10% to 20%.  Incidence of all tumours was 33%
    in female and 46% in male control groups, while the incidences in
    treatment groups ranged from 32% to 42%.  The authors concluded that
    oral administration of quinoxaline-2-carboxylic acid to mice for 19
    months produced no evidence of toxicity (Faccini et al., 1979).

    2.2.3.2  Rats

         Nine male and 9 female Charles River C-D rats were divided into
    groups of 3/sex/dose level.  Rats received quinoxaline-2-carboxylic
    acid in the feed for 2 years.  Weight and food consumption were
    recorded weekly through week 30 and  monthly thereafter.  Drug levels
    were adjusted to provide target doses of 100, 50 or 0 mg/kg b.w./day. 

    Rats received clinical examinations weekly and ophthalmoscopic
    examinations on days 0, 30, 92, 186, 365, 554, and 733.  Haematology
    and urinalysis parameters were evaluated on days 31, 93, 182, 274,
    370, 461, 547, 644, and 730.  Terminal sacrifice was performed on day
    735 of the study.  All rats received gross necropsies and standard
    tissues were evaluated microscopically from each rat.  No treatment-
    related changes were reported.  The authors concluded that
    quinoxaline-2-carboxylic acid was tolerated at up to 100 mg/kg
    b.w./day when given to rats via feed (Coleman, 1971).

         An additional study was conducted to determine whether
    quinoxaline-2-carboxylic acid has tumorigenic potential.  To
    accomplish the study quinoxaline-2-carboxylic acid was administered in
    the diet of Charles River Sprague-Dawley rats (20/sex/dose level) for
    two years to provide target doses of 0, 10, 25, and 50 mg/kg b.w./day. 
    Weight and food consumption were monitored weekly and drug
    concentration adjusted accordingly.  Rats were examined for clinical
    signs weekly.  Ophthalmologic examinations were performed on all rats
    prior to treatment and at 6, 12, 20 and 24 months.  Clinical
    chemistry, haematology and urine parameters were evaluated in 5 rats
    at 6 and 12 months, 2-4 rats at 18 months, and all rats at
    termination.  At 12 months 5 rats/sex/dose and at 24 months all
    remaining rats were sacrificed and received a gross necropsy and
    tissues were obtained for microscopic evaluation.  Organ weights were
    determined for heart, liver, lungs, kidneys, gonads, adrenal, spleen,
    and brain.  A standard array of tissues was examined
    histopathologically.

         No treatment-related effects were noted on any of the parameters
    evaluated.  Cumulative incidences of tumours (based on summing males
    and females with 40 rats/dose) occurring in treatment groups for
    selected organs of interest are summarized in Table 4.  It was
    concluded that quinoxaline-2-carboxylic acid in doses of 10, 25, and
    50 mg/kg b.w./day over a 2 year period to rats does not produce any
    toxicity or elevated tumor incidence (Pfizer, 1971).

    Table 4:  Cumulative incidence of tumours (males and females combined)
              after treatment of rats with quinoxaline-2-carboxylic acid
                                                                        
    Treatment Group       Mammary gland    Pituitary    Pancreas    Liver
                                                                        

    Control                  28%              30%           3%        5%
    10 mg/kg b.w./day        18               23           10         5
    25 mg/kg b.w./day        13               28            3         5
    50 mg/kg b.w./day        33               28            3         3
                                                                        

         A follow-up chronic study was performed using rats obtained from
    the 3-generation study F1 litter.  An additional high dose level, 100
    mg/kg b.w./day, was added for this study.  To accomplish the study
    Charles River C-D rats were divided into groups of 50/sex/dose and
    received 100, 50, 25, or 0 mg/kg b.w./day of quinoxaline-2-carboxylic
    acid in the diet for 29 months.  In addition 10 rats/sex in the
    control and 100 mg/kg b.w./day groups were added for clinical
    chemistry studies at 12 months.  Weights were monitored weekly.  Food
    consumption was recorded weekly during the first 5 months, twice
    during the 6th month, and monthly thereafter, and treatment levels
    were adjusted accordingly.  Clinical examinations were performed
    daily.  Clinical chemistry parameters were evaluated at 6, 12, and 29
    months.  Haematology was evaluated at the conclusion of the study. 
    All rats received a gross necropsy.  Liver and kidneys were weighed
    from rats sacrificed at 12 months and heart, liver, kidneys, brain,
    and testes were weighed during the gross necropsies at 29 months. 
    Tissues from many organs showing macroscopic abnormality plus a
    standard array of grossly normal tissues were evaluated
    microscopically.

         The general health of the rats was unaffected by treatment.  An
    outbreak of sialoacryoadenitis occurred between ages 89 and 97 days. 
    Slightly reduced weight gain (3-5%) was noted in the high-dose groups
    in both sexes.  The cumulative mortality for controls was 56% in males
    and 70% in females, while treatment group cumulative mortality ranged
    from 46% to 74%.  There was no difference in the incidence of
    nonneoplastic lesions among the control and treatment groups, nor did
    treatment shorten the lifespan of treatment groups.  Treatment of rats
    for 2 years did not produce an increase in the incidence of animals
    with malignant or benign tumours compared to the control group.  The
    authors concluded that the NOAEL was 100 mg/kg b.w./day (King, 1979a).

    2.2.4  Reproduction studies

    2.2.4.1  Rats

         The potential reproductive toxicity of quinoxaline-2-carboxylic
    acid was studied in rats.  The study was performed in conjunction with
    a 2-year chronic study.  Treatment route was via feed.  The study
    utilized 2 litters per generation for  3 generations with twenty
    rats/sex/dose level using target doses of 25, 50, and 100 mg/kg
    b.w./day.  The F0 rats were bred following approximately 60 days of
    treatment with quinoxaline-2-carboxylic acid.  Fifty rats/sex/dose
    were selected for the concurrent chronic study from the initial F1
    litter.  Following a 7 day rest period the females were rebred to
    different males to produce the second litter.  The subsequent first
    litters of each generation were sacrificed at weaning while the second
    litters were used to constitute the next generation.  These first
    litters were reduced to 10 pups/litter (5/sex if possible) for
    rearing, then sacrificed, and necropsied at weaning.  The 2nd litters

    were used to produce the next generation.  Subsequent generations of
    rats were mated at 100 days of age.  Dose levels were maintained and
    test animals were treated from weaning throughout the breeding period.

         Rats were inspected daily.  Food consumption and weights were
    determined weekly except for mating periods.  Adults of all
    generations were sacrificed after weaning of the 2nd litter and
    received a gross necropsy with liver weights determined.  Blood levels
    were determined in F0 rats (5/sex/level) just prior to sacrifice. 
    Pups were examined daily until weaning and weighed on days 1, 4, and
    21 after birth.  Gross necropsies were carried out on all pups that
    died or were stillborn and on 5 weanlings/sex/dose.  Reproductive
    parameters evaluated included:  fertility, length of gestation, number
    of livebirths, stillbirths, number alive at 1, 4, and 21 days, number
    weaned, sex ratios, mean pup weights, lactation index, and gross
    abnormalities.  No treatment-related alterations were observed in any
    parameter monitored during the study (King, 1979b).

    2.2.5  Special studies on embryotoxicity/teratogenicity

    2.2.5.1  Rabbits

         The potential embryotoxicity or teratogenicity of quinoxaline-2-
    carboxylic acid was also studied in the New Zealand White Rabbit. 
    Rabbits were randomly assigned to 4 treatment groups of 19 - 20
    animals receiving 0, 25, 50, or 100 mg/kg b.w. quinoxaline-2-
    carboxylic acid via gavage on gestation days 7-18.  Animals were
    observed daily for toxicity and weighed on gestation days 0, 3, 7, 9,
    12, 15, 18, 21, 24, and 28.  Rabbits were sacrificed on day 28 and the
    number of corpora lutea and number and position of live and dead
    implants in the uteri were recorded.  All fetuses were examined for
    external malformations and weighed.  Half the fetuses were cleared,
    stained with alizarin red and examined for skeletal abnormalities. 
    The remaining half were fixed in Bouin Allen fluid and examined by
    serial sectioning.  No treatment related maternal toxicity,
    embryotoxicity or malformations were observed (Holmes, 1976a).

    2.2.6  Special studies on genotoxicity

         The results of genotoxicity studies with quinoxaline-2-carboxylic
    acid are given in table 5. 

    Table 5:  Quinoxaline-2-carboxylic acid genetic toxicology

                                                                          
    Test System    Test Object      Concentration    Results    Reference
                                                                          

    Ames test      S. typhimurium   .008, .032,      Negative   Pfizer, 
                   TA1535           .125, .5,                   1975
                   TA1537           2 mg/plate
                   TA1538
                   TA 1535+S9       .16, .63,        Negative   Pfizer,
                                    2.5 mg/plate                1975

    Chromosomal    Human            50, 100          Negative   Pfizer,
    aberrations    lymphocytes      g/ml                       1975
                   in vitro
                                                                          

    DESOXYCARBADOX

    2.2   Toxicological studies

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Rats

         A long term study was performed to determine the tumorigenic
    potential of desoxycarbadox, a carbadox metabolite.  Four hundred
    Charles River C-D rats (5 weeks of age) were divided into groups of
    50/sex/dose level.  Rats were distributed randomly according to weight
    levels.  Desoxycarbadox was administered at target doses of 0, 5, 10,
    and 25 mg/kg b.w./day continuously in the diet.  Weights were recorded
    weekly.  Food consumption was monitored weekly the first 3 months and
    monthly thereafter.  Treatment levels were adjusted accordingly. 
    Although treatment was planned for 2 years, the test material was
    withdrawn from all rats in the 25 mg/kg b.w./day group and 50% of the
    rats in each sex in the other 2 treatment groups on day 350, due to
    high morbidity and mortality.  Administration of desoxycarbadox was
    stopped completely on day 416.  The study was terminated after 447
    days.

         Clinical examinations were performed twice weekly.  A number of
    treatment-related signs were observed.  These included:  tumours in
    the mammary region of both sexes, small cutaneous nodules, and
    enlargement of the liver with nodules preceded by weight loss and
    polyphagia.  There was also a dose-related decrease in weight gain and
    a dose-related decrease in survival rate.

         Clinical chemistry parameters were evaluated.  Desoxycarbadox
    administration resulted in dose-related increases in plasma enzyme
    activities, urea, and bilirubin.  The abnormalities were consistent
    with hepatic disorders.  The high degree of variability from animal to
    animal and lack of reversibility following drug withdrawal indicates
    a correlation with tumour progression.  A dose-related hypoglycaemia
    was noted in both sexes.  Haematologic parameters were evaluated in
    ten rats/sex/dose at day 413 and the remainder at 447 days.  The two
    primary changes in haematology in the treated rats were a moderate
    hypochromic microcytic anaemia in the 25 mg/kg b.w./day group, a
    slight anaemia in the 10 mg/kg b.w./day group, and a neutrophilia in
    the 25 mg/kg b.w./day group.

         All rats received gross necropsies.  Selected organs were
    weighed.  Histopathologic examination was performed on all grossly
    abnormal tissue and routinely on a standard array of tissues.  There
    was a dose-related increase in pigmentation of renal tubules and
    nephrosis.  The major pathologic findings related to increased tumour
    incidences are summarized in Table 6.  The authors concluded that
    desoxycarbadox was a potent hepatocarcinogen in the rat and that there
    were dose-related increases in other tumours (Reinert, 1976).

    Table 6:  Incidence of tumours after administration of desoxycarbadox
               to rats

                                                                           
    Treatment   Hepatic    Subcutaneous   Haemanigiomas     Mammary
      Group     tumours      fibromas                       tumours

                       Males and females combined            Females only
                                                                        
    Control          0%           0%            0%           12%

    5 mg/kg         75            2             0            24%
    b.w./day

    10 mg/kg        95           10             5            28
    b.w./day

    25 mg/kg       100           18            47            27
    b.w./day
                                                                           

    2.2.5.2  Special Studies on Genotoxicity

         The results of genotoxicity studies with desoxycarbadox are
    summarized in Table 7.


        Table 7:  Desoxycarbadox genetic toxicology
                                                                                                 
    Test System           Test Object         Concentration             Result       Reference
                                                                                                 
    Ames test             TA1535              .0008, .0032,             Negative     Pfizer, 1975
                          TA1537              .0125, 0.05, .125,                     
                          TA1538              .5, 2, 20 mg/plate                     

                          TA1535+S9           .0125, .05, .2, 1,        Negative     Pfizer, 1975
                                              5 ml/plate                             

    Ames test             TA1535              .032, .25, .4, .5         Negative     Pfizer, 1975
                          TA1537              ml/plate  plasma;                      
                          TA1538              .032, .25. .5 ml/plate                 
                                              urine1

    Host mediated         TA1950              500 mg/kg acute           Negative     Pfizer, 1975
    assay                                                                            

    Chromosomal damage    Human lymphocytes   200 g/ml                 Negative     Pfizer, 1975

                          Rat bone marrow     200 mg/kg acute,          Negative     Pfizer, 1975
                                              or 100 mg/kg 5 days                    

                                              10 mg or 25 mg per kg     Positive     Pfizer, 1975
                                              9 months in feed                       

    Host mediated         TA1950              400 or 500 mg/kg          Negative     Pfizer, 1975
    assay in mice

    Host mediated         TA1950              300 mg/kg with 100 mg/    Negative     Pfizer, 1975
    assay in rats                             kg/day previous 31 days

                          TA1535              200 mg/kg                 Negative     
                                                                                                 

    Table 7 (contd)
                                                                                                 
    Test System           Test Object         Concentration             Result       Reference
                                                                                                 
    Yeast assay           S. cerevisiae D3    10 mg/ml                  Negative     Holmes, 1976b
                          & D4

    Host mediated         TA1950              400 mg/kg +DMNA           Negative     Holmes, 1976b
    assay (mouse)                                                                    

    Ames test             TA1537              .01 - 10 mg/plate         Negative     Holmes, 1976b
                          TA100                                                      
                          TA98                                                       

    Ames test             TA1535+/- TA100     .005 - 2 mg/plate         Positive     Holmes, 1976b
                          S9(rat)                                                    

    Ames test             TA1537+S9           .005 - 2 mg/plate         Negative     Holmes, 1976b
                          TA98                                                       

    Ames test             TA1535+S9 from      .005 - 2 mg/plate                      Holmes, 1976b
                          rat                                                        
                          mouse                                         Positive     
                          hamster                                       Positive     
                          dog                                           Negative     
                          monkey                                        Negative     
                                                                        Negative     

    Ames test             TA1535              Quantitative plate        Negative     Holmes, 1976b
                                              assay on plasma, liver                 
                                              & urine at selected                    
                                              intervals from chronic                 
                                              study rats fed carbadox                
                                              or desoxy-carbadox                     

    Cell transformation   BALB/C Swiss 3T3    .1-10 g/ml               Positive     Holmes, 1976b
    test                                                                             
                                                                                                 

    1 Urine and plasma collected from mice or rats administered 400 mg/kg desoxycarbadox administered 
      in 3 hourly doses or 100 mg/kg/day for 5 days.
    

    METHYL CARBAZATE

    2.2  Toxicological studies

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Rats

         Methyl carbazate is a metabolite of carbadox.   The chronic oral
    toxicity of methyl carbazate was studied in the rat.  Wistar rats were
    divided into groups of 12/sex/dose and received methyl carbazate at
    target doses of 1 and 10 mg/kg b.w./day in the feed for 21 months. 
    The colony served as controls.  Food consumption was determined every
    2 days and weights every 2-3 weeks.  Necropsy was performed on all
    animals.  Organ weights were determined for liver, kidney, adrenals,
    testes, and thyroid.  Histology was performed on liver, kidneys,
    adrenals, thyroid, testes, ovaries, and tissues containing tumours
    observed grossly.  Three males and 3 females in the high dose group
    died before termination.  No low dose rats died prior to termination. 
    No histopathologic evidence of toxicity or evidence of an elevation in
    tumours was reported.  The authors concluded that administration of
    methyl carbazate to rats at dose levels of 1 and 10 mg/kg b.w./day in
    the diet produced no evidence of toxicity or carcinogenic potential
    (Rutty, 1972).

         In a second study weanling Wistar rats were divided into groups
    of 24/sex and treated with target doses of 0, 2.5, 5, and 10 mg/kg
    b.w./day methyl carbazate in the feed for 710 days.  Weights were
    determined weekly and food consumption weekly the first 3 months and
    monthly thereafter.  Drug concentrations were adjusted accordingly. 
    Clinical examinations were conducted weekly.  Blood samples for
    clinical chemistry were obtained at terminal sacrifice and from
    moribund animals.  Haematology was also conducted on these samples and
    at 12 months on 6 rats.  Rats were sacrificed and necropsied at 710
    days.  An interim sacrifice was also performed at 12 months.  All
    moribund and dead rats also received a gross necropsy.  Histopathology
    was performed on all gross lesions and the following organs: liver,
    pituitary, thyroid, parathyroid, adrenal, lung, kidney, urinary
    bladder, ovary, and testis.

         There were no treatment-related effects noted in any parameters
    evaluated in the study.  There was histologic evidence of widespread
    chronic respiratory disease in all groups.  It was concluded that
    methyl carbazate had no adverse effect when given to rats in the diet
    for 2 years (Ferrando, 1980).

    2.2.5  Special studies on genotoxicity

         The results of genotoxicity studies with methyl carbazate are
    summarized in Table 8.

        Table 8:  Genetic toxicity studies with methyl carbazate
                                                                                  
    Test System    Test Object    Concentration       Results        Reference
                                                                                  

    Ames test      TA1535         .0005, .002,        Negative       Pfizer, 1975
                   TA1537         .008, .032,
                   TA1538         .125, 0.5, 2, 5
                                  mg/plate

                   TA1535+S9      .0125, .05, .2      Negative       Pfizer, 1975
                                  1, 5, 10
                                  mg/plate

    Host-          TA1950         50, 100, 300        Equivocal      Pfizer, 1975
    mediated                      mg/kg
    assay

    Chromosomal    Human          50, 100             Negative       Pfizer, 1975
    damage         lymphocytes    g/ml
                   in vitro

    Ames test      TA1535+S9      0.5 - 10            Negative       Holmes, 1976b
                                  mg/plate
                                                                                  
    
    HYDRAZINE

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, metabolism, and excretion

         Hydrazine toxicokinetic parameters have been determined in the
    rabbit.  Rabbits received a bolus of 12 mg/kg b.w.  Parameters were
    derived using a one-compartment model, and spectrophotometric serum
    analysis for hydrazine, with 5 rabbits/group.  The serum half-life was
    2.3 hours and the volume of distribution was 0.63 liters/kg (Keller
    et al., 1981).

         The metabolism, distribution, and elimination of hydrazine in
    rats was studied using 15N-labelled hydrazine or a spectrophotometric
    method.  About 15% of the 15N label was found as N2. About 20% and
    30% of the infused hydrazine was recovered in the urine as acetylated
    metabolite and hydrazine, respectively.  The serum half-lives were
    determined, using a two-compartment model, to be 0.74 hours for the
    majority of the hydrazine elimination, and 26.9 hours for a much
    slower, smaller elimination component (Dost et al., 1981).

    2.1.3  Effects on enzymes and other biochemical parameters

         Hydrazine-induced short-term biochemical parameter changes were
    investigated following oral administration of hydrazine in Sprague-
    Dawley rats.  Groups of 5 rats were given doses of hydrazine (0-200
    mg/kg b.w.).  Some rats received pyruvate azine, phenobarbital,
    piperonyl butoxide, or diethylmaleate to more clearly elucidate the
    hepatic effects of hydrazine.  Hydrazine caused a dose-related
    increase in liver triglycerides and liver weight and a decrease in
    hepatic glutathione.  The threshold dose for toxicity was about 10
    mg/kg b.w.  While liver weight and glutathione alterations were
    evident within 30 minutes, triglyceride alteration was not evident for
    4 hours.  Pretreatment with phenobarbital decreased toxicity, while
    pretreatment with piperonyl butoxide increased toxicity suggesting
    hepatotoxicity is due to the parent - not a metabolite.  Depletion of
    hepatic glutathione was without effect on hepatic toxicity, and
    studies with pyruvate azine - a possible oxidative metabolite - also
    supported this conclusion (Timbrell, et al., 1982).

         Hydrazine-induced short-term histologic and ultrastructural
    changes were investigated following oral administration of hydrazine
    to Sprague-Dawley rats.  Groups of 5 rats were given doses of
    hydrazine (0-200 mg/kg b.w.).  Some rats received pyruvate azine,
    phenobarbital, piperonyl butoxide, or diethylmaleate to more clearly
    elucidate the hepatic effects of hydrazine.  A dose of 20 mg/kg caused
    accumulation of lipid, swelling of mitochondria, and the appearance of
    microbodies in periportal and midzonal hepatocytes.  Accumulation of
    lipid droplets and mitochondrial swelling were detected by electron

    microscopy 30 minutes after dosing.  Piperonyl butoxide or
    phenobarbital treatment increased or decreased fatty liver
    respectively, while pyruvate azine was much less effective than
    hydrazine in producing this effect (Scales & Timbrell, 1982).

    2.2  Toxicological studies

    2.2.3  Long-term carcinogenicity studies

    2.2.3.1  Mice

         Oral administration of hydrazine sulfate to Balb/c female mice at
    1.13 mg/day for 46 weeks produced a 100% incidence of lung tumours
    (Biancifiori & Ribacchi, 1962).

         In another study hydrazine was administrated via gavage to 25
    female Swiss mice 5 days/week for 40 weeks at the rate of 0.25 mg/day. 
    Eighty-five mice served as controls. This dose regimen produced a 46%
    incidence of lung tumours in treated mice vs 10% in controls (Roe et
    al., 1967).

         A study with CBA/Cb/Se mice was performed using hydrazine sulfate
    given for 36 weeks by gavage.  Twenty-one 8- week-old rats/sex
    received 1.13 mg/day.  Treated rats had an incidence of 76% and 90%
    lung tumours in males and females, respectively, while controls had
    3%.  Hepatomas were found in 62% of the males and 71% of the females
    treated with hydrazine, while 11% of control males and 4% control
    females had hepatomas (Severi & Biancifiori 1968).

         In another study in mice, 8-week-old CBA mice were divided into
    groups of 40-59 mice/sex.  Hydrazine sulfate was given by gavage daily
    for 150 days at rates of 45, 22, 11, 5.6 and 0 mg/kg b.w./day.  Mice
    were examined at natural death or following sacrifice when moribund. 
    Control male mice had a hepatoma incidence of 10% while females had a
    3.4% rate.  Treated males had hepatoma rates of 60, 48, 28, and 3.8%
    at 45, 22, 11, and 5.6 mg/kg b.w./day, respectively.  Treated females
    had 62.5, 66.6, 8 and 0% hepatomas, respectively.  The author reported
    many lung tumours in treated groups but did not provide the incidence
    rates (Biancifiori, 1970).

    2.2.3.2  Hamsters

         Eight-week old golden hamsters were divided into 3 groups of 23,
    35 and 56 animals which were administered 60 doses of 3.0 mg hydrazine
    sulfate via intubation over a 15 week period, 2.8 mg 100 times during
    a 20 week period or no hydrazine, respectively.  Hepatic lesions
    reported include:  Fibrosis, reticuloendothelial cell proliferation,
    and bile duct proliferation.  Hepatic lesions were found in 60-80% of
    treated hamsters but in none of the controls.  No hepatic tumours were
    reported (Biancifiori, 1970).

         Negative tumorigenicity results were also obtained in a follow up
    chronic study with golden hamsters given 2.3 mg/day over a lifetime. 
    In this study 50 hamsters/sex received hydrazine in drinking water
    from the age of 9 weeks for a lifetime (Toth, 1972).

    2.2.3.3  Rats

         A chronic oral study was performed in conjunction with the above
    mouse study using 8 week old Cb/Se rats.  Aqueous hydrazine sulfate
    was administered daily by stomach tube at doses of 18 mg/rat to 14
    males and 12 mg/rat to 18 females for 68 weeks.  Lung tumours were
    found in 21 and 27% of dosed male and female rats, respectively.  
    Control groups of 28 males and 22 females had no lung tumours. 
    Hepatic tumours were found in 30% of males, while no hepatic tumours
    were found in females or controls (Severi & Biancifiori, 1968).

    2.2.7.5  Special studies on genotoxicity

         The results of genotoxicity studies with hydrazine are summarized
    in Table 9.

        Table 9:  Genetic toxicity studies with hydrazine 
                                                                                 
    Test System    Test Object         Concentration    Results      Reference
                                                                                 

    Bacterial      E. coli WP2         0.5 - 2.0 mole  Positive     von Wright
                   uvr A trp                                         & Tikkanen,
                                                                     1980

    Mouse          L5178Y              0.1 - 5 mmole    Positive     Rogers &
    lymphoma                                                         Back, 1981
    cells

    Ames test      S. typhimurium                       Positive     Ames, 1971

                                                                                 
    
    2.3.  Observations in humans

         No information available

    3.  COMMENTS

         The Committee considered data from short-term, long-term and
    "relay" toxicity studies, and from studies on carcinogenicity,
    mutagenicity, and reproduction with carbadox, together with data from
    mutagenicity and long-term toxicity studies of its metabolites.

         The metabolism of carbadox has been studied in rats, monkeys and
    pigs using [14C]carbadox, labelled in either the phenyl ring or the
    carbonyl group of the side-chain.  The metabolism of carbadox was
    characterized by the rapid reduction of the N-oxide groups, the
    cleavage of the methylcarbazate side-chain, and the liberation of
    respired CO2.  The primary metabolite in the urine was quinoxaline-2-
    carboxylic acid, which was also excreted in conjugated form.  The
    detectable residues in tissues, up to 24 hours after drug withdrawal,
    were carbadox, desoxycarbadox, quinoxaline-1,4-di-N-oxide-2-
    carboxaldehyde and quinoxaline-2-carboxylic acid.  Hydrazine was a
    minor metabolite but would be expected to be present only for a short
    time before undergoing further metabolism.  

         The concentration of carbadox and desoxycarbadox in the tissues
    declined rapidly (half-life about 6 hours), and, after 3 days, was
    less than 5 g/kg.  In a study in pigs, in which carbonyl-labelled
    [14C]carbadox was administered by gavage, the concentration of total
    residues in liver at 5 days withdrawal time corresponded to 0.12 mg/kg
    methyl carbazate equivalent.  However, some of this radioactivity was
    due to the presence of amino acids, which had been labelled by
    incorporation of 14CO2 when ring-labelled [14C]carbadox was
    administered to pigs, extremely low levels of unidentified metabolites
    remained in the liver at withdrawal periods longer than 7 days.  These
    residues were partially released and converted to quinoxaline-2-
    carboxylic acid by alkaline digestion of the liver. 

         The Committee also reviewed recent reports of the effect of
    carbadox on adrenal function in pigs, although the information did not
    effect the outcome of the evaluation.

         Several long-term feeding studies in rats were evaluated which
    demonstrated dose-related increases in the incidence of benign and
    malignant liver tumours at doses of carbadox above 1.0 mg per kg of
    body weight per day.  Doses above 25 mg per kg of body weight per day
    produced pronounced toxic effects which precluded chronic treatment. 
    Data from a variety of mammalian and non-mammalian genotoxicity
    studies were also reviewed.  Positive findings were reported in 14 of
    the 15 tests carried out.  The Committee concluded that carbadox
    appears to be both genotoxic and carcinogenic.

         The Committee evaluated a "relay" toxicity study in which pigs
    were fed carbadox at up to 200 mg/kg in the diet for 30 days and
    killed at zero withdrawal time, their livers were fed to rats as a 10%

    dietary component for 2 years.  Although this study was conducted
    satisfactorily and no treatment-related effects were reported, the
    Committee did not use it for assessing the carcinogenicity of carbadox
    residues  because of the number of animals used and because only small
    quantities of residues can be consumed by rats in this type of study.

         In a long-term study in rats, desoxycarbadox was reported to
    produce an increase in the incidence of tumours.  All rats that
    received 25 mg per kg of body weight desoxycarbadox daily for 10
    months in the diet developed hepatic tumours.  The incidence of
    tumours was increased in all treated groups, at doses of 5-25 mg per
    kg of body weight per day.  While the most pronounced change  occurred
    in the liver, tumour incidence was also elevated at other sites,
    including the skin and mammary glands.

         The mutagenic potential of desoxycarbadox was investigated in a
    range of studies.   The Committee noted that, while desoxycarbadox
    produced negative results in most mutagenicity test systems, positive
    findings were recorded in the cell transformation test and in the Ames
    test using liver microsomes from rats treated with polychlorinated
    biphenyls.  The Committee noted that the tumorigenic potential of
    desoxycarbadox was apparently greater than that of the parent
    compound, and that desoxycarbadox would therefore probably make a
    significant contribution to the tumorigenic activity of carbadox in
    rats.  However, the Committee also noted that the metabolism data for
    carbadox supported the conclusion that desoxycarbadox was a relatively
    short-lived intermediate between carbadox and quinoxaline-2-carboxylic
    acid.

         The Committee also considered information on the carbadox side-
    chain metabolite, methyl carbazate.   This information consisted of
    results from two long-term studies in rats and a number of
    mutagenicity assays.  In the more informative long-term study (in
    which more animals per dose and concurrent controls were used), rats
    received methyl carbazate in the diet for 710 days.  There was no
    increase in the incidence of tumours and no other treatment-related
    effects were observed at 10 mg per kg of body weight per day, which
    was the highest dose tested.  The lack of effect limited the
    usefulness of this study.  The no-observed-effect level for the study
    was 10 mg per kg of body weight per day.  The useful mutagenicity
    studies were reported to be negative.

         The results of studies on the pharmacokinetics, mutagenicity, and
    carcinogenicity of hydrazine were also considered by the Committee. 
    Pharmacokinetic data from rats and rabbits as well as hydrazine's
    known chemical reactivity suggest that it should be rapidly
    eliminated.  The structure of carbadox and its known metabolic
    pathways indicate that hydrazine is probably a further metabolite of
    methyl carbazate.  Hydrazine showed mutagenic and carcinogenic
    potential.  

         In evaluating the potential toxicity of residues of carbadox, the
    Committee also considered toxicity data on the carbadox metabolite
    quinoxaline-2-carboxylic acid to be useful.  In a carcinogenicity
    study in mice in which quinoxaline-2-carboxylic acid was administered
    in the diet for 19 months, no treatment related increases in tumour
    incidence or any other effects on the animals were reported.  The no-
    observed-effect level was 100 mg per kg of body weight per day.

         Several chronic toxicity and carcinogenicity studies in which
    quinoxaline-2-carboxylic acid was administered for 24 months were
    conducted in rats.  There were no effects on the incidence of tumours,
    the only findings being a slight reduction in  weight gain at 100 mg
    per kg of body weight per day.  The no-observed-effect level was 50 mg
    per kg of body weight per day.

         In a three-generation reproduction study in rats, quinoxaline-2-
    carboxylic acid was given in the diet at levels of up to 100 mg per kg
    of body weight per day.  No treatment-related effects were observed. 
    Developmental studies were conducted in rats and rabbits in which the
    compound was administered by gavage at levels of up to  100 mg per kg
    of body weight per day.  There was no evidence of maternal toxicity,
    embryotoxicity, or teratogenicity in either species.  The no-observed-
    effect levels in these studies were 100 mg per kg of body weight per
    day.

    4.  EVALUATION

         With current analytical procedures, quinoxaline-2-carboxylic acid
    is the only carbadox metabolite that can be identified in liver from
    pigs treated according to good practice in the use of veterinary
    drugs.  However, uncertainty remains because of the presence of
    unidentified residues in liver.   Because of the genotoxic and
    carcinogenic nature of carbadox and some of its metabolites, the
    Committee was not able to establish an ADI.

         On the basis of data from studies on the toxicity of quinoxaline-
    2-carboxylic acid, and on the metabolism and depletion of carbadox and
    the nature of the compounds released from the bound residues, the
    Committee concluded that residues resulting from the use of carbadox
    in pigs were acceptable, provided that the recommended MRLs are not
    exceeded.

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    See Also:
       Toxicological Abbreviations
       Carbadox (ICSC)
       Carbadox (JECFA Food Additives Series 51)
       CARBADOX (JECFA Evaluation)