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    NEOMYCIN

    First draft prepared by
    J.E.M. van Koten-Vermeulen and Dr.F.X.R. van Leeuwen
    Laboratory of Toxicology
    National Institute of Public Health and Environmental Protection
    Bilthoven, Netherlands

    1.  EXPLANATION

         The aminoglycosides were evaluated as a group at the twelfth
    meeting of the Joint FAO/WHO Expert Committee on Food Additives when
    an ADI was not established. Because adequate toxicological and residue
    data were not available it was stated that when aminoglycosides are
    used they should not be allowed to give rise to detectable residue
    levels in human food. (Annex I, reference 17).  Since then, data
    specific for neomycin have become available.  The structure of
    neomycin is shown in Figure 1.

    FIGURE 1

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

    2.1.1.1  Guinea-pigs

         Groups of 4 male guinea-pigs were administered single oral doses
    of 5, 10  or 100 mg/kg bw neomycin B sulfate. Serum was collected 1,
    2, 3, and 4 hours after treatment. Neomycin concentrations of 1.5 and
    0.45 g/ml were measured at 1 and 2 hours after dosing with 100 mg/kg
    bw, respectively.  Serum concentrations of neomycin were below
    detectable levels (< 0.10 g/ml) in the low- and mid-dose groups at
    all time points, and in the high-dose group at 3 and 4 hours post-
    administration (Hall  et al., 1983).

    2.1.1.2  Chickens

         Lohman chickens (14 total, weighing approximately 2 kg each) were
    administered a single oral dose of 20 mg neomycin/kg bw.  Neomycin
    could not be detected in blood samples collected up to 8 hours post-
    administration using a microbiological assay (detection limit not
    specified) (Atef  et al., 1986).

         After a single i.v. administration of 20 mg/kg bw neomycin to
    five Lohman chickens, the blood concentration curve showed a
    biexponential decline with a T of about 6 hours for the elimination
    phase and an AUC of approximately 196 g/ml/h.  After a single i.m.
    administration of 20 mg/kg bw to 14 chickens, the peak blood level
    (Cmax) was approximately 17 g/ml and the mean maximum concentration
    was reached 40 minutes after administration. The AUC was approximately
    132 g/ml/h.  The bio-availability following i.m. administration was
    approximately 70% (Atef  et al., 1986).

         In 22 Lohman chickens administered neomycin i.m. at 20 mg/kg bw
    twice daily for 5 days, plateau levels of neomycin (about 6 g/ml)
    were retained for 1-4 days.  Neomycin concentrations were detected in
    the spleen for 3 days, in liver and lung for 6 days, and in kidney for
    10 days post-injection (Atef  et al., 1986).

    2.1.1.3  Sheep

         Serum and milk concentrations of neomycin were determined in
    lactating Awassi ewes (4/group) after a single i.v. (20 mg/kg bw) or
    i.m. injection (10 mg/kg bw). Neomycin was assayed micro-biologically
    using the cylinder cup method (sensitivity limit 1 g/ml).  After i.v.
    administration the T was approximately 3 hours and distribution
    equilibrium was reached within 20 min.  After i.m. administration peak
    concentrations were reached after 1 hour.  Neomycin was not detectable

    in serum 12 hours after treament.  A very small fraction of the dose
    (0.01%-0.02%) was recovered in milk secreted from normal or irritated
    glands (in which the left-side glands of the udder were infused with
    150 ml of hypertonic NaCl solution 24 hours before treatment) during
    the 12 hours after treatment.  Peak milk concentrations of neomycin
    following i.v. or i.m. treatment were less than 2 g/ml (Ziv & Sulman,
    1974).

    2.1.1.4  Pigs

         Two groups (6 pigs/group) of Yorkshire crossed weanling pigs (10
    weeks old, weighing 10 to 15 kg) were orally administered 22 mg
    neomycin sulfate/kg bw/day for 5 consecutive days. Group 1 consisted
    of 3 males and 3 females, while group 2 had 5 males and 1 female.  In
    addition to the oral neomycin treatment, pigs in the second group
    received diets supplemented with sodium sulfate at a rate of 10 g/kg
    feed. Serum neomycin concentrations were measured up to 96 hours after
    dosing.  The mean peak serum concentration (approximately 0.2 g/ml)
    of neomycin was reached in the fourth hour in pigs fed the normal
    ration.  For pigs receiving the supplemented diets, the mean peak
    serum concentration (approximately 0.1 g/ml) was reached at 8 and 24
    hours after dosing (Heath, 1985).

         In another  study by the same author, a single  i.v. dose of 22
    mg/kg bw neomycin sulfate was administered to 6 (3/sex) 10-month old
    pigs.  A peak mean serum level of approximately 30 g/ml was detected
    one hour after dosing, which decreased to 0.01 g/ml by 72 hours
    (Heath, 1985).

         A single i.m. dose (22 mg/kg bw) of neomycin sulfate was
    administered to 12 female pigs weighing 20 to 50 kg.  A peak mean
    serum level of approximately 40 g/ml which was reached one hour after
    dosing, decreased to 0.02 g/ml after 72 hours (Heath, 1985).

    2.1.1.5  Cattle

         Ten castrated Holstein calves (2 to 6 months of age, weighing 80
    to 180 kg) were orally administered 22 mg neomycin sulfate/kg bw/day
    for 5 days.  Serum concentrations were determined up to 96 hours after
    dosing. Mean serum concentrations ranged from 0-0.06 g/ml during the
    1-96-hour dosing period (Heath, 1985).

         A single i.v. dose (22 mg/kg bw) of neomycin sulfate was
    administered to 10 castrated Holstein calves (2 to 6 months old,
    weighing 70 to 170 kg).  A peak mean serum concentration of
    approximately 19 g/ml which was detected one hour after dosing,
    decreased to 0.03 g/ml by 72 hours (Heath, 1985). 

         Four male Holstein calves (3 to 60 days old) were given a single
    oral dose of 30 mg 14C-neomycin/kg bw and then killed after 96 hours.

    The distribution of 14C in excreta and tissues was determined by mass
    spectrophotometric analysis. Most of the radioactivity was recovered
    in faeces (85-97%).  The radioactivity found in urine decreased with
    increasing age of the animal, from 11 to 0.5% of the administered
    dose. Radioactivity found in the kidneys, liver and muscle of calves
    at 3 days of age represented 55, 1.93 and 0.09 ppm of neomycin
    equivalents, respectively.  At least 90% of the 14C in the kidneys of
    3-day old calves was present as neomycin. Seventy to 88% of the 14C
    present in faeces of all calves was also identified as neomycin
    (Aschbacher & Feil, 1991, 1994).

         Serum and milk concentrations of neomycin were measured in 4
    lactating Israeli-Friesian dairy cows following administration of a
    single i.m. dose (10 mg/kg bw).  Neomycin was assayed micro-
    biologically by the cylinder cup method (sensitivity limit 1 g/ml). 
    Peak serum concentrations were reached after 1 hour.  Neomycin was not
    detectable in serum 12 hours after treament. A very small fraction of
    the dose (0.016%-0.022%) was recovered in milk secreted from normal or
    irritated glands (in which the left-side glands of the udder were
    infused with 150 ml of hypertonic NaCl solution 24 hours before
    treatment) during the 12 hours after treatment.  Peak milk
    concentrations of neomycin were less than 2 g/ml (Ziv & Sulman,
    1974).

    2.1.1.6  Humans

         An oral dose of 1.5 g neomycin/day was administered to a 2- year
    old female tuberculosis patient for 15 consecutive days.  A nine-month
    old male tuberculosis patient received 200 mg neomycin/day orally for
    80 days.  Both patients were diagnosed with miliary tuberculosis and
    tuberculous meningitis. A maximum neomycin serum concentration of
    approximately 5.0 g/ml was achieved in both patients.  Cerebrospinal
    fluid concentrations of 1.25 and 2.5 g/ml were measured in the female
    and male patient, respectively (Waisbren & Spink, 1950).

         Poth  et al.  (1951) reported serum concentrations ranging from
    not detectable to <80 g/ml in patients treated with oral neomycin
    (6 g/day) for 3 days. The majority of the administered dose was
    excreted in the faeces unchanged.  Only 3% of the dose was recovered
    from the urine.

         The absorption and excretion of a 2 g oral or rectally
    administered dose of neomycin sulfate was studied in 10 normal
    subjects (healthy physicians who had no history of renal or liver
    disease) and in patients with liver disease, peptic ulcer, regional
    enteritis, or active ulcerative colitis.  Urinary excretion (0.58%) of
    neomycin and serum neomycin concentrations over 48 hours were similar
    in all groups and by both routes.  The percentage of the neomycin dose
    excreted in the faeces following oral or rectal administration was
    comparable (approximately 99%) (Breen  et al., 1972). 

    2.1.2  Biotransformation

         Aminoglycosides as a class are not metabolized in animals (Bevan
    & Thompson, 1983) and are mostly excreted unchanged (Aschbacher &
    Feil, 1994; Keen, 1975). 

    2.1.3  Special studies on binding to macromolecules

         Lactating Israeli-Freisian cows (2 animals) and Awassi ewes (4
    animals) were given single doses of 20 mg/kg bw neomycin i.m. or i.v.
    Resulting serum concentrations ranged from 5-10 g/ml.  Serum protein
    binding, determined by equilibrium dialysis and ultrafiltration
    methods, was approximately 45-50% and 50-55% for cows and ewes,
    respectively (Ziv & Sulman, 1972).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies after oral, i.v., s.c. or
    i.p. administration of neomycin are summarized in Table 1. 

    2.2.2  Short-term toxicity studies

    2.2.2.1  Cats

         Groups of cats (8/sex/group) were orally dosed (gelatin capsule)
    with 0, 6, 12 or 25 mg technical neomycin sulfate/kg bw/day for 1
    year.  The actual administered doses were found by chemical analysis
    to be 72% of the intended doses. Clinical signs, auditory and
    vestibular examinations, body weight, haematology, serum chemistry,
    urinalysis,  organ weights, macroscopy and histopathology were
    evaluated.

         The cause of the death of 7 cats during the study was not
    considered to be treatment-related.  The RBC counts were significantly
    decreased in males at the highest dose at weeks 26 and 52.  Blood urea
    nitrogen levels were significantly increased in high-dose males at 13,
    26 and 52 weeks, but no histopathological evidence of kidney damage
    was observed.  In the high-dose males at termination, potassium,
    chloride and alkaline phosphatase were decreased, and cholesterol
    levels were increased. Relative spleen and pancreas weights were
    significantly decreased at all dose levels in male cats but without a
    dose-related trend.  Bone marrow hypoplasia was seen histogically in
    the high-dose group (3/6 males and 4/8 females) (Kakuk, 1981).


        Table 1.  Acute toxicity of neomycin
                                                                                                          
    Species  Sex    Route  Substance                  Purity          LD50          Reference
                                                                      (mg/kg bw)
                                                                                                          
    Mouse     ?     oral   neomycin sulfate                 ?         approx.       Swoap, 1952c,d
                                                                      2250-2500

    Mouse    M&F    oral   neomycin sulfate bulk        900 g/mg     2250-2298     Swoap, 1952f,g,h 

    Mouse    M&F    oral   neomycin sulfate (bulk       900-1000      >2500         Swoap, 1952b
                           powder from resin process)     g/mg

    Mouse     ?     i.v.   neomycin sulfate                 ?         40-158        Vander Brook, 1949;
                                                                                    Swoap, 1949a,b;
                                                                                    1950a,b,c,d,e,i,j,k;
                                                                                    1951a,b; Cale, 1950;
                                                                                    Gray, 1964

    Mouse     M     i.v.   neomycin sulfate                 ?         64.6-107      Brockie, 1951; Cale,
                                                                                    1950; Swoap,
                                                                                    1950f,g,h 

    Mouse    M&F    i.v.   neomycin sulfate                 ?         >50           Swoap, 1953a

    Mouse     ?     i.v.   neomycin A hydrochloride         ?         97.6          Swoap, 1949a

    Mouse    M&F    i.v.   neomycin in HCH                  ?         74-115        Swoap, 1952a

    Mouse     ?     i.v.   neomycin sulfate bulk        900 g/mg     110           Swoap, 1952e

    Mouse    M&F    i.v.   neomycin sulfate (bulk       910 g/mg     100           Swoap, 1952b
                           powder from resin process)

    Mouse     ?     s.c.   neomycin sulfate                 ?         550           Vander Brook, 1949;
                                                                                    Swoap, 1950c,d,e
                                                                                                          

    Table 1 (contd)
                                                                                                          
    Species  Sex    Route  Substance                  Purity          LD50          Reference
                                                                      (mg/kg bw)
                                                                                                          
    Mouse     ?     s.c.   neomycin sulfate                 ?         400           Brockie, 1951

    Mouse     ?     s.c.   neomycin A                       ?         470           Vander Brook, 1949
                           hydrochloride                                            (51) 

    Mouse     ?     i.p.   neomycin sulfate                 ?         274           Gray, 1964

    Mouse    M&F    i.p.   neomycin sulfate             690 g/mg     310-457       Feenstra, 1958a;
                                                                                    Swoap, 1957a

    Mouse    M&F    i.p.   neomycin B sulfate               ?         277-389       Swoap, 1954a,b;
                                                                                    Shealy, 1956

    Mouse    M&F    i.p.   neomycin B sulfate          1000 g/mg     533           Swoap, 1953c
                                                                                    Shealy, 1956

    Mouse    M&F    i.p.   neomycin sulfate Po.         690 g/mg     356           Swoap, 1958
                           blended USP                                              

    Mouse    M&F    i.p.   neomycin sulfate bulk        658 g/mg     248           Feenstra 1959
                                                                                                          
    

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Rats

         Eight groups of Sprague-Dawley (SD)BR F1a weanling rats (4 groups
    of males, 4 groups of females, 54-56/group) were administered neomycin
    sulfate via the diet at 0, 6.25, 12.5 or 25 mg/kg bw/day until
    terminal sacrifice. The test animals were offspring from the
    F0generation that were mated after consuming similar levels of
    neomycin in the diet for 11 weeks (see section 2.2.4).  After 52 weeks
    of treatment 10 rats/sex/group were selected for an interim kill and
    evaluation.  All male groups were killed after 104 weeks when the
    control group survival reached 20%.   When survival in female groups
    reached 20%, the remaining animals of the group were sacrificed (27 to
    30 months).  Clinical signs, body weight, food consumption,
    haematology, clinical chemistry, urinalysis, ophthalmoscopy, auditory
    and vestibular tests, organ weight and histopathology were evaluated
    in control and high-dose male and female rats. 

         To assess the possible effects of neomycin on the vestibular and
    auditory functions an additional group (6/rats/sex, weanlings selected
    from the control group in the reproduction study) was administered
    neomycin sulfate s.c. at 100 mg/kg bw/day.  Auditory and vestibular
    function tests were scheduled weekly until study termination.  When
    all rats showed hearing loss (week 14), they were killed.  Tissues of
    the inner ear were preserved, but histopathological examinations were
    not carried out.

         Up to week 84, overall survival was about 84% and 92% for males
    and females respectively.  At the end of the study high mortality was
    observed in all groups (33/44, 27/45, 29/46 and 30/45 for males and
    33/46, 34/45, 34/44 and 35/45 for females at 0, 6.5, 12.5 and 25 mg/kg
    bw/day, respectively).  Female body weights were dose-relatedly
    increased during the first three weeks of the study.  Although not 
    statistically  significant  3/15 rats at  25 mg/kg bw/day showed
    hearing loss at the end of the study.  No treatment-related effects
    were observed in any of the other parameters evaluated. There was no
    evidence of carcinogenicity. The NOEL was 12.5 mg/kg bw/day (Kakuk,
    1982).

    2.2.4  Reproductive toxicity studies

    2.2.4.1  Rats

         In a 3-generation reproductive toxicity study groups of Sprague-
    Dawley (SD)BR rats (40-20/sex/group) were administered neomycin
    sulfate via the diet at 0, 6.25, 12.5 or 25 mg/kg bw/day.  After 11
    weeks F0 generation rats were mated to produce F1a offspring which
    were subsequently continued on neomycin sulfate in the life-time
    feeding study described in section 2.2.3.  F0 animals were bred a

    second time to produce F1b offspring for use in the reproduction study
    (through F3b litters).  Through all generations no treatment-related
    effects were observed in any of the parameters evaluated. The NOEL was
    25 mg/kg bw/day (Kakuk, 1980). 

    2.2.5  Special studies on embryotoxicity and teratogenicity

    2.2.5.1  Rats

         Groups of 20 pregnant Sprague-Dawley (SD)BR rats (F2b females
    from the reproductive toxicity study) were maintained on the same dose
    regimen of 0, 6.25, 12.5 or 25 mg/kg bw/day (see section 2.2.4).  From
    gestation days 6-15, doses were increased to 0, 62.5, 125 or 250 mg/kg
    bw/day.  From gestaton days 16-20, doses were returned to 0, 6.25,
    12.5 or 25 mg/kg bw.  Dams were killed on day 20 of gestation.  No
    evidence of maternal toxicity, fetotoxicity or teratogenicity was
    observed (Kakuk, 1980).

    2.2.6  Special studies on genotoxicity

         The results of the available genotoxicity studies on neomycin are
    summarized in Table 2.

    2.2.7  Special study on nephrotoxicity

    2.2.7.1  Mice

         Ten mice/group were given s.c. doses of 0, 30, 100, 300, 600 or
    1000 mg/kg bw/day neomycin A (neamine), neomycin B or neomycin C for
    14 days. None of the mice at the 2 highest doses  survived 14 days. 
    Two mice in the 1000 mg/kg bw/day neamine group died. 
    Histopathological examination of the kidneys was performed.  Based on
    lesion indices the degree of nephrotoxicty was established.  Neomycin
    B and Neomycin C were about equally nephrotoxic.  The observed
    nephrotoxicity in the neamine groups was about 50% of that observed in
    the other neomycin groups (Feenstra, 1954).

    2.2.7.2  Guinea-pigs

         Guinea-pigs (3/sex/group) were administered 10, 20, or 60 mg
    neomycin sulfate/kg bw/day for 3 months (route not specified).  One
    female was kept as a control.  Histopathological evaluation of the
    kidney revealed subacute to chronic focal interstitial nephritis
    associated with degeneration and necrosis at all doses, with a dose-
    related increase in severity (Feenstra, 1950). 


        Table 2.  Results of genotoxicity studies on neomycin
                                                                                                  
    Test system         Test object    Concentration          Results          Reference

     In vitro
                                                                                                  

    Chromosome          human          20, 40 and 80 g/ml    positivea,b      Jaju  et al., 1986
    aberration assay    lymphocytes

    SCE test            human          20, 40 and 80 g/ml    negativeb        Jaju  et al., 1986
                        lymphocytes

     In vivo

    Cytogenetic         Mice bone      50 mg/kg bw            positiveb,c      Manna & Bardhan,
    assay               marrow                                                 1973

                                                                                                  

    a    At doses that significantly inhibit the progression of the cell cycle.
    b    No positive control group was used.
    c    Test was not performed to current standards, i.e., results from 12 fixation 
         times are combined in 6 groups and number and sex of animals not stated.
         Mitotic frequency was decreased; therefore, it was concluded that neomycin 
         reached the bone marrow.
    

    2.2.7.3  Dogs

         Six dogs (3/group) were administered 20 or 60 mg neomycin
    sulfate/kg bw/day for 88 days (route not specified).  All 3 dogs in
    the high-dose group died after 14, 16 and 21 days.  One low-dose dog
    survived.  The other 2 died after 21 and 45 days.  Urine samples from
    all dogs were taken before and after the beginning of treatment and
    analyzed for albumin, blood and the presence of casts. 
    Histopathological examinations of kidneys showed degeneration,
    necrosis and focal interstitial nephritis in all 6 dogs (Feenstra,
    1950).

         Twelve dogs were injected intramuscularly with 24, 48, or 96
    mg/kg bw/day neomycin.  At the highest dose all dogs died within 1-3
    weeks. Blood urea nitrogen was increased and renal function impaired
    as shown by decreased phenolsulfonphthalein excretion. 
    Histopathological evaluation of kidneys revealed renal tubular damage
    characterized by marked epithelial necrosis in the proximal convoluted
    tubules.  Moderate changes in bone marrow and marked congestion of the
    liver were also found.  Inflammation at injection sites was also
    noted.  At the mid-dose the severity of effects were described as
    intermediate.  Dogs exposed to 24 mg/kg bw/day were killed and
    examined after 1 month.  The severity of renal damage in this group
    was described as slight with increased granularity and occasional
    desquamation of epithelial cells being most evident (Nelson  et al.,
    1951).

         No kidney damage was observed in dogs (number/sex/group not
    stated) orally dosed with 100 mg neomycin/kg bw/day for 6 weeks based
    on urinalyses and gross and histopathological examinations of kidneys. 
    No other abnormalities were observed (Poth  et al., 1951).

    2.2.8  Special studies on ototoxicity

    2.2.8.1  Guinea-pigs

         Groups of 3 guinea-pigs were administered i.m. doses of 25, 50,
    100, or 150 mg neomycin/kg bw/day for 30 to 60 weeks.  Parameters
    evaluated included vestibular function, auditory function (Preyer's
    reflex) and histolopathological examination of labyrinths and central
    nervous systems.

         One guinea-pig in the high-dose group died after 22 days on test.
    Vestibular function was reported as normal or slightly impaired across
    all treatment groups with no dose-response relationship. Preyer's
    reflex was abolished in all guinea-pigs receiving 100 and 150 mg/kg
    bw/day. Histopathological examinations of animals in which the
    Preyer's reflex was abolished revealed marked destruction of hair
    cells and sometimes complete destruction of the organ of Corti.
    Animals with intact hearing had normal cochleae, cristae, and maculae.

    Reversible as well as irreversible degeneration of the ganglion cells
    was seen in animals with abolished hearing, while animals with intact
    hearing showed no definite pathological changes (Riskaer  et al.,
    1956) .

         Hearing was not affected (determined by audiometry) after the
    installation of 3 mg/kg bw neomycin into the middle ear of 18 guinea-
    pigs.  Six of the test animals had either one or both tympanic
    membranes perforated prior to treatment (Riskaer  et al., 1956). 

         Groups of guinea-pigs (50/sex/group) were orally admini-stered
    neomycin B sulfate at 0, 1, 5, or 10 mg/kg bw/day for 90 days. Two
    positive control groups (20/sex/group) received 10 or 100 mg/kg bw/day
    s.c. for 90 and 34 days, respectively.  Parameters evaluated included
    clinical signs, body weight, auditory function test (Preyer pinna
    reflex), gross necropsy, and histopathology (including cochleae to
    score the loss of hair cells from the organ of Corti).

         At 100 mg/kg bw/day s.c., ototoxic changes in the Preyer pinna
    reflex thresholds and cochlear hair cell counts were observed.  These
    changes were not seen in the low-dose s.c. group nor in any of the
    groups after oral administration.  The NOEL for orally administered
    neomycin in this study was 10 mg/kg bw/day (Brummett  et al., 1985).

    2.2.8.2  Cats

         A clinical neomycin formulation (containing mostly neomycin B
    sulfate), neomycin A hydrochloride and neomycin B sulfate were
    administered s.c. to groups of cats (number/sex/group not stated) in
    daily doses of 80 mg/kg bw/day for 5 or 15 days.  The clinical
    formulation was also administered in doses of 20, 40, or 100 mg/kg
    bw/day s.c. for periods of 90, 60 and 30 days, respectively.  Crude
    neomycin (about 70% pure) was orally administered at 2 daily doses of
    500 mg/kg bw (1 g/animal/day) for 30 days.  Vestibular function was
    studied by electrical recording of nystagmus evoked by rotating the
    animal on a motor-driven turntable.  Hearing ability was followed and
    the cochlear function was studied electrophysically. 
    Histopathological examination of the kidneys was performed of all
    cats.

         Vestibular function was affected only in cats receiving the
    highest dose of the clinical formulation (100 mg/kg bw/day) for 30
    days.  One of these cats showed a gradual loss of nystagmus which was
    entirely absent by day 35, 5 days after the last treatment. Depression
    of cochlear function was observed in all groups of cats and was most
    severe in cats receiving the clinical preparation s.c.  Degeneration
    of external hair cells in the organ of Corti and internal hair cells
    was also observed.  A dose-related increase in the severity of damage
    to renal tubular epithelium occurred in all cats receiving the
    clinical preparation s.c. and in cats orally dosed with crude neomycin
    (Hawkins, 1952; Hawkins  et al., 1953). 

         Ototoxicity was studied in 5, 8, 3, and 4 cats who received oral
    doses of 0, 6.25, 12.5, or 25 mg neomycin/kg bw/day, respectively, for
    1 year.  No evidence of auditory or vestibular dysfunction was
    observed.  In surface preparations of the organ of Corti
    (cytocochleograms) ototoxicity was present microscopically as loss of
    outer hair cells from the extreme lower portion of the basal turn of
    the cochlea.  Severe ototoxicity was observed in 3/4 high-dose cats. 
    Hair cell loss of a lesser degree was also observed in 7/8 and 1/3
    cats in the low- and mid-dose groups, respectively (Kakuk, 1981).  Due
    to serious shortcomings in the histological technique and the absence
    of a clear dose-related effect, the Committee considered this study
    inadequate for the safety evaluation of neomycin. 

    2.2.9  Special studies on irritation and sensitization

         No significant irritation was observed 24 hours after the
    intrapleural administration of single doses of solutions of 1 ml
    neomycin sulfate (0-100 mg/ml) to guinea-pigs (3-4/dose) (Swoap,
    1951c).

         A group of 9 neomycin-sensitive guinea-pigs was evaluated with
    the patch test for cross sensitivy to kanamycin, streptomycin,
    dihydrostreptomycin and bacitrin.  Positive results were obtained for
    kanamycin (1/9) and streptomycin (8/9) (Epstein & Wenzel, 1962).

    2.2.10  Special studies on microbiological effects

         Neomycin is active against most gram-positive and gram-negative
    rods, many gram-positive cocci, and such acid-fast pathogens as
     Mycobacterium tuberculosis.

         Neomycin reacts with 30S ribosomal subunits of procaryotic cells
    by electrostatic attraction causing a change in the conformation of
    the ribosomal binding protein.  This results in mRNA reading errors
    and disrupted protein synthesis.

    2.2.10.1  In vitro

         Minimum Inhibitory Concentration (MIC) values for different
    bacterial strains obtained from animal or human isolates are listed in
    Tables 3 and 4, respectively.

         In a special study, strains of various bacterial genera, mostly
    of human origin, were tested for their susceptibility to neomycin
    sulfate. MIC values were estimated on two agar types, Wilkens
    Chalgren/glucose medium (WCG) and supplemented blood medium (SB) with
    2 inoculum densities. Estimations were made under anaerobic and
    aerobic conditions  (E. coli) by the agar dilution method. The
    results are listed in Table 5 (Thurn  et al., 1994).

         The adsorption of neomycin to dog faeces was calculated as the
    difference between the amount of neomycin added to dilute suspension 
    of dog  faeces and the  amount of neomycin  measured (microbioloigal
    assay) in the supernatant after the mixture was incubated and
    centrifuged. About 75% adsorption of neomycin was observed.  After
    acid extraction 47% of the added neomycin was still bound to the
    faecal solids (Wagman  et al., 1974). 

         Several concentrations of neomycin were evaluated for adsorption
    to human faeces.  Faecal samples were obtained from 8 healthy human
    volunteers.  The binding of neomycin to the faeces was calculated as
    the difference between the amount added to a dilute suspension of
    faeces and the amount measured (micro-biological assay) in the
    supernatant after the mixture was incubated for 1 hour and
    centrifuged.  Approximately 83-98% faecal binding was observed
    (Hazenberg  et al., 1983b, 1984).

         Neomycin solutions of approximately 0.1-30 mg/ml were mixed with
    faecal suspensions prepared from faeces of 8 healthy human volunteers. 
    Neomycin antimicrobial activity (tested with  Enterobacter cloacae)
    remaining in the supernatants was measured and used for calculating
    the "% inactivation" relative to the amount of added neomycin.  The
    percentage of added neomycin that was biologically inactivated
    decreased with increasing concentrations of neomycin.  Nearly 100%
    inactivation due to binding occurred when the ratios of neomycin to
    faecal weight were < 5 mg neomycin to 1 g wet weight of faeces in an
    incubation mixture (Veringa & Van der Waaij, 1984).

    2.2.10.2  In vivo

         Oral administration of neomycin to 37 healthy mongrel dogs (400
    mg/day for 2, 4, or 10 days) and 24 healthy humans (6 g/day for 1 or
    3 days) caused a marked reduction or complete elimination of coliform
    bacilli from faeces in both species within 24 to 48 hours.  In 4/10
    dogs receiving a dose of 400 mg/day for 10 days resistant strains of
     E. coli appeared on the 5th-6th day of treatment.  Resistance was
    not observed in  E. coli isolates from humans treated with neomycin
    in this study (Schweinburg  et al., 1952).

         Groups of 3-5 human flora associated (HFA) mice (germ-free mice
    colonized with bacteria from human faecal suspensions) were
    administered 1, 2, 3 or 4 g/l neomycin in their drinking-water. 
    Faecal samples were cultured on days 0, 1, 3, 5, 7, 14, 21, 28 and 35
    of treatment.  A control group of HFA mice received no neomycin
    treatment. The number of obligate anaerobes, gram negative obligate
    anaerobes,  E. coli and  Enterococci did not change over 35 days
    in the 1 g neomycin/l drinking-water group.  In the 2 and 3 g
    neomycin/l groups,  Escherichia coli and  Enterococci were
    eliminated, and the percentage of gram-negative obligate anaerobes was
    increased. The NOAEL in this study is 1 g/l, equal to 125 mg/kg bw/day
    (Hazenberg  et al., 1983a). 


        Table 3.  Summary of MIC values for different bacterial species obtained from animal isolates
                                                                                                      
                                                    MIC (g/ml)

    Species                           No. of        Mean or Range      MIC50     Reference
                                      strains
                                                                                                      

     Staphylococcus aureus                          0.5                          Moellering, 1983

     Escherichia coli                               8.0                          

     Klebsiella pneumoniae                          2.0                          
                                                     
     Proteus mirabilis                              8.0                          

     Proteus morganii                               8.0                          

     Proteus rettgeri                               8.0                          

     Proteus vulgaris                               4.0                          

     Escherichia coli (calves)                                            16     Hewett, 1990
     Escherichia coli (pigs)                                              16     
     Escherichia coli (chickens)                                           1     

     Salmonella sp. (calves)                                               1
     Salmonella sp. (pigs)                                               0.5

     Bacteroides nodosus              68            16 - >256           >256     Duran  et
                                                                                  al. , 1991
     Bacteroides putredinis           36            16 - >256            128     

     Bacteroides buccae               16            2 - >256             128     

     Bacteroides sp.                  21            8 - >256            >256     

                                                                                                      

    Table 3 (contd)
                                                                                                      
                                                    MIC (g/ml)

    Species                           No. of        Mean or Range      MIC50     Reference
                                      strains
                                                                                                      


     Fusobacterium necrophorum        10            16 - >256            128     

     Fusobacterium sp.                19            2 - >256             128     

     Peptostreptococus sp.            35            <0.06 - >256          64     Piriz  et al., 1992

                                                                                                      
    

        Table 4.  Summary of MIC values for different bacterial species obtained from 
              human isolates
                                                                                             
                                                    MIC (g/ml)

    Species                           No. of        Mean or range         Reference
                                     strains
                                                                                             

     Escherichia coli                   10            0.25 - 12.5         Schweinberg  et al.,
                                                                          1952

     Enterococci                         5            3.2 - 15

     Sphaerophorus necrophorus           7            200 - 1600          Finegold  et al.,
                                                                          1967

     Bacteroides fragilis               55            1600 - >25 600

     Bacteroides melaninogenicus        20            <100 - 400            

     Bacteroides oralis                 16            <100 - 400

     Fusobacterum sp.                   22            100 - 3200           

     Bifidobacterium adolescentis       11            12.5 - 400          Miller & Finegold,
                                                                          1967

     Bifidobacterium bifidum             5            400 - 1600            

     Bifidobacterium longum             11            200 - 1600            

     Bifidobacterium sp.                 6            200 - 1600            

     Clostridium novyi Type A           16            32 - 512            Dornbusch  et al.,
                                                                          1975

     Clostridium novyi Type B            7            64 - 512              

     Clostridium bifermentans            9            64 - 512              

     Clostridium sordellii               6            64 - 512

     Clostridium sporogenes             18            32 - 512              
                                                                                             

    Table 4 (contd)
                                                                                             
                                                    MIC (g/ml)

    Species                           No. of        Mean or range         Reference
                                     strains
                                                                                             



     Propionicum agnes                  38            6.25 - 25           Hoeffler & Pulverer,
                                                                          1976

     Escherichia coli                    2            16                  Hazenberg  et al.,
                                                                          1983b

     Escherichia coli                    3            1 - 32              Hazenberg  et al.,
                                                                          1984
                                                                                             

    Table 5.  Summary of MIC values under different agar medium and inoculum density conditions
                                                                                       

                                                            MIC50 (g/ml)a

    Bacterial species/genus          Strains       Low densityb        High densityc

                                                   SBd      WCGd       SBd      WCGd
                                                                                       

     Bacteroides                      15e,f        >128     >128       >128     >128

     Bifidobacterium                  12e                     16                 128

     Clostridium                      11e, 5f      >128      128                >128

     Enterococcus                     10e, 2f      >128      128       >128     >128
                                                                         
     Escherichia                      13e                     16                  64

     Escherichia - Aerobic            13e                   >128                >128

     Eubacterium - Anaerobic          9e                       8                >128

     Fusobacterium                    5e, 3f         16       32       >128      128

     Lactobacillus                    15e, 2f      >128       32       >128       64

     Peptostreptococcus/              16e, 14f     >128       32       >128      128
     Peptococcus
                                                                                       

    a    MIC50 values are for the multiple strains included in the assay.
    b    Low density inocula had cell concentrations of approx. 1 x 108 cells/ml.
    c    High density inocula had cell concentrations of approx. 1 x 1010 cells/ml.
    d    SB = Supplemented blood medium; WCG = Wilkins-Chalgren/glucose medium
    e    Number of tested strains of which MIC values for WCG were used to 
         calculate the summary values
    f    Number of tested strains of which MIC values for SB were used to 
         calculate the summary values
    
    2.2.10.3  Special studies on potential effects on microorganisms used
              for industrial food processing

         Thirty strains of  Propionobacterium sp., used to manufacture
    Emmenthal and related cheese varieties, showed no resistance to 30
    g/disc neomycin but at 5 g/disc moderate sensitivity was observed
    (8/30 strains were resistant) (Reddy  et al., 1973).

         Forty-two strains of mesophilic lactic  Streptococci,
     Enterococci, Lactobacilli, Leuconostoc, Staphylococci  and
    other dairy- and food-related microorgansims were tested for their
    sensitivity to neomycin at 5 and 30 g/disc.  Elliker's lactic agar
    served as the growth medium; incubations temperature and times were
    varied according to individual culture growth requirements.

         Most of the strains of the starter streptococci  (S. lactis,
     S.cremoris, S. diacetilactis and S. thermophilus) and  Lactobacillus
     bulgaricus were sensitive to neomycin. Strains of  S. faecalis,
    exhibited resistance at 5 and 30 g/disc (Reinbold & Reddy, 1974). 

         In another study, 24/29 strains of  Lactobacillus bulgaricus
    and all 15 tested strains of  Streptococcus thermophilus were
    resistant to 5 g/disc neomycin.  The tests for resistance were
    carried out with commercially available sensitivity disks.  At higher
    doses of neomycin (30 g/disc) resistance in 14/15 strains of
     S.thermophilus and 10/29 strains of  L. bulgaricus was observed
    (Sozzi & Smiley, 1980).

         The effect of residues of neomycin in milk on bacterial starter
    cultures used in the production of fermented milk products was
    investigated.  Milk samples spiked with either 4 or 40 g/ml neomycin
    were serially diluted with antibiotic-free milk to provide
    concentrations ranging from 0.063-4 g/ml.  Actual concentrations
    measured with a microbiological cylinder plate assay ranged from
    <0.20-3.12 g/ml (detection limit: 0.20 g/ml).  The starter culture
    types included a group of buttermilk/sour cream cultures containing
    strains of  Lactococcus lactis spp. and  spp. cremoris or a
    mixture of lactic acid producers and citric acid fermenters, 2 groups
    of Italian cheese cultures containing strains of  Streptococcus
    thermophilus, another group of Italian cheese cultures containing
    strains of  Lactobacillus helveticus, and a group of yogurt cultures
    containing  Streptococcus thermophilus and  Lactobacillus debruckii
     spp. bulgaricus. "Time to clot" ratios greater than 2 indicated
    that cultures were adversely affected by the presence of neomycin. 
    The only cultures adversely affected were the yogurt starter cultures
    at a neomycin concentration of 4 g/ml (equivalent to 3.12 g/ml).  In
    this study neomycin concentrations in milk <2 g/ml (equivalent to
    1.42 g/ml) had no effect on the growth of the bacteria in any of the
    starter cultures (Hallberg  et al., 1994). 

    2.3  Observations in humans

    2.3.1  Nephrotoxicity

         A clinical investigation of 63 patients (34 males and 29 females
    ranging in age from nine months to 63 years (22 of them >60 years) on
    neomycin therapy was undertaken.  Renal toxicity and ototoxicity
    toxicity were reported as treatment-related effects in some patients.
    Twenty-four out of 32 patients, in whom serial urinalyses were
    performed, showed fine granular casts in their urine either during or
    just after neomycin therapy (Waisbren & Spink, 1950).

         Powell and Hooker (1956) reported several cases of severe tubular
    damage found in kidneys at autopsy of patients treated intramuscularly
    with neomycin. 

         Oral administration of decreasing doses of neomycin (9.4- 1.5 g/d
    (total 46 g over 11 days) to a patient produced acute renal failure
    (Greenberg & Momary, 1965).

    2.3.2  Ototoxicity

         Ototoxic effects determined by audiograms and vestibular
    functional tests were observed in patients treated with neomycin at
    doses ranging from 1.5-2 g/day i.m. for up to 9 days.  Hearing loss
    was reported in 5 patients and loss of vestibular function occurred in
    2 patients (Waisbren & Spink, 1950). 

         Deafness with histopathological inner ear changes were reported
    in patients receiving high doses of neomycin orally (18 to 633 g total
    dose) for unspecified periods of time (Lindsay  et al., 1960;
    Halpern & Heller, 1961; Greenberg & Momary, 1965).  

         Severe deafness occurred in a 1-year old female, treated for
    enteritis for 10 days with a neomycin-containing medication.  The
    total dose of neomycin was less than 2 g (King, 1962). 

         Severe deafness was reported in a 75 year-old female patient
    receiving colonic and rectal irrigations with a neomycin solution for
    2 months. A 5% solution was administered initially, then decreased to
    a 1% solution for the majority of the treatment period.  The total
    neomycin dose administered was estimated at >900 g.  The patient had
    a hearing deficit but did not require a hearing aid prior to treatment
    (Fields, 1964).

    2.3.3  Hypersensitivity

         From a survey of 675 patients with skin infections treated with
    neomycin it was concluded that the sensitizing index for neomycin was
    very low (Kile  et al., 1952).  However, cases of contact dermatitis

    from neomycin (e.g., eardrops and eyedrops) were reported as dermal
    delayed (tuberculin-type) and not epidermal sensitivity (Epstein,
    1956; Calnan & Sarkany, 1958). In 55-75% of patients with neomycin
    sensitivity, evidence of atopy (a genetic predisposition to mounting
    large immune responses to antigenic stimulation) was established
    (Epstein, 1965).

         Results of patch tests with 21 antibiotics performed on a large
    number of patients sensitive to neomycin showed cross-sensitivity to
    bacitracin (80%), framycetin (91%), paromomycin (up to 99%) and
    kanamycin (61%) (Pirila & Rouhunkoski, 1962).  Cross-sensitization
    with neomycin in streptomycin-sensitized patients was also described,
    although contradictory results were obtained with respect to the
    neomycin sensitivity in streptomycin-sensitive patients 
    (Sidi  et al., 1958; Calnan & Sarkany, 1958).

         A patient treated with ear drops containing neomycin (5 mg/ml in
    distilled water) developed an acute exacerbation of otitis externa
    after previously having shown a highly satisfactory response to the
    use of the neomycin ear drops (Baer & Ludwig, 1952).

    2.3.4  Special studies on the malabsorption syndrome

         Morphologic alterations in the jejunal mucosa of patients,
    similar to but less marked than those seen in patients with idiopathic
    steatorrhea, were seen in 11/12 biopsy specimens from patients treated
    with neomycin (4-12 g/day) for at least 4 days. Eighteen days after
    the last dose of neomycin the jejunal morphology was restored
    (Jacobson  et al., 1960). 

         During the oral administration of 4-6 g neomycin/day for 6-8 days
    plasma carotene concentrations were decreased in 6/8 patients despite
    the administration of supplemental carotene (10 000 units/day). In
    addition, d-xylose excretion in urine was decreased in 3/4 subjects. 
    Faecal fat excretion doubled during the treatment period in 1 patient
    (Jacobson & Faloon, 1961).  

         Neomycin sulfate was administered orally (12 g/day for 4 days) as
    well as by inlying tube directly into the jejenum and ilieum in 11
    subjects. Urine and faeces were evaluated for fat, calcium, sodium,
    potassium and nitrogen content.  Steatorrhea was produced after oral
    administration, but when neomycin was administered more distally, the
    faecal fat content progressively diminished.  Lowest urinary calcium,
    sodium and potassium levels were measured after oral administration.
    Faecal sodium, potassium and nitrogen levels were less affected by
    this administration route, whereas the faecal calcium content was
    greatest after jejunal instillation. The steatorrhea could not be
    reversed by the administration of bile salts, sodium bicarbonate or
    pancreatic enzymes (Gordon  et al., 1968).

         Three healthy male volunteers were orally administered 8 g
    neomycin sulfate/day for 7 days.  Lactose malabsorption was induced
    after 3 days of treatment.  Biopsy specimens from the small bowel
    taken after 7 days of treatment showed slight villous blunting of the
    mucosa and depressed disaccharidase activity.  The lamina propria was
    infiltrated with plasma cells and eosinophils.  Enzyme levels and
    histology of the mucosa and lamina propria of the small bowel returned
    to normal 10 to 14 days after the treatment was stopped.  No
    treatment-related effects on stool lactic acids were observed (Cain
     et al., 1968). 

    2.3.5  Special studies on microbiological effects

         In a study with 37 hospitalized patients with various types of
    intestinal lesions, the effect of oral neomycin treatment on bacteria
    from the intestinal tract was determined. Treatment regimes of
    neomycin administered prior to surgery (duration not stated) were as
    follows: 1.5 g 4 times/day; 2 g 3 times/day; or 1 g every hour for
    four doses the first day, then 1.5 g 4 times/day.  After the
    administration of neomycin the only clinical observations were
    occasional nausea and vomiting. In stool specimens from patients
    treated with 1.5 g 4 times/day (30 mg/kg bw/day) most of the aerobic
    bacteria ( E. coli, A. aerogenes, S. faecalis, Proteus and
     Pseudomonas) disappeared within 1-4 days, whereas anaerobic
    organisms ( Bacteroides and  Clostridium) remained. No resistant
    strains of  Mycrococcus pyopgenes were found.  Similar results were
    seen in patients receiving 2 g 3 times/day.  In the group receiving 1
    g hourly for four doses followed by 1.5 g 4 times/day, aerobic
    bacteria were removed from the intestinal tract within two to three
    days, while anaerobes ( Bacteroides and  Clostridium) persisted
    (Deering & Needham, 1953).

         The intestinal flora of 14 patients hospitalized for non-
    gastrointestinal diseases was studied before and after the use of
    neomycin. Four patients received 4 g neomycin orally for 7 days and 10
    patients were given 2 g neomycin for 6 days. The bacterial populations
    of faecal specimens collected before, during and after neomycin
    treatment were enumerated. A decrease in all bacterial species was
    noted.  Most affected were  Enterococci, followed by  E.coli,
    lactose non-fermenters,  Lactobacilli, Clostridia and  Proteus
    sp. Yeast and  Klebsiellae were increased (Daikos  et al., 1968).

         Seven healthy adult volunteers received 1 g neomycin orally 3
    times a day for 5 days (equivalent to 50 mg/kg bw/day). Fresh stool
    samples were collected before, during and after treatment, and
    bacterial counts recorded.  Neomycin treatment did not affect
     Bacteroides or  Clostridia counts.  E. coli counts were
    significantly reduced in 2/7 volunteers (Arabi  et al., 1979).

    3.  COMMENTS

         The Committee considered data on pharmacokinetics, acute
    toxicity, short-term and long-term toxicity, reproductive and
    developmental toxicity, genotoxicity and carcinogenicity, effects in
    humans and effects on antimicrobial activity.  An evaluation report,
    as requested for veterinary drugs with a long history of use (Annex 1,
    reference 104) was also provided.

         Neomycin is poorly absorbed after oral administration, whereas
    after parenteral administration it is readily bioavailable.  In
    calves, oral absorption was estimated at 1-11%, depending on age. In
    humans, up to 3% of an oral dose was recovered in urine, indicating
    low absorption. Of the neomycin present in blood, 45-55% was bound to
    plasma proteins in cows and ewes and the remainder was present in
    nonionized form and may cross tissue barriers. After oral
    administration the compound is mainly excreted unchanged in the
    faeces. After parenteral administration neomycin is mainly excreted in
    the urine.

         Single doses of neomycin were slightly toxic to mice after oral
    administration (LD50 = 2250 mg/kg bw), but single i.v. doses in mice
    were highly toxic (LD50 <100 mg/kg bw).

         Nephrotoxic effects were observed in mice after repeated s.c.
    administration of 30-300 mg neomycin/kg bw/day, in guinea-pigs after
    repeated s.c. doses of 10-60 mg neomycin sulfate/kg bw/day, and in
    dogs after repeated i.m. doses of 24-96 mg neomycin/kg bw/day.  No
    kidney damage was observed in dogs receiving 100 mg/kg bw/day orally
    for 6 weeks. Ototoxicity was observed after parenteral administration
    of neomycin ranging from 25 to 150 mg/kg bw/day in guinea-pigs, and
    20 to 80 mg/kg bw/day in cats.  No ototoxic effects were observed in
    guinea-pigs following oral administration of up to 10 mg neomycin
    sulfate/kg bw/day for 90 days. In cats orally administered 6.25, 12.5
    or 25 mg of neomycin/kg bw/day for one year, auditory function was not
    affected, but upon histopathological examination changes in the organ
    of Corti were reported at all dose levels.

         The Committee concluded that, although ototoxicity was observed
    in the one-year study in cats at the lowest dose tested  (6.25 mg/kg
    bw/day), this study was inadequate for the safety evaluation of
    neomycin because of serious shortcomings in the histological technique
    and the absence of a clear dose-related effect.  It, however, accepted
    the NOEL of 10 mg neomycin sulfate/kg bw/day, equivalent to 6 mg
    neomycin/kg bw/day, for ototoxicity from the study in guinea-pigs.

         Only a limited number of mutagenicity studies were available,
    which had been poorly performed. The available  in vitro 
    genotoxicity tests indicated that neomycin causes chromosomal
    aberrations. 

         The tumour incidence was not increased in a two-year toxicity and
    carcinogenicity study with rats orally administered neomycin up to a
    dose level of 25 mg/kg bw/day.  In the high-dose males only a slight
    but statistically non-significant impairment of hearing was observed.
    Because the Committee regarded the observed impairment of hearing as
    treatment-related, the NOEL was 12.5 mg/kg bw/day.

         In a multi-generation reproductive toxicity study in rats in
    which neomycin was administered orally, no effects on reproductive
    parameters were observed up to a dose of 25 mg/kg bw/day, which was
    the highest dose administered.  A teratogenicity study with an
    unconventional protocol was conducted with F2b female rats from the
    reproductive toxicity study.  Neomycin was administered in the feed at
    0, 6.25, 12.5 or 25 mg/kg bw/day from days 0-6 and 16-20 of gestation. 
    Dose levels were raised to 62.5, 125 or 250 mg/kg bw/day from days 6-
    15 of gestation.  No malformations, feto-toxicity or maternal toxicity
    were observed.

         Skin reactions due to hypersensitivity have been observed in
    humans after the therapeutic use of neomycin. Nephrotoxic effects and
    ototoxicity have been observed in humans after oral therapeutic use of
    neomycin.

         From several  in vitro microbiological studies with different
    bacteria, mostly isolated from humans, an MIC50 of 64 g/ml was
    derived for the most relevant sensitive bacterial organisms
    ( Escherichia coli and  Lactobacillus spp.) under conditions of
    high inoculum density. In human-flora-associated mice the NOEL for
    antibacterial activity was 125 mg/kg bw/day.  Studies on the effect of
    neomycin on human gut flora in patients revealed effects at oral doses
    >30 mg/kg bw/day.

         Applying the formula developed at the thirty-eighth meeting of
    the Committee (Annex 1, reference 97) an ADI based on anti-microbial
    activity could be calculated as follows:

                             Concentration without effect                       daily faecal
                         of ADI on human gut flora (g/ml)a        x        bolus (g)

    Upper limit     =                                                                             
    of ADI               fraction of oral       x  safety          x        Weight of human (60kg)
                         dose availableb           factorc

                         64  x  150
                    =             
                         1 x 1 x 60

                    =    160 g/kg bw

    a    The MIC50 value of 64 g/ml was measured in the most relevant
         sensitive species under conditions of high inoculum density.  No
         adjustment was deemed necessary.

    b    A conservative estimate of 100% availability to human gut flora
         was selected. Experimental data were inadequate to correct for
         inactivation of neomycin as a result of binding to gut contents.

    c    A substantial amount of data covering a variety of organisms,
         including anaerobes isolated from the human gut, were available.
         Also in view of the applied conservative factor for the availability
         of neomycin to the gut flora a safety factor of 1 was adopted.
    
         In reviewing the available toxicological and antimicrobial data
    the Committee concluded that the toxicological data provided the most
    appropriate endpoint for the evaluation of neomycin.

         Only a limited set of genotoxicity tests was available, with gene
    mutation studies in eukaryotic cells being absent.  The available
    information indicates that neomycin causes chromosomal aberrations.
    However, the Committee noted that the long-term study in rats did not
    provide evidence for a carcinogenic potential of neomycin.

         The Committee established a temporary ADI of 0-30 g/kg bw based
    on the NOEL of 6 mg/kg bw/day for ototoxicity in the guinea-pig and a
    safety factor of 200. The ADI was made temporary in view of the
    deficiencies in the genotoxicity data. 

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    ATEF, M., EL-GENDI, A.Y.I., EL-SAYED, M.G.A. & A.M.M.AMER (1986).
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    DURAN, S.P., VALERA, R.G., MANZANO, J.V. & MACHOTA, V. (1991).
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    EPSTEIN, S. (1965). Neomycin sensitivity and atopy.  Dermatology,
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    EPSTEIN, S. & WENTZEL, F.J. (1962). Cross-sensitivity to various
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    FEENSTRA, E.S. (1950). Interoffice memorandum of pathology report -
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    See Also:
       Toxicological Abbreviations
       Neomycin (JECFA Food Additives Series 51)
       Neomycin (WHO Food Additives Series 38)
       NEOMYCIN (JECFA Evaluation)