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        INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

        WORLD HEALTH ORGANIZATION



        TOXICOLOGICAL EVALUATION OF CERTAIN
        VETERINARY DRUG RESIDUES IN FOOD



        WHO FOOD ADDITIVES SERIES 41





        Prepared by:
          The 50th meeting of the Joint FAO/WHO Expert
          Committee on Food Additives (JECFA)



        World Health Organization, Geneva 1998




    SARAFLOXACIN

    First draft prepared by
    Dr P.L. Chamberlain
    Center for Veterinary Medicine
    Food and Drug Administration, Rockville, Maryland, USA

    1.   Explanation
    2.   Biological data
         2.1  Biochemical aspects
              2.1.1     Absorption, distribution, and excretion
              2.1.2     Biotransformation
         2.2  Toxicological studies
              2.2.1     Acute toxicity
              2.2.2     Short-term toxicity
              2.2.3     Long-term toxicity and carcinogenicity
              2.2.4     Genotoxicity
              2.2.5     Reproductive toxicity
              2.2.6     Special studies on microbiological effects
              2.2.7     Special studies on ecotoxicity
         2.3  Observations in humans
    3.   Comments
    4.   Evaluation
    5.   Acknowledgements
    6.   References

    1.  EXPLANATION

         Sarafloxacin is a fluoroquinolone antibacterial agent that acts
    by inhibiting the activity of DNA gyrase. It is used for treatment and
    control of bacterial infections in poultry caused by  Escherichia 
     coli and  Salmonella spp. Sarafloxacin has not previously been
    reviewed by the Committee.

         The structure of sarafloxacin is shown in Figure 1.

    FIGURE 1

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

          Mice

         Three groups of 12 female mice were treated with
    14C-sarafloxacin base, as follows: Animals in the first two groups
    were given a single dose of 10 mg/kg bw, one group intravenously and
    the other by gavage; animals in the third group were given a dose of
    100 mg/kg bw by gavage. Urine and faeces were collected from the mice
    daily for three days. Compliance with the principles of GLP was not
    required for this study. The quality and design of the study was
    consistent with current scientific standards.

         Absorption of the parent drug was estimated from the content in
    0-24-h urine samples: 48% (range, 27-73%) of the parent drug was
    absorbed after the 10 mg/kg bw dose and 34% (range, 29-38%) after the
    100 mg/kg dose. Within three days of administration of the single
    intravenous dose, 49% was excreted in the urine and about 44% was
    eliminated in the faeces. After oral administration of the same dose,
    urinary excretion accounted for about 25% and faecal excretion for
    80%. Mice given 100 mg/kg bw orally eliminated 18% in the urine and
    74% in the feces. Almost all of the radiolabel was excreted during the
    first 24 h after oral or intravenous administration (Merits & Bopp,
    1985a).

          Rats

         Five groups of 18 Sprague-Dawley rats of each sex were treated
    with sarafloxacin as follows: One group received a single intravenous
    dose of 20 mg/kg bw; three groups received a single oral dose of 20,
    75, or 275 mg/kg bw; and animals in the fifth group received an oral
    dose of 1000 mg/kg bw daily for 14 consecutive days. Blood samples
    were collected from four rats in each group just before treatment and
    0.5, 1, 2, 4, 6, 8, 12, and 24 h after treatment on day 1 for the
    groups receiving the single dose and on days 1 and 14 for the 14-day
    treatment group. Plasma and urine samples were assayed for
    sarafloxacin base by high-performance liquid chromatography. The
    pharmacokinetic parameters determined from this study are presented in
    Table 1. A comparison of the 0 to infinity area under the
    concentrationtime curve (AUC) after a single intravenous or oral dose
    of 20 mg/kg bw sarafloxacin indicated that its bioavailability was
    about 12%. A plot of the AUC against dose was linear up to 275 mg/kg
    bw but deviated from linearity at 1000 mg/kg bw. Compliance with the
    principles of GLP was not required for this study. The quality and
    design of this study were consistent with current scientific standards
    (Patterson, 1985).


        Table 1. Pharmacokinetic parameters of sarafloxacin in rats

                                                                                                               

    Dose        Route         Vd        Elimination   Tmax    Cmax      Ka      Ke           Apparent
    (mg/kg                    (L/kg)    t1/2 (h)      (h)     (g/ml)   (h-1)    (h-1)       body clearance 
    bw)                                                                                      (ml/min per kg)
                                                                                                               

    20          i.v.          5.3       2.0           -       -         -       0.3          30
    20          Oral          60        3.0           1.0     0.3       3.0     0.3          270
    75          Oral          70        2.0           2.0     0.6       1.0     0.4          470
    275         Oral          250       7.0           2.0     0.9       2.0     0.1          420
    1000        Oral          400       6.0           1.0     2.0       2.0     0.1          820
    1000        Oral once/    110       6.0           2.0     8.0       1.0     0.1          200
                d for 14 d
                                                                                                               

    i.v., intravenous
    
         In a summary report of another study, Sprague-Dawley rats (number
    and sex not stated) were given 10 mg/kg bw 14C-sarafloxacin orally.
    Within three days, about 37% of the radiolabelled dose had been
    excreted in the urine, while about 52% was recovered in faeces (Bopp,
    1985a)

          Rabbits

         The absorption, metabolism, and excretion of 14C-labelled
    sarafloxacin was studied in three-month-old female New Zealand white
    rabbits. Two groups of three animals per group were treated orally by
    gavage with 10 mg/kg bw of 14C-sarafloxacin base. A third group of
    three animals received the same dose by intravenous administration.
    Blood samples were collected 1, 3, 6, 12, and 24 h after oral
    administration from animals in one of the groups, and urine and faeces
    were collected daily for five days from animals in the other groups.
    Compliance with the principles of GLP was not required. The quality
    and design were consistent with current scientific standards.

         Within five days of oral administration, about 11% of the dose
    was eliminated in the urine and about 79% in the faeces. Urinary
    excretion after intravenous administration indicated that about 16% of
    the oral dose had been systemically absorbed (Merits & Bopp, 1985b).

          Dogs

         Three groups of 14 dogs (strain, age, and sex not stated) were
    given daily oral doses of 5, 25, or 125 mg/kg bw sarafloxacin base by
    capsule. After one month, six dogs from each group were killed, and
    plasma and cerebrospinal fluid were collected for high-performance
    liquid chromatography. The remaining dogs continued to be treated
    daily for a total of 90 days. The pharmacokinetic parameters
    determined from this study are presented in Table 2. Compliance with
    the principles of GLP was not required. The quality and design were
    consistent with current scientific standards. It was also shown that
    the dispositional kinetics of sarafloxacin in the dog are independent
    of dose and treatment duration, while absorption of sarafloxacin
    becomes less efficient with increasing dose (Granneman, 1985a) .

         The tissue distribution of 14C-sarafloxacin base after a single
    oral dose of 10 mg/kg bw was studied in four adult male beagle dogs.
    The concentrations of radiolabel in tissues 2 and 6 h after treatment
    are shown in Table 3. Compliance with the principles of GLP was not
    required for this study. The quality and design were consistent with
    current scientific standards (Bopp, 1985b).

         The bioavailability of an oral dose of 200 mg sarafloxacin base,
    equal to 20 mg/kg bw, was studied in six adult female dogs. The
    compound was administered as a suspension, a solution, or a capsule.
    Compliance with the principles of GLP was not required for this study.
    The quality and design were consistent with current scientific
    standards.


        Table 2. Pharmacokinetics of sarafloxacin base after oral administration to dogs

                                                                                                                  

    Dose           Mean half-life (h)a               AUC (g h/ml)                          Mean ratio
    (mg/kg bw)                                                                              cerebrospinal
                   2 doses    24 doses   79 doses    2 doses      24 doses     79 doses     fluid:plasmab
                                                                                                                  

    5              5          6          6           9            9            10           0.2

    25             5          5          6           30           31           30           0.2

    125            5          6          6           104          108          106          0.2
                                                                                                                  

    a Samples taken 1, 3, 6, and 24 h after treatment
    b Samples taken about 24 h after treatment
    

         The bioavailability of the suspension and capsule were similar.
    The values for the 0-32-h mean AUC for these formulations were 27 and
    23 g h/ml, respectively. The mean AUC for the solution was 52 g
    h/ml. The author cited the results of other studies which showed that
    the bioavailability of an oral 10 mg/kg bw dose of the solution in
    comparison with an equal intravenous dose was 58-70%. The relationship
    between dose and bioavailability for the capsule formulation appears
    to be non-linear or log-linear. The suspension resulted in AUC values
    that were about one-half those obtained with the solution; however, in
    a study with a dose of 10 mg/kg bw, the formulations were equivalent.
    The basis for these differences was not readily apparent (Granneman,
    1985b).

         In another study, reported as a summary, dogs (breed, sex, and
    number not stated) were given an oral or intravenous dose of 10 mg/kg
    bw dose of 14C-sarafloxacin base. The extent of absorption was
    estimated to be 73% on the basis of the AUCs and 89% on the basis of a
    comparison of volumes of distribution. About 54% of the radiolabelled
    dose was recovered from urine and about 27% from faeces. About 30% of
    the intravenous dose was also eliminated in the faeces, suggesting
    that biliary secretion is a factor in the disposition of the compound
    (Bopp, 1985c).


    Table 3. Concentrations (g equivalents/g or ml) of 
    radiolabel in tissues of male dogs after oral 
    administration of 14C-sarafloxacin at a dose of 
    10 mg/kg bw

                                                       

    Tissue            2h           6h           24h
                                                       

    Liver             14           12           2
    Kidney            16           14           1
    Lung              6            5            1
    Brain             0.4          0.7          0.3
    Fata              0.6          0.5          0.6
    Musclea           5            6            1
    Boneb             3            3            2
    Retina/uvea       15           43           45
    Blood             3            3            0.4
    Plasma            3            3            0.4
    Bile              154          454          420
    Urine             89           412          188
                                                       

    a   Percent dose in muscle and fat calculated by
        assuming that those tissues represent 46% and
        10% of the body weight, respectively
    b   Rib including marrow

          Humans

         Grannemann (1985b) cited data from a clinical study in which
    capsules of the same lot given to the dogs were administered to
    humans. The recoveries in urine ranged from 24% at 1.3 mg/kg bw to 10%
    at 10 mg/kg bw. As the recoveries of sarafloxacin in human urine are
    considered to provide an approximate estimate of absorption, since the
    urine is the predominant route of elimination in humans, the results
    indicate that the absorption rates in humans are considerably lower
    than those in dogs.

         A single oral dose of 100, 200, 400, or 800 mg sarafloxacin was
    administered to 22 healthy male volunteers ranging in age from 20 to
    39 years. Blood samples were taken before treatment and 0.25, 0.5, 1,
    1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 28, 32, and 48 h after treatment.
    Urine was collected at 0-4, 4-8, 8-12, 12-16, 16-24, 24-32, and 32-48
    h. Compliance with the principles of GLP was not required for this
    study. The quality and design were consistent with current scientific
    standards.

         The plasma concentrations peaked 1.5-4 h after treatment and
    declined biphasically, the terminal phase becoming dominant about 12 h
    after treatment. The means of the individual peak concentrations after
    the 100, 200, 400, and 800 mg doses were 140, 180, 240, and 350 ng/ml,
    respectively. The corresponding dose-normalized peak concentrations
    were 106, 62, 44, and 34 ng/ml per mg/kg. The average dose-normalized
    AUC values for the 100, 200, 400, and 800 mg doses were 860, 570, 410,
    and 350 ng h/ml per mg/kg, respectively. The decreases in the values
    of the dose-normalized AUC and peak concentrations as a function of
    dose provide evidence that the efficiency of absorption deceased by a
    factor of about 3 as the dose was increased. The average terminal
    phase half-lives were 9, 9, 10, and 11 h at the 100, 200, 400, and 800
    mg doses, respectively

         Elimination occurred mainly by renal excretion of unchanged drug.
    The average renal clearances of the 100, 200, 400, and 800 mg doses
    were 280, 290, 290, and 260 ml/min, respectively. Little difference
    was seen between the groups. The average urinary recovery of unchanged
    drug was 19, 14, 10, and 7% of the administered 100, 200, 400, and 800
    mg doses, respectively. The extent of absorption of sarafloxacin
    decreased from about 27-34% for the 100-mg dose to 11-13% for the
    800-mg dose (Granneman, 1985c).

    2.1.2  Biotransformation

          Mice

         The biotransformation of sarafloxacin after oral and intravenous
    administration to mice in the study described above is shown in Table
    4 (Merits & Bopp, 1985a).


        Table 4. Metabolites of sarafloxacin identified in 24-h excreta of mice

                                                                                                            

    Identity                    Mean percent of 14C dose                           Mean percent of 14C dose 
                                of 10 mg/kg bw                                     of 100 mg/kg bw
                                                                                   (oral)
                                Oral                     Intravenous                                        
                                                                            
                                Urine        Faeces      Urine       Faeces        Urine       Faeces
                                                                                                            

    Unknown                     0.3          0.2         1           ND            0.4         1
    Unknown                     0.1          0.1         0.3         0.2           ND          ND
    Sarafloxacin glucoronide    6            ND          9           ND            5           0.5
    Sarafloxacin-N-acetyl       0.2          0.1         1           < 0.1         0.1         ND
    Sarafloxacin                15           79          32          43            11          71

    Sum                         21.6         79.4        43.3        43.2          16.5        72.5
    Total                             101                       86.5                           89
                                                                                                            

    ND, not detected
    

          Rabbits

         The biotransformation of sarafloxacin after oral and intravenous
    administration to rabbits in the study described above is shown in
    Table 5 (Merits & Bopp, 1985b).


    Table 5. Metabolites of sarafloxacin identified in excreta of rabbits

                                                                       

    Identity                    Mean percent of 14C dose
                                                                       
                                Oral                 Intravenous
                                                                         
                                Urine     Faeces     Urine      Faeces
                                                                       

    Sarafloxacin glucoronide    0.8       ND         3          ND
    Unknown                     0.3       ND         2          ND
    Unknown                     < 0.1     ND         0.2        ND
    Sarafloxacin-3'-oxo         0.2       < 0.1      2          0.2
    Sarafloxacin-N-acetyl       0.3       ND         3          ND
    Sarafloxacin                9         76         61         24
                                                                       

    ND, not detected

          Dogs

         In the summary report cited in section 2.1.1, about 79% of the 10
    mg/kg bw dose of 14C-sarafloxacin base was excreted as unmetabolized
    parent drug in urine and faeces. In bile, the unchanged parent drug
    and its glucuronide were found in about equal proportions (Bopp,
    1985c).

          Humans

         The pharmacokinetics and metabolism of sarafloxacin were studied
    in two groups of six volunteers given a single oral dose of 100 or 200
    mg sarafloxacin and two groups of five volunteers given a single oral
    dose of 400 or 800 mg. Compliance with the principles of GLP was not
    required for this study. The quality and design were consistent with
    current scientific standards.

         The metabolism of sarafloxacin appears to involve mainly
    oxidative degradation of the piperazinyl substituent, first producing
    3'-oxo-sarafloxacin. Subsequent oxidation produces an ethylene
    diamine-substituted quinolone, which in turn is oxidized to an
    aminoquinolone. The plasma concentrations of the ethylene
    diamine-substituted quinolone parallel those of the parent drug, but
    the average AUC for the quinolone was consistently only about 6% that
    of sarafloxacin. The concentration of the aminoquinolone in plasma and

    urine was considerably lower than that of the ethylene
    diamine-substituted quinolone. Owing to its weak fluorescence,
    3'-oxo-sarafloxacin was not detected in plasma. In urine, the major
    drug-related peak was sarafloxacin, accounting for 75-80% of all
    urinary metabolites. After sarafloxacin, the predominant metabolite in
    urine was tentatively identified as 3'-oxo-sarafloxacin, which
    occurred at concentrations that were typically one-third to one-fourth
    those of sarafloxacin. The total urinary recovery of parent drug plus
    metabolites was low and dose-dependent, decreasing from 24 to 10% as
    the dose increased from 100 to 800 mg. The extent of the decrease was
    similar to that in the dose-normalized AUC. Collectively, the
    aminoquinolone, the ethylene diamine-substituted quinolone, and their
    conjugates accounted for < 7% of the urinary excretion (Granneman,
    1985c).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         The results of studies of the acute toxicity of sarafloxacin are
    presented in Table 6. Compliance with the principles of GLP was not
    required for these studies. The quality and design were consistent
    with current scientific standards.


    Table 6.  Acute toxicity of orally administered 
    sarafloxacin in male rodents

                                                                
    Species    Formulation       LD50           Reference
                                 (mg/kg bw)
                                                                

    Mouse      Suspension        18 000         Hahn (1991)
               Suspension        > 8000         Hahn (1991b)
               Capsule           > 8000         Hahn (1991c)
               Suspension        > 8000         Majors (1985)
               Suspension        > 5000         Hahn (1991)
               Suspension        > 5000         Hahn (1991e)

    Rat        Suspension        > 8000         Hahn (1991a)
                                                                

    2.2.2  Short-term toxicity

          Mice

         In a study of dietary palatability, sarafloxacin was administered
    to four groups of five CD-1 mice of each sex, four to five weeks old,
    as a dietary admixture for 15 consecutive days. The diets contained
    about 5000, 10 000, 25 000, or 50 000 mg/kg feed of sarafloxacin,
    providing doses equivalent to 1250, 2500, 6250, or 12 500 mg/kg bw per
    day, respectively, of the base. An untreated control group of five

    males and five females was fed a basal diet. Compliance with the
    principles of GLP was not required for this study. The quality and
    design were consistent with current scientific standards.

         Survival, general condition, clinical signs, body weight, food
    consumption, and body-weight gain were evaluated. No overt signs of
    toxicity or mortality were reported in animals consuming diets
    containing up to 10 000 mg/kg feed. Decreased feed consumption and
    body-weight gain were the only treatment-related effects observed in
    rats consuming the diets containing 25 000 and 50 000 mg/kg feed
    (Weltman, 1989).

          Rats

         In a study of dietary palatability, sarafloxacin was administered
    to four groups of five CD rats of each sex, four to five weeks old, as
    a dietary admixture for two weeks. The diets contained about 1000,
    5000, 10 000, and 50 000 mg sarafloxacin base per kg feed, providing
    doses equal to 15, 480, 850, or 3000 mg/kg bw per day, respectively.
    An untreated control group of five males and five females was fed a
    basal diet. Compliance with the principles of GLP was not required for
    this study. The quality and design were consistent with current
    scientific standards.

         Clinical signs, body weight, food consumption, and body-weight
    gain were evaluated. No overt signs of toxicity or mortality were
    reported in animals consuming diets containing up to 10 000 mg/kg
    feed. Alopecia, emaciation, dehydration, decreased feed consumption,
    and body-weight gain were treatment-related effects observed in rats
    consuming 50 000 mg/kg feed (Weltman, 1988).

         In a range-finding study, sarafloxacin was administered to six
    groups of four CD rats of each sex, six weeks old, by gavage at doses
    of 20, 50, 125, 275, 650, or 1500 mg/kg bw per day for 14 or 15 days.
    The untreated controls received 10-ml doses of the vehicle, 0.2%
    hydroxypropylmethylcellulose. Compliance with the principles of GLP
    was not required for this study. The quality and design were
    consistent with current scientific standards. Clinical signs,
    ophthalmological parameters, body weight, food consumption, and
    clinical and anatomical (gross and microscopic) pathological
    appearance were evaluated. No overt signs of toxicity or deaths were
    observed in rats treated at up to 650 mg/kg bw. At the highest dose,
    the only abnormality noted was light-coloured faeces (Fort & Buratto,
    1984).

         A 90-day study with a one-month interim kill was conducted in
    four groups of 25 Sprague-Dawley rats of each sex, which were given
    sarafloxacin at doses of 20, 75, 280, or 1000 mg/kg bw per day by
    gavage. Animals in an untreated control group received 10-ml doses of
    the vehicle, 0.2% hydroxypropylmethylcellulose. After one month of
    treatment, 10 animals of each sex per group were randomly selected for
    the interim kill. The remaining animals continued receiving the drug
    daily for a total of 90 days, when 10 rats of each sex per group were

    killed and necropsied. The remaining animals were killed and
    necropsied after a 30-day recovery period. This study was conducted in
    accordance with the principles of GLP.

         Clinical observations, body weight, food consumption, and
    ophthal-moscopic changes were evaluated, and urinalysis, haematology,
    clinical chemistry, organ weights, and anatomical (gross and
    microscopic) examinations were carried out. The only treatment-related
    effect observed in animals treated for one month was grossly enlarged
    caeca in those at the intermediate and high doses, but no microscopic
    alterations were detected in these enlarged caeca. The
    treatment-related effects in animals treated for 90 days included
    grossly enlarged caeca in males at 75 mg/kg bw per day and higher and
    in females at 280 mg/kg bw per day and higher. No microscopic
    alterations were detected in these enlarged caeca. Caecal enlargement
    was not present in the rats that were necropsied at the end of the
    one-month recovery period. At gross necropsy, swollen ears were
    reported in rats treated for 90 days, in two rats at the low dose, one
    at 75 mg/kg bw per day, one at 280 mg/kg bw per day, and three at the
    high dose. Auricular chondritis was seen histologically in the three
    females with swollen ears at the high dose. Microscopically, the
    auricular chondritis was characterized by nodular cartilagenous
    proliferation and by a cellular infiltrate of predominantly
    mononuclear cells. Swollen ears were not reported in treated or
    control animals at the one-month interim kill, but they were observed
    during clinical examinations of control male and female animals
    throughout the treatment period. As data were not available on
    individual animals, it was not possible to determine how many animals
    per group had swollen ears on clinical examination. At necropsy, the
    incidences were 0, 2, 1, 1, and 3 animals in the control group and at
    20, 75, 280, and 1000 mg/kg bw per day, respectively. In the absence
    of a clear doseresponse relationship for the incidence at necropsy,
    this finding could not be attributed to treatment. Auricular
    chondritis was observed histologically in three females at the high
    dose. Three males at the high dose died during the study, and one of
    these deaths may have been related to treatment; however, the cause of
    death could not be determined owing to the presence of moderate
    autolysis in several tissues from this animal. The other two deaths
    were attributed to treatment accidents. The NOEL was 280 mg/kg bw per
    day on the basis of auricular chondritis at 1000 mg/kg bw per day
    (Creighton & Pratt, 1985a,b).

          Dogs

         In a range-finding study, six groups of two young adult (age not
    stated) beagle dogs of each sex were given sarafloxacin in gelatin
    capsules at doses of 8, 20, 50, 125, 300, or 800 mg/kg bw per day for
    two weeks. Two positive control groups were included: in one, dogs
    received nalidixic acid in a gelatin capsule at a dose of 50 mg/kg bw
    per day, and in the other dogs received nalidixic acid at a dose of
    125 mg/kg bw per day for two weeks. The negative control group
    received empty gelatin capsules. Compliance with the principles of GLP

    was not required for this study. The quality and design were
    consistent with current scientific standards.

         Clinical signs, body weight, food consumption, clinical
    pathology, and anatomical (gross and microscopic) pathology were
    evaluated. The treatment-related effects in the sarafloxacin-treated
    groups included lachrymation and emesis (at 8-125 mg/kg bw per day);
    emesis, salivation, lachrymation, reduced activity, dehydration,
    increased serum activity of alanine and aspartate aminotransferases
    and alkaline phosphatase, a biliary sediment characteristic of
    glucuronated drug in the gall-bladder, and centrilobular necrosis of
    the liver (at 300 mg/kg bw per day). Evidence of hepatotoxicity was
    also found microscopically in the livers of dogs at 125 mg/kg bw per
    day. One female at the highest dose died, and the liver of this animal
    showed moderate vacuolar degeneration. Flattening of the angle of the
    radial-carpal joint when the limb is in a weight-bearing position was
    observed from day 7-8 until the end of the study in both front legs of
    both dogs at 800 mg/kg bw per day. No microscopic articular lesions
    were observed. Decreased food consumption and body-weight gain,
    increased serum alanine aminotrasferase activity and biliary sediment
    characteristic of glucuronated drug in the gall-bladder were also
    observed in this group.

         The animals treated with 50 mg/kg bw per day nalidixic acid had
    emesis, loose stools, bilirubinuria, increased serum activity of
    alanine and aspartate aminotransferases and alkaline phosphatase,
    lachrymation, dehydration, and centrilobular necrosis of the liver.
    Emesis, salivation, lachrymation, soft stools, dehydration,
    bilirubinuria, increased serum alanine aminotransferase activity,
    weight loss, centrilobular necrosis of the liver, and decreased
    activity were observed in both dogs at 125 mg/kg bw per day nalidixic
    acid. In addition, the male experienced tremors, seizures, dyspnoea,
    and flattening of the angle of the radialcarpal joint in both front
    feet from day 7 until the end of the study. No microscopic articular
    lesions were observed (Kimura & Pratt, 1984).

         In another range-finding study, five groups of one
    three-month-old beagle dog of each sex were given sarafloxacin in
    gelatin capsules at doses of 2, 20, 50, 125, or 300 mg/kg bw per day
    for two weeks. Two positive control groups were included, receiving
    nalidixic acid at a dose of 50 or 125 mg/kg bw per day. The negative
    control group received empty gelatin capsules. Compliance with the
    principles of GLP was not required for this study. The quality and
    design were consistent with current scientific standards.

         Clinical signs, food consumption, body weight, and the results of
    ophthalmological examinations and clinical and anatomical (gross and
    micro-scopic) pathology were evaluated. In the sarafloxacin-treated
    groups, one male at 300 mg/kg bw per day was killed on day 8 in a
    moribund condition, and decreased food consumption and body-weight
    gain were observed in animals at this dose. Emesis and flattening of
    the angle of both radial-carpal joints were observed at 125 and 300
    mg/kg bw per day. Moderate to severe vesicular arthropathic changes of

    the articular cartilage were observed microscopically in animals at
    300 mg/kg bw per day. These lesions were present on the proximal and
    distal femoral extremities and the proximal humeral and tibial
    surfaces. The female at the high dose experienced a convulsion-like
    state on day 8. In the nalidixic acid-treated groups, one male at 125
    mg/kg bw per day was killed on day 12 in a moribund condition. The
    treatment-related effects in both dose groups included hepatotoxicity
    (increased activity of serum liver enzymes and mild-to-severe liver
    necrosis and degeneration), flattening of the radial-carpal joints,
    and vesicular arthropathic changes of the articular cartilage, which
    were of equal severity and in the same locations as in the
    sarafloxacin-treated dogs (Dudley & Buratto, 1984).

         A 90-day study of toxicity with a one-month interim kill was
    conducted in groups of seven 9-14-month-old beagle dogs of each sex
    which received sarafloxacin base in gelatin capsules at doses of 5,
    25, or 125 mg/kg bw per day. A control group received empty gelatin
    capsules. After 30 days of treatment, three dogs of each sex per group
    were killed and necropsied; the remaining animals continued to receive
    treatment for 90 days before they were killed and necropsied. The
    study was conducted in accordance with the principles of GLP. Clinical
    signs, body weight, and food consumption were evaluated, and
    electroretinography, ophthalmoscopy, electrocardiography, clinical
    pathology, organ weighing, and anatomical (gross and microscopic)
    pathology were carried out.

         Males treated for one month had a dose-related decrease in
    body-weight gain. Food consumption was difficult to assess owing to
    numerous instances of spilled food, so it was not known whether the
    decrease in body-weight gain was due to decreased food intake or to
    treatment. The mean serum globulin concentration was decreased from
    control values in all treated males and females, and the differences
    were statistically significant for males at all doses; however, the
    decreases did not show a doseresponse trend in animals of either sex,
    nor were the lower concentrations outside the normal range for this
    species. Decreased globulin concentrations have been reported
    elsewhere in animals treated with antimicrobial drugs and have been
    attributed to reduced stimulation of the immune system after reduction
    of the resident microbial population. The finding is thus considered
    to be a treatment-related effect.

         Numerous instances of food spilling by both treated and control
    animals were reported during treatment for 90 days, and animals at the
    intermediate and high doses were involved in a greater number of
    instances; however, no significant difference in body-weight gain was
    seen between days 0 and 91. The mean serum globulin concentrations
    were comparable to those of controls for males at the low and
    intermediate doses but were slightly decreased for males at the high
    dose. The mean serum globulin concentrations of females at the
    intermediate and high doses were statistically significantly lower
    than the control values, whereas the value for those at the low dose
    was comparable to that of controls. The NOEL was 5 mg/kg bw per day on
    the basis of decreased serum globulin concentrations in females at the

    intermediate and high doses after 90 days of treatment (Kimura &
    Tekeli, 1985a,b).

         A 90-day study was conducted in groups of six four-month-old
    beagle dogs which received gelatin capsules containing sarafloxacin
    base at 0 or 200 mg/kg bw per day. An additional two groups of four
    animals of each sex were given gelatin capsules containing 10 or 50
    mg/kg bw per day. The study was conducted in accordance with the
    principles of GLP. During the first two weeks of the study,
    sarafloxacin, as the hydrochloride salt, was administered as the
    actual weight, without regard to the concentration of free base. This
    resulted in actual doses of the free base that were about 80% of those
    intended, i.e. 8, 40, and 160 mg/kg bw per day. For the remainder of
    the study, the doses were adjusted to the intended original doses of
    10, 50, and 200 mg/kg bw per day (US Food and Drug Administration,
    1995). The Committee considered that the lower values corresponded to
    actual intake during the study. Clinical signs, body weight, and food
    consumption were evaluated, and ophthalmos-copy, electrocardiography,
    electroretinography, urinalysis, haematology, clinical chemistry,
    organ weighing, and anatomical (gross and microscopic) pathology were
    carried out.

         The treatment-related effects included erythema of the ear flaps
    and muzzle in animals at the intermediate and high doses. During weeks
    9-14, 50% of animals at the high dose were affected, and generalized
    erythema was observed in one male at this dose. Swelling around the
    eyes, eyelids, and earflaps was seen in two animals of each sex at the
    high dose during the first three weeks of treatment. The swelling
    occurred 2-6 h after treatment but was not evident the next day
    (before treatment). An increased incidence of ocular discharge was
    observed in females at the high dose. No other treatment-related
    effects were found. A slight decrease in mean serum globulin
    concentrations was observed in males at the high dose; in females, a
    statistically significant decrease in mean serum globulin
    concentration was observed at the intermediate and high doses; the
    mean serum globulin concentration of females at the low dose was
    comparable to that of controls. The investigator proposed that the
    decreases in serum globulin were due an effect on the gastrointestinal
    flora. Thus, a treatment-related reduction in the flora may have
    caused a secondary reduction in immunoglobulin and acute-phase protein
    synthesis in response to diminished antigenic stimulation. The
    Committee considered the effect to be related to treatment. The NOEL
    was 8 mg/kg bw per day of sarafloxacin base on the basis of the facial
    swelling and erythema observed in males and females at the
    intermediate and high doses and the decreased serum globulin
    concentrations in females at these doses (Kiorpes, 1991).

    2.2.3  Long-term toxicity and carcinogenicity

          Mice

         Sarafloxacin was administered to groups of 60 CD-1 mice of each
    sex as a dietary admixture at 1000, 5000, or 20 000 mg/kg feed
    (equivalent to 150, 750, and 3000 mg/kg bw per day). An additional 10
    animals of each sex were included in each group for haematological
    evaluations and sacrifice at 52 weeks. The carcinogenicity phase was
    terminated at 78 weeks because of high mortality. The study was
    conducted in accordance with the principles of GLP. Mortality,
    clinical signs, body weights, food consumption, haematology and
    anatomical (gross and microscopic) pathology were evaluated.

         Mortality was increased in mice of each sex at the intermediate
    and high doses, the survival at 78 weeks being 23% for males at the
    high dose, 28% for females at the high dose, 45% for males at the
    intermediate dose, and 40% for females at the intermediate dose; the
    survival of mice at the low dose was comparable to that of controls.
    Abdominal distension and increased faecal volume and moisture were
    noted consistently in animals that died prematurely. Nephrotoxic
    effects (epithelial vacuolation with tubular dilatation and chronic
    interstitial nephritis) were observed in females at the intermediate
    and high doses. Gall-bladder calculi and urolithiasis were found in
    males at the high dose. Caecal dilatation was observed in males and
    females at all doses, and caecal torsion was also observed in males
    and females at the intermediate and high doses. The effect was
    attributed to a local effect of large doses of the compound on caecal
    microflora. No treatment-related pathological effects were observed in
    animals at the low dose. There was no evidence of carcinogenicity
    (Procter et al., 1991).

          Rats

         Sarafloxacin was incorporated into the feed of Sprague-Dawley
    rats at concentrations of 1000, 10 000, or 25 000 mg/kg. Twenty rats
    of each sex were used to test toxicity (52 weeks) and 65 of each sex
    to test carcinogenicity  (104 weeks). Two control groups of the same
    numbers of rats of each sex were included in both phases of the study.
    Five satellite groups consisting of 10 rats of each sex (two control
    groups and three treated groups) were included for analysis of the
    plasma concentrations of the drug after about 52 and 103 weeks and for
    laboratory investigations after about 13 and 38 weeks of treatment.
    The study was conducted in accordance with the principles of GLP.

         Clinical examinations, mortality, food consumption, and
    body-weight gain were evaluated in the toxicity phase, with
    ophthalmoscopic examinations, urinalyses, clinical chemistry,
    haematology, organ weighing, and anatomical (gross and microscopic)
    pathology. Drug intake over 52 weeks was equal to 61, 670, and 1700
    mg/kg bw per day. A treatment-related decrease in mean body-weight
    gain was observed in animals at the high dose, despite the fact that
    overall (weeks 1-52) food intake was greater than in controls by 5% in

    males and 11% in females. Statistically significant increases in blood
    urea nitrogen and creatinine concentrations were observed in females
    at the high dose at week 52 and in males at the high dose at week 51.
    The total protein values were statistically significantly decreased in
    comparison with controls for males at all doses at each sampling
    period (weeks 25/26 and weeks 51/52). The decreased values were
    characterized by a statistically significant decrease in globulin and
    a relatively unchanged albumin concentration. Total protein and
    globulin concentrations were statistically significantly decreased in
    females at the intermediate and high doses at week 51/52 only. The
    absolute and relative kidney weights of females at the high dose were
    statistically significantly increased in comparison with controls, and
    a statistically significant increase in relative pituitary weights was
    observed in these animals, with gross enlargement of the pituitary in
    6/20 rats. A slight increase in relative pituitary weight was observed
    in females at the intermediate dose. Dilatation of the caecum was
    observed in nearly all rats treated with 10 000 or 25 000 ppm
    sarafloxacin. The treatment-related histopathological findings
    included tubular nephropathy in 10/20 males and females at the high
    dose and in 1/20 females at the intermediate dose. Tubular nephropathy
    was characterized by the presence of filamentous crystalline material
    in the collecting ducts with associated basophilia and dilitation of
    both cortical and medullary tubules. The severity varied, the most
    severe cases being associated with interstitial inflammatory
    infiltrates, fibrosis, rare focal tubular necrosis, and occasional
    apoptosis or mitosis in the tubules. In addition, minimal glomerular
    sclerosis and focal granulomatous inflammation were observed. The
    tubular nephropathy was different from that of chronic progressive
    nephropathy, which was characterized by dilated renal tubules with
    proteinaceous casts, mononuclear infiltration, and focal fibrosis with
    atrophic tubules. Other changes seen in chronic progressive
    nephropathy were mineralization of the pelvis, occasional cysts, and
    sclerotic glomeruli. No histopathological changes were observed in
    caecae that were grossly dilated. Therefore, as in the 90-day study,
    this effect was attributed to a local effect of large doses of the
    compound on caecal microflora. The NOEL was 61 mg/kg bw per day on the
    basis of the nephrotoxicity observed in males and females at the
    intermediate and high doses and the decreased body-weight gain of
    males and females at the high dose (Smith, 1990).

         The parameters evaluated in the carcinogenicity phase were
    clinical signs, body-weight gain, food consumption, ophthalmological
    and haematological parameters, organ weights, and anatomical (gross
    and microscopic) pathological changes. The mean intake of sarafloxacin
    in the carcinogenicity phase was 54, 580, and 1500 mg/kg bw per day.
    Reduced body-weight gain and tubular nephropathy were seen in females
    at the high dose, which were considered to be related to treatment.
    Tubular nephropathy similar to that seen in the 52-week study was also
    observed in males at the intermediate and high doses. Increased
    relative and absolute kidney weights were observed in males and
    females at the high dose, and a treatment-related increase in relative
    kidney weight was observed in females at the intermediate dose.
    Treatment-related dilatation of the caecum was observed in males and

    females at all doses. No histopathological changes were seen in caecae
    that were grossly dilated, but the Committee considered this to be a
    treatment-related effect. There was no evidence of carcinogenicity
    (Smith et al., 1991).

    2.2.4  Genotoxicity

         The results of assays for the genotoxicity of sarafloxacin are
    summarized in Table 7. All of the studies were conducted in accordance
    with the principles of GLP.

    2.2.5  Reproductive toxicity

          (i)  Multigeneration reproductive toxicity

          Rats

         A three-generation study of reproductive toxicity was conducted
    in Sprague-Dawley rats, each generation consisting of 30 males and 30
    females per group. The animals were treated orally by gavage with
    sarafloxacin base at 75, 275, or 1000 mg/kg bw per day, beginning a
    minimum of 70 days before breeding. A control group consisting of 30
    males and 30 females received daily 10-ml doses of the vehicle, 0.2%
    hydroxypropylmethylcellulose. Each generation was permitted to produce
    up to two litters. The study was conducted in accordance with the
    principles of GLP. Adult animals were observed for clinical signs,
    mortality, reproductive performance, body weights (weekly during
    gestation and lactation), food consumption (weekly during gestation
    and lactation), and organ weights; anatomical (gross and microscopic)
    examinations were made  post mortem. The litter parameters evaluated
    were live birth and viability indices, sex ratios, general physical
    condition, deaths, live litter size, and body weights; weaned pups
    that were not selected for breeding in the subsequent generation were
    necropsied.

         Soft stools and/or whitish faeces resulting from faecal excretion
    of sarafloxacin were observed in the parental animals at the
    intermediate and high doses in all three generations. Gross necropsy
    of the F0 animals that died during the study or were killed as
    scheduled revealed red contents in the gastrointestinal tract and/or
    red foci in the stomach; however, most of these observations were made
    in animals that had died prematurely. In animals killed at scheduled
    sacrifice, the occurrence of these lesions did not show a clear
    doseresponse relationship and was sporadic in all groups, including
    the controls (one animal). These lesions were not considered to be
    related to treatment. Microscopic examinations were not performed on
    the grossly affected intestinal portions of the animals that died or
    on the affected stomachs. The parental animals of the second
    generation at the high dose that died also had red contents in the
    intestine, but histopathological examination was not performed. Some
    animals at the low and intermediate doses had gastric and intestinal
    lesions at scheduled sacrifice, but these also were not examined
    microscopically. In female parental animals of the first generation at


        Table 7. Results of genotoxicity testing of sarafloxacin

                                                                                           

    Test system        Test object        Concentration   Results           Reference
                                                                                           

    In vitro
    Forward mutationa  Chinese hamster    100-1000        Positive (+S9)    Young (1985)
                       ovary cells        g/ml           Negative (-S9)
                       (hprt locus)

    Unscheduled DNA    Rat primary        1-500 g/ml     Positive          Cifone (1985)
    synthesis          hepatocytes

    Chromosomal        Chinese hamster    120-2000        Positive (+S9)    Hemalatha
    aberrations        ovary cells        g/ml                             (1988)
                                          50-800 g/ml    Negative (-S9)

    In vivo/in vitro
    Unscheduled DNA    Rat primary        250-2500        Negativec         Cifone (1988)
    synthesis          hepatocytes        mg/kg orallyb

    Micronucleus       Mouse bone         2000, 4000,     Negative          Diehl (1994)
    formationd         marrow             8000 mg/kg
                                          bw orallye
                                                                                           

    S9, 9000  g fraction from rat liver
    a   With and without S9
    b   Three rats per concentration
    c   Justification for use of the solvent/vehicle was not provided, nor were the criteria
        by which the doses were selected (e.g. preliminary range finding study).
    d   Five males and five females at the low and intermediate doses, and bone-marrow cells 
        harvested after 24 h; 15 males and 15 females at the high dose, and cells harvested
        at 24, 48, and 72 h after treatment
    e   The high dose represented the maximum applicable dose.  Neither general nor bone-marrow
        toxicity was observed.
    

    the intermediate and high doses, the absolute and relative liver
    weights were significantly decreased in comparison with controls. The
    relative liver weights were also significantly decreased in male and
    female parental animals of the second generation and in males of the
    third generation at the intermediate and high doses. Parental females
    of the third generation at the high dose had decreased relative liver
    weights. Because this effect was seen in all three generations and was
    dose-related, it is considered to be related to treatment with 275
    mg/kg bw per day and higher. The NOEL for maternal toxicity was 75
    mg/kg bw per day on the basis of decreased liver weights in rats at
    the intermediate and high doses. No treatment-related effects were
    observed on reproductive parameters, litter parameters, or fetal
    morphology at doses up to 1000 mg/kg bw per day (Lehrer, 1991).

          (ii)  Developmental toxicity

          Rats

         The developmental toxicity of sarafloxacin was evaluated by daily
    oral administration of the compound to pregnant rats during days 615
    of gestation. Four groups of 20 pregnant Sprague-Dawley rats were
    treated orally by gavage with 20, 75, 280, or 1000 mg/kg bw per day. A
    control group of 20 pregnant females received a daily 10-ml dose of
    the vehicle, 0.2% hydroxymethyl-cellulose, on the same schedule as the
    treated animals. On day 20 of gestation, the rats were killed and the
    fetuses removed. The study was conducted in accordance with the
    principles of GLP. Dams were evaluated for clinical signs, body weight
    (on gestation days 6, 9, 12, 15, and 20), food consumption (on
    gestation days 6-9, 9-12, and 12-15), numbers of implants, and percent
    of nonviable implants. The parameters evaluated in the litters were
    sex ratio, group mean weights, and external, visceral, and skeletal
    morphology.

         No maternal toxicological effects were seen, and there was no
    evidence of teratogenicity. The investigator noted that the results of
    a study of the bioavailability of sarafloxacin in rats (see above)
    indicated that at 20 mg/kg bw per day about 12% of a single oral dose
    of sarafloxacin was absorbed. Comparable figures for absorption of the
    doses of 75, 275, and 1000 mg/kg bw per day would be about 6.5, 7, and
    4%, respectively. Therefore, the intended 50-fold range of test doses
    in this study more closely approximated 16-fold (2.3-37 mg/kg bw per
    day). Although no maternal toxic effects were seen, the highest dose
    administered was considered to be sufficiently high to conclude that
    sarafloxacin is not teratogenic in rats. The NOEL for maternal
    toxicity and teratogenicity was 1000 mg/kg bw per day (Lehrer, 1985;
    Patterson, 1985).

          Rabbits

         A study of developmental toxicity was conducted in three groups
    of 18 artifically inseminated New Zealand white rabbits given
    sarafloxacin by gavage once daily on gestation days 6-18 at doses of
    15, 35, or 75 mg/kg bw per day. A concurrent control group of 18

    females received a daily 2-ml dose of 0.5% aqueous methylcellulose
    vehicle. The dams were observed for clinical signs and survival. The
    ovaries were examined for the number of corpora lutea, and the uteri
    were examined for the location of viable and nonviable fetuses, early
    and late resorptions, and the total number and distribution of
    implantation sites. The litter parameters evaluated were group mean
    weight, sex ratio, and external, visceral, and skeletal morphology.

         Fourteen females aborted between gestation days 21 and 29; three
    of the females were receiving the low dose, four the intermediate
    dose, and seven the high dose. These abortions were considered to be
    related to treatment as a secondary effect of the maternal toxicity.
    On gestation day 6, all females at the high dose and most of those at
    the low and intermediate doses showed treatment-related decreases in
    defaecation and urination. Soft stools were observed in two animals at
    the low dose, four at the intermediate dose, and 10 at the high dose.
    Diarrhoea was observed in single animals at the low and intermediate
    doses and in four animals at the high dose. Body-weight gain was
    slightly decreased in animals at the low dose in comparison with
    control values during the last six days of treatment and during the
    first six days after cessation of treatment. Animals at the
    intermediate dose lost weight throughout the treatment period and
    during the initial six days after cessation of treatment. Loss of body
    weight throughout the gestation period was severe in animals at the
    high dose.

         External examination showed that six fetuses from one litter at
    the high dose had malformations, reported as carpal and/or tarsal
    flexure. Visceral examination revealed that five fetuses from one
    litter at the high dose had malformations, reported as hydrocephaly.
    Six fetuses from one litter at the high dose had skeletal
    malformations, reported as cartilagenous skeletal anomalies. Three
    malformations (two external and one skeletal) were observed in two
    litters at the intermediate dose. The numbers of malformations and the
    numbers of litters affected at the low dose were comparable to those
    in controls. The only parameters not affected by treatment were the
    mean numbers of corpora lutea, implantation sites, viable fetuses per
    litter, and mean postimplantation loss at scheduled removal of
    fetuses. A dose-related decrease in mean fetal weight occurred at
    doses of 35 and 75 mg/kg bw per day. The NOEL for teratogenicity and
    fetotoxicity was 15 mg/kg bw per day. The teratogenic effects were
    considered to be secondary to maternal toxicity and not directly
    attributable to treatment. No NOEL for maternal toxicity was
    identified (Lehrer & Tekeli, 1986).

    2.2.6  Special studies on microbiological effects

          In vitro

         In a study of several microbiological end-points, the minimum
    inhibitory concentrations (MICs) of sarafloxacin were determined
    against human clinical bacterial isolates (Table 8); the effect of
    inoculum size on the potency of sarafloxacin against human clinical

    isolates was investigated  in vitro (Table 9); and the effect of pH
    on the potency of sarafloxacin against human clinical isolates of
    aerobic (Table 10) and anaerobic bacteria (Table 11) was investigated
     in vitro. Compliance with the principles of GLP was not required for
    this study. The quality and design were consistent with current
    scientific standards (Prabhavathi, 1984).

         The microbiological activity of four metabolites of sarafloxacin,
     N-acetyl sarafloxacin,  N-formylsarafloxacin, 3'-oxosarafloxacin,
    and a sulfamic acid conjugate of sarafloxacin, was determined in MIC
    assays with  Staphylococcus spp.,  Streptococcus spp.,
     Micrococcus spp.,  E. coli,  Klebsiella pneumoniae, Providencia 
     stuartii, Pseudomonas spp.,  Acinetobacter calcoaceticus, and
     Lactobacillus cosei. The MIC50 values for the metabolites varied
    with the organism tested but were in general significantly higher than
    those seen with sarafloxacin against  E. coli. Therefore, the
    metabolites were less microbiologically active than the parent
    compound (Dr Stephan Schutte, Global Project and Registration Manager,
    Fort Dodge Animal Health Holland, personal communication, 1998).

         The frequency of the development of resistance to sarafloxacin by
    human clinical isolates of  E. coli Juhl,  S. aureus 730a, and
     P. aeruginosa 5007 was studied in two ways. In one method, overnight
    broth cultures were grown from single colonies. A 0.1-ml sample of the
    undiluted culture and 0.1 ml of 10-fold dilutions of the cultures were
    plated on agar plates containing four or eight times the MIC of
    sarafloxacin. Viable cells were counted on drug-free plates. The
    plates were incubated for 72 h at 35C, and the colonies on plates
    containing the drug were counted. Resistant colonies were picked and
    streaked on medium containing four or eight times the MIC of
    sarafloxacin in order to confirm resistance, and the MIC of
    sarafloxacin against resistant mutants was then determined. Finally,
    the resistant mutants were subcultured for 10 consecutive transfers on
    drug-free medium to determine the stability of the resistance.

         The second method involved transfer of the test organisms to
    broth containing increasing concentrations of sarafloxacin. The
    inoculum used for each step of the serial transfer was a 10-3
    dilution of the broth from the well containing the highest
    concentration of antibiotic that allowed growth about equal to that in
    the control well, containing no antibiotic. This procedure was
    repeated for 10 transfers, and each organism was also streaked for
    isolation on agar plates to verify its purity and presumptive
    identity. After the final transfer, the susceptibility of the organism
    to sarafloxacin was determined by the agar dilution method. Resistant
    mutants obtained by this procedure were subcultured to drug-free broth
    medium for 10 consecutive transfers in order to determine the
    stability of the resistance, and the MICs were determined by the agar
    dilution method. The results of these studies are shown in Tables 12
    and 13. Compliance with the principles of GLP was not required for
    this study. The quality and design were consistent with current
    scientific standards (Prabhavathi, 1984).

    Table 8. Minimum inhibitory concentrations (MICs) for sarafloxacin
    against human clinical isolates (104 bacteria/inoculation)

                                                                    

    Organism                                No.     MIC50    MIC90
                                                    (g/ml)  (g/ml)
                                                                    

    Staphylococcus aureus                   70      0.25     0.25
    Staphylococcus epidermidis              43      0.25     0.5
    Staphylococcus spp.                     12      0.25     0.5
    Streptococcus pyogenes (group A)        13      0.5      2
    Streptococcus agalactiae (group B)      13      1        2
    Streptococcus (group C)                 3       0.5      1
    Streptococcus (group D; enterococcus)   58      2        4
    Streptococcus (group G)                 5       0.5      1
    Streptococcus pneumoniae                5       1        2
    Corynebacterium spp.                    10      2        16
    Pseudomonas aeruginosa                  53      0.25     1
    Pseudomonas spp.                        3       0.125    0.5
    Pseudomonas cepacia                     1       2        2
    Pseudomonas maltophilia                 7       1        4
    Acinetobacter anitratus                 8       0.5      0.5
    Achromobacter xylosoxidans              1       16       16
    Acinetobacter spp.                      5       0.125    0.125
    Aeromonas hydrophila                    2       <0.031   <0.031
    Cedecea davisae                         1       0.062    0.062
    Chromobacterium spp.                    1       0.25     0.25
    Miscellaneous gram-negative bacteria    1       0.062    0.062
    Escherichia colia                       140     <0.031   0.062
    Enterobacter aerogenesa                 10      0.062    0.125
    Enterobacter cloacaea                   18      <0.031   0.5
    Enterobacter agglomerans                1       0.062    0.062
    Klebsiella penumoniae                   34      0.062    0.125
    Klebsiella oxytoca                      4       <0.031   4
    Klebsiella rhinoscleromatis             2       <0.031   <0.031
    Citrobacter freundii                    6       <0.031   0.5
    Citrobacter diversus                    2       <0.031   <0.031
    Citrobacter spp.                        7       0.062    4
    Proteus mirabilis                       43      0.25     0.5
    Morganella morganii                     12      0.062    0.25
    Providencia rettgeri                    7       0.125    0.25
    Providencia stuartii                    13      0.062    0.25
    Provindencia spp.                       5       0.25     0.5
    Serratia marcescens                     1       0.25     0.5
    Serratia liquefaciens                   3       0.25     0.25
    Shigella flexneri                       4       <0.031   0.062
    Shigella dysenteriae                    1       <0.031   <0.031
    Shigella boydii                         1       0.5      0.5
    Shigella sonnei                         3       <0.031   <0.031
                                                                    

    Table 8 (continued)

                                                                    

    Organism                                No.     MIC50    MIC90
                                                    (g/ml)  (g/ml)
                                                                    

    Salmonella typhimurium                  5       <0.031   <0.031
    Salmonella choleraesuis                 1       0.62     0.062
    Salmonella arizonae                     2       <0.031   <0.031
    Salmonella spp.                         10      <0.031   0.062
    Yersinia enterocolitica                 2       <0.031   <0.031
    Hafnia alvei                            4       <0.031   0.125
    Haemophilus influenzae                  21      0.125    0.125
    Neisseria gonorrhoeae                   8       <0.015   0.5
    Campylobacter fetus                     4       2        4
    Legionella spp.                         3       0.25     0.25
    Bacteroides fragilisa                   17      2        8
    Bacteroides disiensa                    1       4        4
    Bacteroides melaninogenicusa            1       2        2
    Bacteroides thetaiotaomicrona           4       4        8
    Bacteroides vulgatusa                   1       8        8
    Bacteroides spp.a                       2       4        16
    Clostridium difficile                   1       8        8
    Clostridium perfringensa                8       0.5      1
    Fusobacterium spp.a                     1       2        2
    Peptostreptococcus spp.a                3       0.125    1
    Peptococcus spp.a                       2       0.5      1
    Propionibacterium spp.a                 1       2        2
    Veillonella spp.                        1       4        4

                                                                    

    a Possible constituent of human intestinal microflora

    Table 9. Effect of inoculum size on potency of sarafloxacin in vitro

                                                                  
    Organism             No. of      Geometric mean MIC (g/ml)
                         strains                                  

                                     105 CFUs/ml   107 CFUs/ml
                                                                  

    E. colia             7           0.018         0.05
    K. pneumoniae        3           0.04          0.19
    S. marcescens        3           0.12          0.25
    C. freundii          2           0.015         0.02
    E. cloacae           3           0.03          0.19
    P. mirabilis         2           0.25          0.25
    P. vulgaris          1           0.06          0.12
    M. morganii          2           0.08          0.25
    P. stuartii          2           0.17          0.17
    P. rettegeri         1           0.12          0.25
    Acinetobacter spp.   3           0.04          0.05
    P. aeruginosa        8           0.16          0.35
    S. aureus            5           0.12          0.21
    S. epidermidis       3           0.12          0.25
    S. faecalis          5           1.3           7
                                                                  

    CFU, colony-forming units
    a Possible constituent of human intestinal microflora

    Table 10. Effect of pH on the potency of sarafloxacin against 
    aerobic bacteria in vitro

                                                                  

    Organism             No. of      Geometric mean MIC (g/ml)
                         strains                                  
                                     pH 6.5    pH 7.2     pH 8.0
                                                                  

    E. colia             7           0.07      0.03       0.03
    K. pneumoniae        3           0.06      <0.03      <0.03
    S. marcescens        3           0.63      0.32       0.15
    C. freundii          2           0.09      0.04       0.03
    E. cloacae           3           0.08      0.04       0.04
    P. mirabilis         2           0.5       0.25       0.25
    P. vulgaris          1           0.5       0.12       0.12
    M. morganii          2           0.25      0.09       0.12
    P. stuartii          2           0.98      0.35       0.17
    P. rettegeri         1           0.5       0.12       0.06
    Acinetobacter spp.   3           0.25      0.12       0.20
    P. aeruginosa        8           1         0.55       0.65
    S. aureus            5           0.16      0.16       0.16
    S. epidermidis       3           0.12      0.12       0.15
    S. faecalis          5           1.51      1.15       1.15
                                                                  

    a Possible constituent of human intestinal microflora


    Table 11. Effect of pH on the potency of sarafloxacin against 
    anaerobic bacteria in vitro

                                                                  
    Organism               No. of     Geometric mean MIC (g/ml)
                           strains                                 
                                      pH 6.6    pH 7.3    pH 8.1
                                                                  

    B. fragilisa           6          5.6       1.6       1.4
    Bacteroides spp.a      7          7.1       3.8       3.9
    Fusobacterium spp.a    2          2.2       3.1       1.6
    Clostridiuma           3          0.4       0.4       0.4
    C. Difficilea          1          6.2       6.2       6.2
    Peptococcus and        5          0.4       0.6       0.7
    Peptostreptococcus
    spp.a
                                                                  

    a Possible constituents of human intestinal microflora

    Table 12. Frequency of spontaneous resistance of human clinical
    isolates to sarafloxacin

                                                                
    Organism (strain)           Selected at       Selected at
                                4  MIC           8  MIC
                                                                

    E. coli Juhl                1.0  10-8        <2.0  10-9
    S. aureus CMX730a           1.2  10-7        1.3  10-8
    P. aeruginosa 5007          3.2  10-6        3.9  10-7
                                                                

         The effects of sarafloxacin on five  E. coli strains of human
    origin were assessed in a gastrointestinal model system  in vitro. 
    The study was conducted in accordance with the principles of GLP. The
    strains of  E. coli were obtained from the National Collection of
    Type Cultures, London, United Kingdom, and Abbott Laboratories, North
    Chicago, USA. The strains were  E. coli NCTC 8603, 8761, 8783, 9437,
    and ATCC 25922. The model was designed to simulate possible
    inactivation of sarafloxacin by degradation and protein binding after
    residues of food are ingested. Thus, 1 ml of an aqueous solution of
    sarafloxacin was added to 9 ml of cooked meat medium, and the mixture
    was incubated for 0.5 h at 37C to simulate binding of residues to
    tissues. Gastric fluid (sodium chloride, pepsin, and hydrochloric
    acid) was then added in order to decrease the pH of the mixture to
    about 3, and the mixture was incubated for 1 h at 37C to simulate
    conditions in the stomach. Intestinal fluid (monobasic potassium
    phosphate, sodium hydroxide, pancreatin, and sodium hydroxide) and
    bile salts (sodium cholate and sodium deoxycholate) were then added to
    the mixture, which raised the pH to about 7. This mixture was allowed
    to equilibrate under anaerobic conditions for 4-6 h at 37C before
    being inoculated with about 106 bacteria from a fresh overnight
    culture of  E. coli. This mixture was incubated at 37C for 18 h
    under strictly anaerobic conditions.

         Plate counts were performed on aliquots of the organism
    suspension that was used as inoculum, the model contents immediately
    after inoculation, and the model contents 18 h after inoculation. For
    each strain, 10 individual colonies were picked off and subcultured.
    The MIC of sarafloxacin was determined for each isolate by inoculation
    of about 105 bacteria into 1 ml of broth. The medium contained
    sarafloxacin at 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.625, 0.0313, 0.0156,
    or 0.0078 g/ml. The inoculated tubes were incubated aerobically
    (unless otherwise stated) at 37C overnight and assessed visually for
    growth. The MIC was defined as the lowest concentration of
    antimicrobial agent that resulted in complete inhibition of visible
    growth. The MIC50 was defined as the concentration of the
    antimicrobial agent at which at least 50% of the tested strains were
    inhibited. On the basis of the results of a range-finding study, the
    doses selected for the definitive study were 0, 0.025, 0.07, 0.1, and
    0.4 g/ml.

         The model gastrointestinal tracts were supplemented with
    sarafloxacin over the range of 0.025-0.4 g/ml. A later
    high-performance liquid chromatography analysis of the model contents
    revealed that the actual sarafloxacin concentrations were 10-fold
    higher than the target doses (i.e. 0.25, 0.7, 1, and 4 g/ml). No
    strains survived at the highest concentration after 18 h of
    incubation. Strains NCTC 8761, 8783, and 9434 survived at 1 g/ml,
    strain NCTC 8603 survived at 0.7 g/ml, and strain ATCC 25922 survived
    at 0.25 g/ml. The MICs of test isolates from each strain selected
    from models containing the highest concentration of sarafloxacin that
    permitted survival were determined. The survival of bacteria in the
    model system and changes in the sensitivity of the isolates to
    sarafloxacin are shown in Table 14 (McConville, 1992a,b).

         The effects of sarafloxacin on five strains of  Bacteroides 
     fragilis and five strains of  Bifidobacterium spp. of human origin
    were assessed in the same gastrointestinal tract model system
    described above. The study was conducted in accordance with the
    principles of GLP. Table 15 shows the survival of  B. fragilis 
    strains in model gut supplemented with sarafloxacin over the range of
    2-16 g/ml. Strains NCTC 9343, NCTC 9344, and NCTC 11625 survived at
    concentrations < 16 g/ml. Strains NCTC 8560 and NCTC 10581
    survived at 8 g/ml. All of the strains grew well in the absence of
    sarafloxacin. The MICs of isolates of each strain obtained from the
    model gastrointestinal tracts containing the highest concentration of
    sarafloxacin that permitted survival were then determined.

         For  Bifidobacterium spp., the doses were chosen such that a
    toxic effect of sarafloxacin could be observed and selective pressure
    applied to surviving bacteria.  B. adolenscentis,  B. infantis, 
     B. breve, and  B. longum all survived at concentrations < 16
    g/ml;  B. angulatum survived at concentrations < 8 g/ml,
    although survival was about 0.01% of the inoculum. The MICs of these
    isolates and the MIC50 values are shown in Table 16. The
    investigators proposed that the similar sensitivity of the isolates is
    due to saturation of the model at concencentrations of sarafloxacin
    > 8 g/ml.

         The results show that sarafloxacin is more active against
    facultative anaerobe strains of  E. coli than against the obligate
    anaerobes  B. fragilis and  Bifidobacterium spp. The finding that
    strains grew in the presence of higher concentrations of sarafloxacin
    in the model than in broth culture suggests that sarafloxacin was
    partially unavailable in the model. The 'unavailability' factor ranged
    from 3 to 12 for  E. coli and from 2 to 4 for  B. fragilis and was
    essentially 1 for  Bifidobacterium spp. (McConville, 1992b).


        Table 13. Susceptibility of resistant mutants obtained from experiments designed to determine the frequency of spontaneous resistance to
    sarafloxacin

                                                                                                                                              

    Organism             Susceptibility of resistant  Susceptibility of resistant   Susceptibility of resistant   Susceptibility of resistant
                         mutants from studies of      mutants after 10 subcultures  mutants obtained by           mutants from serial
                         frequency of spontaneous     in quinolone-free medium      culturing in increasing       subculture in quinolone-
                         resistance                                                 concentrations of             containing medium after
                                                                                    quinolones                    10 subcultures in quinolone-
                                                                                                                  free medium
                                                                                                                                               

                         No. of        MIC            No. of         MIC            No. of         MIC            No. of         MIC
                         mutants       (g/ml)        mutants        (g/ml)        mutants        (g/ml)        mutants        (g/ml)
                                                                                                                                              

    E. coli Juhl         1             0.5a           1              0.5            NR             0.25           NR             0.12
    S. aureus CMX 730a   5             2b             4              2-4            NR             2              NR             2
    P. aeruginosa 5007   6             8c             5              8              NR             4              NR             2
                                                                                                                                              

    NR, not reported
    a MIC before induction of resistance was < 0.03 g/ml
    b MIC before induction of resistance was 0.25 g/ml
    c MIC before induction of resistance was 0.5 g/ml

    Table 14. MIC (in g/ml) determination on E. coli isolates from model gastrointestinal tracts 
    containing sarafloxacin

                                                                                                        
    E. coli strain    MIC of parent strain        MIC50 (g/ml) of 10 isolates     Concentration of
                      (g/ml)                     after incubation in model        sarafloxacin (g/ml)
                                                                                   in model from which
                      Aerobic     Anaerobic       Without         With             isolates were
                                                  sarafloxacina   sarafloxacin     obtained
                                                                                                        

    NCTC 8603         0.0625      0.0625          0.0625          0.0313           0.7
    NCTC 8761         0.5         0.25            1               0.5              1
    NCTC 8783         0.125       0.125           0.0625          0.125            1
    NCTC 9434         0.125       0.125           0.0625          0.0625           1
    ATCC 25922        0.0625      0.0625          0.0625          0.125            0.25
                                                                                                        

    a Only one isolate tested


    Table 15. MIC (in g/ml) determination on B. fragilis isolates from model gastrointestinal tracts 
    containing sarafloxacin

                                                                                                        

    B. fragilis       MIC of parent strain        MIC50 (g/ml) of 10 isolates     Concentration of
    strain            (g/ml)                     after incubation in model        sarafloxacin (g/ml)
                                                                                   in model from which
                                                  Without         With             isolates were
                                                  sarafloxacina   sarafloxacin     obtained
                                                                                                        
    NCTC 8560         4                           4               4                8
    NCTC 9343         8                           2               4                16
    NCTC 9344         4                           4               4                16
    NCTC 10581        4                           2               4                8
    ATCC 11625        4                           4               2                16

                                                                                                        

    a Only one isolate tested

    Table 16. MIC (in g/ml) determination on Bifidobacteria isolates from model gastrointestinal tracts 
    containing sarafloxacin

                                                                                                        

    Bifidobacteria       MIC of parent strain     MIC50 (g/ml) of 10 isolates     Concentration of
    strain               (g/ml)                  after incubation in model        sarafloxacin (g/ml)
                                                                                   in model from which
                                                  Without         With             isolates were
                                                  sarafloxacina   sarafloxacin     obtained
                                                                                                        
    B. adolescentis      8                        8               8                16
    B. infantis          8                        8               8                16
    B. angulatum         8                        8               8                8 and 4b
    B. breve             16                       16              16               16
    B. longum            > 16                     > 16            > 16             16
                                                                                                        

    a Only one isolate tested
    b Five isolates each were obtained from model gastrointestinal tracts containing 4 and 8 g/ml 
      sarafloxacin.
    

         The activities of five quinolones - ciprofloxacin, lomefloxacin,
    oxofloxacin, sparfloxacin, and DU-6859 - against 320 anerobic
    bacterial strains isolated from human patients with infections were
    determined by an agar dilution method  in vitro. The tested organisms
    were 50 strains of  Peptostrepto-coccus, 30 strains of  Clostridium 
     perfringens, 50 strains of  Clostridium difficile, 100 strains of
     Bacteroides fragilis, 50 strains of  Prevotella and
     Porphyromonas, and 40 strains of  Fusobacterium. The most sensitive
    strains to all of the quinolones tested were  Clostridium 
     perfringens (MIC50, 0.008-1 g/ml) and  Peptostreptococcus 
    (MIC50, 0.008-4 g/ml) (Nord, 1996).

    2.2.7  Special studies on ecotoxicity

         Because use of sarafloxacin as a therapeutic agent in fish
    farming has been considered, various aspects of its ecotoxicity have
    been evaluated. Adsorption and desorption and the effect on bacteria
    present in sediments are components of such an assessment that may be
    relevant to the microbiological assessment of foodborne residues. The
    results of those studies are summarized in an environmental report
    submitted to the Committee as part of the dossier. The study of
    sorption and desorption was conducted in accordance with the
    principles of GLP, but the study on the effect of sarafloxacin on
    bacteria in sediment was not.

         A study of adsorption and desorption was conducted in silty clay
    loam (pH 5.4), sandy clay loam (pH 6.0), and loam (pH 8.3). The
    results indicate that sarafloxacin hydrochloride is readily sorbed and
    would be considered immobile on the three soil types tested. The
    adsorption coefficients  (Kd) for sarafloxacin were 8400 in silty
    clay loam, 7400 in sandy clay loam, and 143 000 in loam. The
    desorption capacity of the compound in all three soils was negligible,
    with average values of 0.03, 0.02, and 0.63%, respectively.

         The effect of sarafloxacin on bacteria present in sediment was
    tested in ocean sediment from a source where there had been no
    previous sarafloxacin use. The numbers of bacteria from samples
    containing 0, 3, 30, or 300 g/ml of sarafloxacin hydrochloride
    equalled 106 to 107 colony forming units, indicating no inhibitory
    effect. The observed lack of inhibition could be due to the strong
    sorption of sarafloxacin hydrochloride and its consequent
    unavailability to bacteria present in the sediment. The unavailability
    of sarafloxacin hydrochloride in sediment to bacteria could also help
    explain the apparent resistance of sarafloxacin to biodegradation
    (Duke, 1990).

    2.3  Observations in humans

         The safety of single oral doses of sarafloxacin was studied in
    groups of healthy male volunteers. Six subjects received 100 mg
    sarafloxacin, six received 200 mg, five received 400 mg, and five
    received 800 mg. The adverse events reported most frequently were
    dizziness and asthenia, although the increase in incidence was not

    dose-related. Emotional lability, somnolence, and hiccoughs were the
    only adverse events reported by those receiving the lowest dose.
    Compliance with the principles of GLP was not required for this study.
    The quality and design were consistent with current scientific
    standards (Tolman, 1986).

         The safety of oral administration of sarafloxacin for seven
    consecutive days was studied in groups of six healthy male volunteers
    who received doses of 100 or 200 mg twice daily, as a slurry to
    maximize exposure of the surface of the stomach. Compliance with the
    principles of GLP was not required for this study. The quality and
    design were consistent with current scientific standards. Asthenia,
    vasodilatation, anxiety, dizziness, and nervousness were reported by
    the treated subjects but not those receiving a placebo. Somnolence was
    the most frequently reported adverse event in both the treated and
    placebo groups. There were no clinically significant changes in
    haematological, clinical chemical, coagulation, or urinary parameters,
    nor were clinically significant changes seen in physical,
    ophthalmological, or neurological examinations or on
    electrocardiograms or electroencephalograms (Tolman, 1986).

         The safety of oral administration of sarafloxacin for seven
    consecutive days was also studied in groups of six healthy male
    volunteers who received 100 mg every 12 h, 200 mg every 12 h, or
    100 mg every 6 h. The most frequently reported adverse events were
    asthenia (eight reports, 20%) and dizziness (six reports, 15%). The
    most frequently reported adverse events in the group receiving placebo
    were asthenia (six reports, 17%) and somnolence (four reports, 11%).
    Compliance with the principles of GLP was not required for this study.
    The quality and design were consistent with current scientific
    standards (Tolman, 1988).

    3.  COMMENTS

         The Committee considered data from studies of pharmacodynamics,
    pharmacokinetics, metabolism, acute and short-term toxicity,
    carcinogenicity, genotoxicity, reproductive toxicity, and
    developmental toxicity, special studies on microbiological effects and
    ecotoxicity, and observations in humans. All of the studies were
    conducted according to appropriate standards for study protocol and
    conduct.

         The absorption, metabolism, and excretion of sarafloxacin were
    studied in mice, rats, rabbits, dogs, and humans. After oral
    administration, the percent absorption ranged from a low of 10% in
    humans given a single 800-mg dose to a high of 70% in dogs given a
    single 10-mg/kg bw dose. In mice, rats, and rabbits, the primary route
    of excretion was the faeces. The parent drug accounted for 80-90% of a
    radiolabelled dose in urine and faeces of mice, rats, rabbits, and
    dogs given an oral dose of 10 mg/kg bw, indicating that sarafloxacin
    undergoes little metabolism in these species. In humans given single
    oral doses of 100-800 mg, sarafloxacin accounted for 75-80% of total
    urinary recovery. A metabolite, 3'-oxo-sarafloxacin, comprised about

    15% of the recovery. In all species studied, a decreased fraction of
    the dose was absorbed at high doses.

         Orally administered sarafloxacin was slightly hazardous in
    studies of acute toxicity in mice and rats, with LD50 values on the
    order of > 5000 to > 8000 mg/kg bw.

         In a 90-day study of toxicity with a one-month interim kill, rats
    were treated with sarafloxacin at 0, 20, 75, 280, or 1000 mg/kg bw per
    day by gavage. The only treatment-related effect observed in animals
    treated for one month was grossly enlarged caeca in animals at the
    intermediate and high doses. In animals treated for 90 days, the
    treatment-related effects included grossly enlarged caeca in males at
    doses > 75 mg/kg bw per day. No microscopic pathological changes
    were detected in these enlarged caeca. At necropsy, swollen ears were
    reported in rats treated for 90 days, with none in controls, two
    animals at the low dose, one each at the two intermediate doses, and
    three animals at the high dose. The Committee concluded that this
    finding was of no toxicological significance. Auricular chondritis was
    observed histologically in three females at the high dose. Three
    deaths occurred in this study, all among rats at the high dose; one
    may have been related to treatment, but the presence of autolysis in
    several tissues from this animal made it impossible to determine the
    cause of death. The NOEL was 20 mg/kg bw per day on the basis of
    grossly enlarged caeca at doses > 75 mg/kg bw per day.

         The potential for sarafloxacin to induce arthropathy in dogs was
    evaluated in two two-week pilot studies. Arthropathy, characterized by
    flattening of the angle of the radial-carpal joint, with no
    microscopic evidence of articular lesions, was observed in young adult
    (age not stated) dogs given sarafloxacin at 800 mg/kg bw per day in
    gelatine capsules. Similar arthropathy was seen in three-month-old
    dogs given 125 or 300 mg/kg bw per day in gelatine capsules for two
    weeks. Moderate to severe vesicular arthropathic changes of the
    articular cartilage were observed microscopically in dogs receiving
    300 mg/kg bw per day. The NOEL was 50 mg/kg bw per day on the basis of
    the arthropathic effect of sarafloxacin in young dogs.

         A 90-day study with a one-month interim kill was conducted in
    914-month-old dogs. Three groups of seven dogs of each sex received
    sarafloxacin at 0, 5, 25, or 125 mg/kg bw per day in gelatine
    capsules. Decreases in mean serum globulin concentration were observed
    in males and females at all doses after 28 days of treatment. After 90
    days of treatment, statistically significant decreases were seen in
    females at the intermediate and high doses and in males at the high
    dose. A dose-related decrease in body-weight gain was observed in
    males. The NOEL was 5 mg/kg bw per day on the basis of decreased mean
    serum globulin concentrations at higher doses.

         A 90-day study was conducted in groups of four four-month-old
    dogs of each sex, which received 10 or 50 mg/kg bw per day in gelatine
    capsules; a third group of six dogs of each sex received 200 mg/kg bw
    per day. During the first two weeks of the study, sarafloxacin (as the

    hydrochloride salt) was administered as the actual weight, without
    regard to the concentration of free base. This resulted in actual
    doses that were about 80% of the intended free base doses (i.e. 8, 40,
    and 160 mg/kg bw per day). For the remainder of the study, the doses
    were adjusted to the target doses. After 90 days of treatment, a
    significant decrease in mean serum globulin was observed in females at
    the intermediate and high doses. The mean serum globulin concentration
    of females at the low dose was comparable to the control value.
    Treatment-related toxicity included erythema of the earflaps and
    muzzle in males and females at the intermediate and high doses.
    Generalized erythema was observed in one male at the high dose.
    Swelling around the eyes, eyelids, and earflaps was also seen in dogs
    at the high dose. The NOEL was 8 mg/kg bw per day on the basis of
    decreased serum globulin, facial swelling, and erythema at higher
    doses.

         A study of carcinogenicity was conducted in groups of 60 mice of
    each sex which received sarafloxacin in the diet at 0, 1000, 5000, or
    20 000 mg/ kg feed. The study was terminated after 78 weeks owing to
    high mortality in the animals at the intermediate and high doses. The
    treatment-related toxicity included nephrotoxicity in females at the
    intermediate and high doses and gall-bladder calculi and urolithiasis
    in males at the high dose. Caecal dilatation was observed in all
    treated males and females, and caecal torsion was observed in males
    and females at the intermediate and high doses. No treatment-related
    toxicity was observed in those given the low dose. There was no
    evidence of carcinogenicity.

         A combined long-term study of toxicity and carcinogenicity was
    conducted in rats. Sarafloxacin was incorporated into the feed at 0,
    1000, 10 000, or 25 000 mg/kg of feed. The toxicity phase (52 weeks)
    included 20 animals of each sex per group, and the carcinogenicity
    phase (104 weeks) included 65 animals of each sex per group. In the
    toxicity phase, the drug intake was equal to 61, 670, or 1700 mg/kg bw
    per day. A treatment-related decrease in mean body-weight gain was
    observed in males and females at the high dose. Increased blood urea
    nitrogen and creatinine concentrations were observed in females and
    males at the high dose at weeks 51 and 52, respectively. Total protein
    values were decreased in comparison with controls for males at all
    doses at each sampling period. These decreased values were
    characterized by a significant decrease in globulin concentration with
    a relatively unchanged albumin concentration. Total protein and
    globulin concentrations were statistically significantly decreased in
    females at the intermediate and high doses at week 51/52 only. The
    absolute and relative kidney weights were significantly increased in
    females at the high dose. Tubular nephropathy was seen in 10/20 males
    and females at the high dose and in 1/20 females at the intermediate
    dose. Dilatation of the caecum was observed in most rats treated with
    sarafloxacin at 10 000 or 25 000 mg/kg of feed. No histopathological
    changes were observed in caeca that were grossly dilated. The NOEL was
    61 mg/kg bw per day. In the carcinogenicity phase, drug intake was
    equal to 54, 580, or 1500 mg/kg bw per day. The signs of toxicity were

    similar to those found in the toxicity phase of the study. There was
    no evidence of a carcinogenic effect.

         Sarafloxacin induced mutation in Chinese hamster ovary cells
     in vitro, unscheduled DNA synthesis in rat primary hepatocytes, and
    chromosomal aberrations in Chinese hamster ovary cells. It did not
    induce unscheduled DNA synthesis in rat primary hepatocytes   in 
     vitro/in vivo or micronuclei in mouse bone marrow  in vivo. The
    Committee concluded that sarafloxacin is genotoxic  in vitro but not
     in vivo.

         The reproductive toxicity of sarafloxacin was studied in a
    three-generation study in rats treated  by gavage with 0, 75, 275, or
    1000 mg/kg bw per day beginning a minimum of 70 days before breeding.
    Each generation was comprised of 30 males and 30 females per group.
    The relative liver weights were significantly decreased in males and
    females of the second parental generation at the intermediate and high
    doses, in males of the second parental generation at the intermediate
    and high doses, and in females of the second generation at the high
    dose. No treatment-related effects were observed on reproductive or
    litter parameters or fetal morphology at doses up to 1000 mg/kg bw per
    day, the highest dose tested. The NOEL for parental toxicity was 75
    mg/kg bw per day on the basis of decreased liver weights in males and
    females at higher doses.

         The developmental toxicity of sarafloxacin was evaluated by daily
    oral administration of the compound to pregnant rats during days 615
    of gestation. Four groups of 20 pregnant rats were treated by gavage
    with sarafloxacin base at doses of 0, 20, 75, 280, or 1000 mg/kg bw
    per day. There was no evidence of maternal toxicity or teratogenicity
    at any dose.

         In a study of developmental toxicity, rabbits were given doses of
    0, 15, 35, or 75 mg/kg bw per day by gavage during days 6-18 of
    gestation, but the highest dose was considered to be inappropriate for
    evaluating teratogenicity owing to excessive maternal toxicity,
    manifested by decreased body weight, abortions, and decreased
    defaecation and urination. Maternal toxicity was also observed in
    animals at the low and intermediate doses. External, visceral, and
    skeletal malformations and fetotoxicity were observed in fetuses at
    the intermediate and high doses. The NOEL for teratogenicity and
    fetotoxicity was 15 mg/kg bw per day. No NOEL for maternal toxicity
    was identified. The teratogenic effects in this study were considered
    to be secondary to maternal toxicity and not directly attributable to
    treatment with sarafloxacin.

         Data on humans reviewed by the Committee consisted of reports of
    side-effects in healthy male volunteers enrolled in clinical trials of
    the safety of oral doses of sarafloxacin in comparison with placebo.
    The doses ranged from 100 to 800 mg/person per day for one to seven
    consecutive days. Effects on gastrointestinal microflora were not
    evaluated in these studies. The reported side-effects included

    asthenia, vasodilation, anxiety, dizziness, and nervousness or
    somnolence, which were observed sporadically at all doses.

         Sarafloxacin belongs to a group of antimicrobial fluoroquinolones
    that are primarily active against aerobic gram-negative bacteria. In
    humans, this characteristic is used therapeutically for selective
    elimination of potential aerobic and facultative anaerobic pathogens
    from the gastrointestinal tract while preserving the predominant
    anaerobic bacterial intestinal flora that protect the gastrointestinal
    tract from invasion or overgrowth by potentially pathogenic bacteria.

         Several studies on the microbiological activity of sarafloxacin
     in vitro were evaluated by the Committee. In one study, MIC50 and
    MIC90 values were determined for 735 human clinical isolates of 65
    genera at an inoculation density of 104 bacteria. The effect of
    sarafloxacin against 210 bacterial strains was assessed, 14 of which
    were identified as possible constituents of the human intestinal
    microflora. The most sensitive were  Escherichia coli, and
     Enterobacter cloacae, each with MIC50 values < 0.031 g/ml. The
    most sensitive relevant organism was  Peptostreptococcus spp., with
    an MIC50 value of 0.125 g/ml. The least sensitive relevant organism
    was  Bacteroides vulgatus, with an MIC50 value of 8 g/ml. In a
    study designed to evaluate the effect of inoculum size on the potency
    of sarafloxacin  in vitro, the geometric mean MIC value was
    calculated for sarafloxacin against 50 clinical isolates of human
    origin. At inoculation densities of 105 and 107 colony-forming
    units per ml, geometric mean MIC values of 0.018 and 0.05 g/ml,
    respectively, were reported for  E. coli, the most sensitive
    organism.

         The microbiological activities of four potential metabolites of
    sarafloxacin were determined. The MIC50 values varied with the
    species tested, but were generally significantly higher than those for
    sarafloxacin against  E. coli. Therefore, the Committee concluded
    that the microbiological activity of the metabolites against relevant
    strains of bacteria found in the human gastrointestinal tract would be
    significantly lower than that of sarafloxacin.

         The frequency of spontaneous resistance of human clinical
    isolates to sarafloxacin was studied in  E. coli, S. aureus, and
     P. aeruginosa cultured on agar plates containing sarafloxacin at
    four and eight times the MIC and by repeatedly transferring organisms
    into broth containing increasing concentrations of sarafloxacin.
    Stable, resistant mutants arose.

         The effects of sarafloxacin against five strains each of
     E. coli, B. fragilis, and  Bifidobacterium spp. of human origin
    were tested in an in-vitro gastrointestinal model system designed to
    simulate possible inactivation of sarafloxacin by degradation and
    binding in food.  E. coli strains grew in the presence of higher
    concentrations of sarafloxacin in the model than in broth culture. The
    'unavailability' factor ranged from 3 to 12, indicating that
    sarafloxacin binds to organic matter in the gastrointestinal model and

    decreases the sensitivity of the  E. coli strains to the compound.
    The factor ranged from 2 to 4 for  B. fragilis and was essentially 1
    for  Bifidobacterium spp. The results of this study suggest that
    sarafloxacin is less available in the gastrointestinal model than in
    broth culture.

         The effect of pH on the potency of sarafloxacin  in vitro was
    studied with aerobic and anaerobic bacterial isolates of human origin.
    In general, the geometric mean MIC value of those organisms considered
    to be potential constituents of the human gastrointestinal tract
    increased as the pH decreased.

         The Committee noted that the organisms most sensitive to the
    antimicrobial effects of sarafloxacin, namely  E. coli and
     Enterobacter cloacae, comprise approximately 1% of the total
    gastrointestinal bacterial population and are considered to make a
    minimal contribution to colonization resistance in the
    gastrointestinal tract. The available microbiological data did not
    permit a full evaluation of the relevant bacteria of the human
    gastrointestinal tract, as very few such strains were tested. On the
    basis of a published report on the activity of related
    fluoroquinolones against relevant human intestinal anerobic bacteria,
    however,  Clostridium perfringens and  Peptostreptococcus spp. were
    shown to be the most sensitive strains tested. In relevant bacteria,
    the lowest MIC50 for sarafloxacin was seen in  Peptostreptococcus 
    spp., with a value of 0.125 g/ml. The Committee considered these data
    to be limited because only three strains rather than the preferred 10
    were tested, but they were sufficient to support an ADI.

    4.  EVALUATION

         The upper limit of the ADI based on the antimicrobial activity of
    sarafloxacin was calculated on from the formula described on p. 28 as
    follows:

                   Upper limit     0.125 g/ga  220 g
                                =  
                      of ADI       0.70b  2c  60 kg

                                =  0.33 g/kg bw

    a  For the purpose of this evaluation, this is the MIC50 for three
         strains of human isolates of  Peptostreptococcus spp., which was
         the most sensitive strain of the relevant bacteria of human
         gastrointestinal microflora tested with sarafloxacin.

    b  The fraction of the dose available to act upon microorganisms in
         the colon was based on studies in humans in which approximately
         70% of a 100-mg oral dose of sarafloxacin was not absorbed.

    c  A safety factor of 2 was used because of the limited MIC data
         available on the sensitive, relevant bacteria of the human
         gastrointestinal tract.

         The Committee established an ADI of 0-0.3 g/kg bw on the basis
    of the microbiological activity of sarafloxacin against the most
    sensitive, relevant constituent of the human gastrointestinal flora
    for which limited adequate microbiological data were available, i.e.
     Peptostreptococcus spp. This ADI provides a margin of safety of
    17 000 when compared with the lowest toxicological NOEL of 5 mg/kg bw
    per day in the 90-day study in dogs.

    5.  ACKNOWLEDGMENTS

         The following individuals at the US Food and Drug Administration
    are acknowledged for their assistance with the preparation of the
    first draft of this monograph: Dr Carl Cerniglia, Microbiologist,
    National Center for Toxicological Research; Dr Robert Condon,
    Biostatistician, Center for Veterinary Medicine; Dr Anna Fernandez,
    Toxicologist, Center for Veterinary Medicine; Dr Louis T. Mulligan,
    Toxicologist, Center for Veterinary Medicine; Dr Terry Peters,
    Pathologist, Center for Drug Evaluation and Research; and Dr Leonard
    Schechtman, Genetic Toxicologist, Center for Veterinary Medicine.

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    See Also:
       Toxicological Abbreviations
       SARAFLOXACIN (JECFA Evaluation)