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    TILMICOSIN

    First draft prepared by
    Dr G. Roberts
    Commonwealth Department of Health and Family Services
    Canberra, Australia

    1.   Explanation

    2.   Biological data
         2.1   Biochemical aspects
               2.1.1   Excretion
               2.1.2   Biotransformation
         2.2   Toxicological studies
               2.2.1   Acute studies
               2.2.2   Short-term toxicity studies
               2.2.3   Long-term toxicity/carcinogenicity studies
               2.2.4   Reproductive toxicity studies
               2.2.5   Special studies on embryotoxicity and
                       teratogenicity
               2.2.6   Special studies on genotoxicity
               2.2.7   Special study on the immune system
               2.2.8   Special studies on microbiological activity
               2.2.9   Special studies on pharmacology
         2.3   Observations in humans

    3.   Comments

    4.   Evaluation

    5.   References

    1.  EXPLANATION

         Tilmicosin is a macrolide antibiotic with the chemical name of
    20-deoxo-20-(3,5-dimethyl piperidin-1-yl) desmycosin. It is
    structurally similar to tylosin. Tilmicosin is a mixture of one cis
    and two trans isomers in the approximate ratio 85:15.

         Tilmicosin is available as an injectable formulation for the
    treatment of respiratory diseases in cattle and sheep (10 mg/kg bw)
    and as a feed premix for the treatment and control of respiratory
    diseases in pigs (200 to 400 mg/kg in the feed). It had not been
    previously evaluated by the Committee. The molecular structure of
    tilmicosin is shown below.

    CHEMICAL STRUCTURE 6

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Excretion

         Pigs were administered a dose of 110 mg 14C-tilmicosin in the
    diet over the course of one day. The recovery of radioactivity was 15%
    in the urine and 80% in the faeces (Giera & Thomson, 1986).

         In two studies, pigs were given a dose of 154 or 400 mg
    14C-tilmicosin in the diet following a similar dose given for 5
    days. The recovery of radioactivity was 4 to 6% in urine and 62 to 75%
    in faeces. Radioactivity was detected in the bile but was not
    quantified (Donoho & Thomson, 1988; Donoho  et al., 1993).

    2.1.2  Biotransformation

    2.1.2.1  Rats

         Tilmicosin, labelled with 14C in both the desmycosin macrolide
    ring and the piperidine ring, was given orally to 15 male and 15
    female Fischer-344 rats. The dosage was 20 mg/kg bw per day for 3
    days. In the liver, radiolabel corresponded to tilmicosin and a
    desmethyl derivative, T1(demethylated in the mycaminose ring). The
    single radioactive substance identified in urine was unchanged
    tilmicosin, while in faeces the major peak was parent compound with
    lesser amounts of desmethyl tilmicosin and a high molecular weight
    compound known to be present as an impurity in the administered
    substance, T2 (consisting of two macrolide rings and one piperidine
    ring) (Donoho, 1988).

         Fischer-344 rats (10 males and 10 females) were given gavage
    doses of 50 mg/kg bw per day 14C-tilmicosin for 5 days. An analysis
    of faecal radioactivity for the presence of the sulfate metabolite
    that was found in the faeces of pigs revealed the presence of a
    similar compound, but quantification was not undertaken (Donoho &
    Kennington, 1993).

    2.1.2.2  Sheep

         Beulah cross lambs were administered a single subcutaneous dose
    of 20 mg/kg bw per day 14C-tilmicosin. The major radioactive
    component in the liver, kidneys and urine was the parent drug,
    together with lesser amounts of T1 and T2, and minor amounts of other
    unidentified substances (Hawkins  et al., 1993).

    2.1.2.3  Pigs

         Pigs were fed diets containing 14C-tilmicosin, which resulted
    in daily doses of 400 mg, for 5 days. Of the radiolabel in liver and
    kidney, approximately 60 to 70% was in the form of the parent drug and
    there were small amounts of T1. Similarly in urine and faeces, there
    were high levels of tilmicosin and low levels of T1. A further
    metabolite was detected that comprised 14% of faecal and 25% of
    urinary radioactivity and that was consistent with reduction of one
    double bond in the macrolide ring followed by sulfation
    (Donoho  et al., 1993).

    2.1.2.4  Cattle

         In a summary of results obtained in cattle injected with
    14C-tilmicosin, it was reported that the radioactivity profile in
    the liver of treated rats was similar to that in the faeces. In
    animals treated with a highly purified sample of tilmicosin,
    metabolite T2 was not detected in the liver, suggesting that its
    presence was a result of direct administration as a component of the
    drug substance. Radioactivity in the kidneys was essentially in the
    form of unchanged tilmicosin (Donoho, 1988).

    2.2  Toxicological studies

    2.2.1  Acute studies

         Major signs of toxicity in mice and rats were leg weakness,
    hypoactivity, lethargy, ataxia, poor grooming and coma. Monkeys given
    20 mg/kg vomited during the first day but were subsequently normal.
    The single monkey given 30 mg/kg vomited, exhibited hypoactivity,
    laboured respiration, vocalization and ataxia, and died within 2
    hours.

         New Zealand white rabbits (five males and five females) received
    5000 mg/kg bw tilmicosin onto clipped skin for a period of 24 hours,
    under a non-occlusive dressing. There were no deaths and no signs of
    skin irritation (Jordan  et al., 1987).

         A volume of 0.1 ml (17 mg) of tilmicosin was instilled into one
    eye of three male and three female New Zealand White rabbits.
    Conjunctival hyperaemia and chemosis were noted for several days,
    clearing by the end of one week (Jordan  et al., 1987).

         Hartley albino guinea-pigs (6 to 12 females) were given 10
    intracutaneous induction doses of tilmicosin (0.05 to 0.1 ml of a
    50 mg/ml solution). Fourteen days after the induction phase each
    animal was injected intracutaneously with 0.05 ml tilmicosin. This
    challenge dose did not result in an enhancement of skin reactions,
    suggesting that a skin sensitization response had not been elicited
    (Jordan et  al., 1989b).

    2.2.2  Short-term toxicity studies

    2.2.2.1  Rats

         Groups of 20 male and 20 female Fischer-344 rats were given
    gavage doses of 0, 50, 175 or 600 mg/kg bw per day tilmicosin
    (purity 88%) in an aqueous vehicle. Drug administration was continued
    for 2 weeks, during which there was no mortality or clinical signs of
    toxicity. Food consumption was lower in males and females at 600 mg/kg
    and body weight gain was depressed in males throughout the study and
    in females during the first few days only.

        Table 1.  Results of acute toxicity studies with tilmicosin
                                                                                              

    Species         Route           Vehicle       Sex          LD503             Reference
    (strain)                                                   (mg/kg bw)
                                                                                              

    Mouse           sc              aqueous       M            97                Jordan
    (ICR)                                         F            109               et al., 1986a

    Rat1            oral            aqueous       M            850               Jordan
    (Sprague-                                     F            800               et al., 1986b
    Dawley)

    Rat2            oral            aqueous       M & F        > 2000            Piroozi
    (Fischer-344)                                                                et al., 1993

    Rat             sc              aqueous       M            185               Jordan
    (Fischer-344)                                 F            440               et al., 1986c

    Rat             inhalation      aerosol       M & F        > 494 (0/20)      Jordan
    (Fischer-344)                                              < 4053 (14/20)    et al., 1987

    Rabbit          dermal          -             M & F        > 5000            Jordan
    (NZ White)                                                                   et al., 1987

    Monkey          im              aqueous       ?            > 20 (0/2)        Jordan &
    (Rhesus)                                                   30 (1/1)          Shoufler, 1990

    Sheep           sc              aqueous       ?            > 150 (0/5)       Cochrane &
                    iv              aqueous       ?            > 7.5 (1/5)       Thomson, 1990
                                                                                              

    1    Animals were fasted
    2    Animals were non-fasted
    3    Figures in brackets represent the incidence of deaths
    
         In the 600 mg/kg bw per day groups, there were increases in
    haematocrit owing to an increase in corpuscular volume and in serum
    levels of alanine aminotransferase and hepatic  p-nitroanisole
     O-demethylase activity. Thrombocyte counts were slightly decreased
    at this dose. Urinalysis was unaffected. At the highest dose, there
    were slight decreases in the weights of kidneys, spleen and ovaries,
    increased adrenal weight and multifocal inflammation in the liver of
    some animals. All treated animals exhibited caecal distension, which
    is a typical response to high doses of an antibiotic compound
    (Jordan, 1986).

         Groups of 20 male and 20 female Sprague Dawley (Crl:CD) rats
    received 0, 50, 250 or 1000 mg/kg bw per day tilmicosin (purity 88%)
    in an aqueous vehicle, by gavage for 3 months. Signs of overt toxicity
    were noted at 1000 mg/kg and included thinness, ventral soiling,
    chromorhinorrhea, chromodacryorrhea, alopecia and poor grooming.
    Mortality was increased at this highest dose and in females given
    250 mg/kg bw per day.

         Although food consumption was depressed in the 1000 mg/kg bw per
    day group (males only), body weight gain was lower in males and
    females at 1000 mg/kg bw per day and females at 250 mg/kg bw per day.
    At 1000 mg/kg bw per day, serum alanine aminotransferase was increased
    in males, blood urea nitrogen (BUN) was increased in males and
    females, urinary pH was slightly lower in females and the presence of
    occult blood was greater in males and females. There were no effects
    on ophthalmology, haematology or hepatic  p-nitroanisole
     O-demethylase activity.

         The weights of kidneys, liver and heart were increased in females
    at 1000 mg/kg bw per day and adrenal weight was increased at 250 mg/kg
    bw per day (females only) and 1000 mg/kg bw per day (males and
    females). Gross pathology revealed caecal enlargement or distension at
    250 mg/kg bw per day or more and small spleens in a few rats at
    1000 mg/kg bw per day. Slight nephrosis was noted in two males in each
    of the 250 and 1000 mg/kg bw per day groups. Other pathological
    alterations were seen only in males and females of the 1000 mg/kg bw
    per day group, i.e. hypertrophy of the zona fasciculata of the adrenal
    cortex in most rats, necrosis of individual skeletal muscle fibres in
    some rats; myocardial degeneration was increased and lymphoid
    depletion was evident in the spleen and thymus of some animals. The
    NOEL was 50 mg/kg bw per day (Jordan, 1988).

    2.2.2.2  Dogs

         Beagle dogs (two males and two females per group) were exposed by
    inhalation to aerosols of 0, 12, 47 or 251 mg/m3 tilmicosin
    (purity 83%). The treatment was administered for 4 hours per day on 12
    days out of a 16-day period (excluding weekends). Particle median
    equivalent aerodynamic diameters were between 1.2 and 1.5 m. The mean
    serum concentrations of tilmicosin were 0.23 to 0.33 and 1.67 to
    2.46 g/ml at 47 and 251 mg/m3 respectively. Drug levels at
    12 mg/m3 were below the detection limit of 0.1 g/ml.

         One male given 251 mg/m3 died on the final day of exposure but
    no other overt signs of toxicity were observed. Heart rates were
    increased in dogs at 251 mg/m3. Food intake, body weight,
    haematology and blood chemistry were unaffected. At autopsy, lung
    weight was increased in females given 251 mg/m3 and inflammation was
    noted in the respiratory tract at 47 mg/m3 (females only) and
    251 mg/m3 (males and females) (Jordan  et al., 1991).

         Groups of four male and four female beagle dogs were given 0, 6,
    20 or 70 mg/kg bw per day tilmicosin (purity 88%) in capsules for 3
    months. The daily dose was administered in two equal amounts, 6 hours
    apart. Serum concentrations of tilmicosin were measured 3 hours and 24
    hours after the first of the two daily doses in weeks 2, 3, 5, 9 and
    13. At increasing doses, the mean concentrations were 0.1 to 0.2, 0.74
    to 1.49 and 3.24 to 6.05 g/ml at 3 hours and 0, 0.26 to 0.59 and 1.72
    to 3.96 g/ml at 24 hours.

         Half the 70 mg/kg bw per day males and females died during the
    first month. Clinical signs prior to death were pale mucous membranes
    in two dogs and ataxia in one dog. Food intake, body weight, haemato-
    logy and urinalysis were unaffected. Serum alanine aminotransferase
    activity was progressively increased at 70 mg/kg bw per day, while
    hepatic  p-nitroanisole  O-demethylase activity was increased
    in females at this level.

         Two of the four surviving dogs at 70 mg/kg bw per day showed
    bilateral multifocal areas of subretinal fluid concentrated along
    arterioles in the tapetal region of the eye. The changes were claimed
    to be consistent with those associated with systemic hypertension. One
    of these dogs also had retinal degeneration and the other showed
    miosis with normal pupillary light responses.

         Heart rate increases were dose related, being marked at 70 mg/kg
    bw per day and moderate to severe at 20 mg/kg bw per day. The increase
    in males given 6 mg/kg bw per day was not significant. At the highest
    dose, examination of the electrocardiogram revealed depression of the
    ST segment.

         At 70 mg/kg bw per day, heart weight was increased in males and
    females, and liver and kidney weights were increased in females. Gross
    necropsy showed small spleens in the females that died during the
    study and an enlarged heart in a surviving male at 70 mg/kg bw per
    day. Slight diffuse mucosal oedema was seen in the gall bladders of
    two dogs at 70 mg/kg bw per day. Despite the physiological changes in
    the eyes and heart, there were no associated pathological alterations.
    The NOEL was 6 mg/kg bw per day (Jordan, 1987).

         Groups of four male and four female beagle dogs were given 0, 4,
    12 or 36 mg/kg bw per day tilmicosin (purity 86%) in capsules for 12
    months. The daily dose was administered in two equal amounts, 6 hours
    apart. There were no deaths. Peripheral redness was seen sporadically
    in some animals of all groups, in particular at 12 and 36 mg/kg bw per
    day.

         Body weight gains were lower at > 12 mg/kg bw per day. There
    were no effects on ophthalmology, haematology, blood chemistry and
    urinalysis. Heart rates were markedly increased at 36 mg/kg bw per day
    with sporadic depression of the ST segment in some dogs.

         Heart weight was increased at 36 mg/kg bw per day and four males
    and one female at this dose showed enlarged hearts. Mild chronic
    dermatitis was noted in the external ears of dogs from all treated
    groups. The findings included minimal thickening of the epidermis,
    foci of acantholysis and inflammatory cell infiltration in the dermis.
    These changes were only slight and non-dose-related, and therefore of
    questionable relationship to treatment. The NOEL was 4 mg/kg bw per
    day (Jordan & Bernhard, 1989).

    2.2.3  Long-term toxicity/carcinogenicity studies

         Tilmicosin has not been tested in toxicological studies longer
    than 12 months in duration and hence the carcinogenic potential of
    this drug has not been directly determined. There are a number of
    observations which are relevant in assessing the carcinogenicity of
    tilmicosin:

    a)   The results of available toxicological studies with tilmicosin
         have not identified lesions or proliferative changes which could
         be considered suggestive of potential neoplasia.

    b)   Tilmicosin has been tested in a wide range of genotoxicity
         assays. All results were negative and it was concluded that the
         compound has no genotoxic activity.

    c)   Tilmicosin is a macrolide antibiotic. This class of chemical has
         been in widespread usage in humans for many years but there is no
         evidence of carcinogenicity. Tylosin is the closest structural
         analogue and this chemical was reviewed by the thirty-eighth
         meeting of JECFA (Annex 1, reference 97).

    2.2.3.1  Reconsideration of tylosin tumorigenicity

         Tylosin was tested in 2-year feeding studies in rats. The
    findings revealed an association between drug administration at doses
    of 150 and 300 mg/kg bw per day in the diet and an increased incidence
    of pituitary adenomas in males. While it was claimed that the increase
    in the incidence of tumours was an indirect result of the ability of
    tylosin to increase survival and weight gain, supporting data were not
    available to the Committee and an ADI was not established.

         New information, not considered at the thirty-eighth meeting,
    indicates that spontaneous pituitary tumours occur in male rats at
    variable rates with increased rates occurring in animals with higher
    body weights (Gries & Young, 1982). In the tylosin studies, 12-month
    body weights were somewhat higher in treated males rats and mortality
    was increased in control animals due to respiratory infection. The
    highest observed incidence of tumours in tylosin-treated groups was
    comparable to the upper limit of the historical control range (23%) in
    the test facility.

    2.2.4  Reproductive toxicity studies

    2.2.4.1  Rats

         A dose-ranging study was carried out in groups of 10 female
    Sprague-Dawley (Crl:COBS CD) rats given gavage doses of 0, 50, 125,
    250, 500 or 750 mg/kg bw per day tilmicosin (purity unknown). The
    females were treated from 14 days before mating with untreated males
    until the end of study on post-partum day 4.

         Excess salivation was dose-related at > 250 mg/kg bw per day.
    Chromodacryorrhea, urine-stained fur and alopecia were seen in some
    rats at 750 mg/kg bw per day; the three most severely affected died
    during the study. Prior to death, these animals exhibited reduced food
    intake, lost weight and were emaciated. Body weight gain was depressed
    in surviving rats at 750 mg/kg bw per day during the pre-mating period
    while food intake was reduced at > 250 mg/kg bw per day in week 1
    and at 750 mg/kg bw per day in week 2. The pregnancy rate was
    decreased at > 500 mg/kg bw per day. Duration of gestation, litter
    size and weight, pup survival and weight gain to post-partum day 4
    were unaffected (Dearlove  et al., 1987).

         Groups of 30 male and 30 female Sprague Dawley (Crl: COBS CD BR)
    rats were administered 0, 10, 45 or 200 mg/kg bw per day tilmicosin
    (purity 87%) by gavage in an aqueous vehicle. Treatment commenced 70
    days (males) and 14 days (females) before the first mating period and
    was continued through two litters per generation for two generations.
    F1 litters were culled to five pups per sex on post-partum day 4 and
    F1b pups (40 males and 40 females per group) were bred to produce
    the following generation. F2a and F2b pups were killed on
    post-partum day 4.

         In adult animals, salivation was noted in males and females at
    200 mg/kg bw per day, but there were no other signs of toxicity. Body
    weight gain and food consumption were depressed in females of the 45
    and 200 mg/kg bw per day groups during the pre-mating period only.
    During the production of the four litters in this study there were no
    effects on mating performance, pregnancy rates, duration of gestation,
    litter size and weight and the weight gain of offspring. In both F1
    litters at 200 mg/kg bw per day, pup mortality was slightly increased
    up to post-partum day 4, but the finding was not duplicated in either
    F2 litter. The NOEL was 10 mg/kg bw per day in adult rats (Christian
    & Hoberman, 1989).

    2.2.5  Special studies on embryotoxicity and teratogenicity

    2.2.5.1  Rats

         Groups of 25 presumed pregnant female Sprague Dawley (Crl: CD)
    rats were administered gavage doses of 0, 10, 70 or 500 mg/kg bw per
    day tilmicosin (purity 86%) in an aqueous vehicle. The dams were
    treated on gestation days 6 to 15 and were killed on gestation day 20.
    Increased salivation was seen at 70 and 500 mg/kg bw per day and
    alopecia at 500 mg/kg bw per day. There were no deaths or abortions.
    Body weight gain was reduced at 70 and 500 mg/kg bw per day, and food
    intake was reduced at 500 mg/kg bw per day, during gestation days 6 to
    10.

         The number of resorption or live fetuses, fetal weight, sex ratio
    and the incidence of fetal malformations were similar between groups.
    The incidences of total skeletal and visceral anomalies were increased
    in treated groups, but there was no dose-response relationship and the
    findings were within the historical control values for the laboratory.
    The NOEL for maternotoxicity was 10 mg/kg bw per day (Jordan & Higdon,
    1988).

    2.2.5.2  Rabbits

         Groups of 15 presumed pregnant female Japanese White-NIBS rabbits
    were given gavage doses of 0, 8, 19 or 48 mg/kg bw per day tilmicosin
    (purity unknown) in an aqueous vehicle. Does were treated on gestation
    days 6 to 18 and were killed on gestation day 28. One female given
    48 mg/kg bw per day aborted on gestation day 26 and died. Reduced
    faeces were seen at 19 and 48 mg/kg bw per day with only a transient
    effect at 8 mg/kg bw per day. During the treatment period food intake
    was depressed in a dose-related manner and body weight loss was
    observed at 19 and 48 mg/kg bw per day.

         Fetal and placental weight tended to decrease at 19 and 48 mg/kg
    bw per day but did not achieve statistical significance. There were no
    meaningful effects on the incidence of resorptions or fetal deaths or
    on sex ratio. Open eyelids were observed in 11/91 and 16/68 fetuses
    from the 19 and 48 mg/kg bw per day groups, respectively, and some of
    these fetuses showed cleft palate or club foot. The affected fetuses
    had low body weights and the seven litters involved were derived from
    does that had lost body weight during drug administration. Skeletal
    examination revealed retardation of fetal development at 19 and
    48 mg/kg bw per day. Similar effects have been seen in this laboratory
    in dietary restricted rabbits, suggesting the effects on the fetus
    were secondary to maternal malnutrition (Noda, 1993).

    2.2.6  Special studies on genotoxicity

        Table 2.  Results of genotoxicity studies on tilmicosin
                                                                                              

    Test system1        Test object           Concentration        Results       Reference
                                                                                              

    Reverse             S. typhimurium        1 - 100 g/plate     negative      Jordan
      mutation2         TA98, TA100,          ( S9)                             et al., 1986f
                        TA1535, TA1537,
                        TA1538

    Reverse             E. coli               1 - 100 g/plate     negative      Garriott
      mutation2         WP2uvrA               ( S9)                             et al., 1993

    Forward             L5178Y mouse          100-900 g/ml        negative      Jordan
      mutation2         lymphoma cells        (- S9)                             et al., 1986e
                                              200-1000 g/ml
                                              (+ S9)

    Forward             HGPRT+ Chinese        25-250 g/ml         negative      Jordan
      mutation2         Hamster ovary         (- S9)                             et al., 1989c
                        cells                 50-300/ g/ml
                                              (+ S9)

    Unscheduled         primary cultures      0.5-10 g/ml         negative      Jordan
      DNA synthesis     of rat hepatocytes                                       et al., 1985
      assay

    Sister chromatid    Chinese hamster       200-1800  1         negative      Jordan et al.,
      exchange          bone marrow           mg/kg bw oral                      1986d, 1989a
      assay

    Chromosome          rat bone marrow       180-1800  1 and     negative      Jordan &
      aberrations                             100-1000  5,                      Ivett, 1989
                                              mg/kg bw per
                                              day oral
                                                                                              

    1    Positive controls used
    2    Both with and without liver microsomal activation
    
    2.2.7  Special study on the immune system

         Groups of eight male CD-1 mice were given gavage doses of 0,
    10, 250, 500 or 1000 mg/kg bw per day tilmicosin (purity 87%) in
    an aqueous vehicle. Drug treatment was continued for 10 days.
    After the third dose mice were injected intravenously with sheep
    red blood cells. At the end of the study, serum from each mouse
    was assayed for antibodies (haemagglutinin). There was one death
    at 500 mg/kg bw per day and six at 1000 mg/kg bw per day, but
    antibody production was not affected at any level (McGrath
     et al., 1988a).

    2.2.8  Special studies on microbiological activity

    2.2.8.1  In vitro

         The antibacterial activity of tilmicosin was determined
    against a range of organisms representative of human intestinal
    flora. Cultures were initiated using inoculum sizes of between
    3  105 and 1.6  106 cells per spot on blood agar plates and
    incubated anaerobically at 37C.

         The median MIC values for the clinical isolates of
     Bifidobacterium and  Peptostreptococcus, respectively, were
    0.015 and 0.5 g/ml. The above results were obtained at pH 7.7.
    At pH 6.6 the MIC values were one to two orders of magnitude
    higher (Scott  et al., 1993).

         The antibacterial activity of tilmicosin was examined
    against a number of organisms used in industrial food processing
    and originally obtained from dairy products. Cultures were
    initiated in blood agar plates using three strains of
     Propionibacterium and four strains of  Lactobacillus. Under
    anaerobic conditions the MICs were between 8 and 64 g/ml and
    under microaerophilic conditions the values were between 4 and
    32 g/ml (McLaren, 1994).

    2.2.8.2  Rats

         In two separate studies, female germ-free rats were orally
    dosed with 1 ml of 10% (w/v) pooled human faecal suspension.
    Three weeks later groups of two males and two females were given
    gavage doses of 0, 0.12 or 0.4 mg/kg bw per day tilmicosin
    (purity 87%) for 5 days. Rat faecal samples were collected before
    treatment, daily during treatment and once during each of the 2
    weeks following cessation of dosing. Body weight gain remained
    unaffected.

         The animals in each study received faecal suspensions
    obtained from different donors. Results from different studies
    varied widely, possibly reflecting differences between the
    bacterial populations used. Total anaerobes were not reduced by
    tilmicosin treatment. Total enterobacterial counts and the
    proportion of enterobacteria with respect to total anaerobe
    counts were transiently increased at 0.4 mg/kg bw per day in one
    study only. Tilmicosin-resistant enterobacteria were not
    significantly increased in either study. Spiramycin, at a dose of
    0.5 mg/kg bw per day, increased total enterobacterial counts and
    the number of spiramycin-resistant enterobacteria; the latter
    appeared to remain high after the cessation of treatment. The
    NOEL for tilmicosin was 0.4 mg/kg bw per day (Rumney, 1993).

    2.2.9  Special studies on pharmacology

        Table 3.  Results of pharmacological assays with tilmicosin
                                                                                              

    Test system      Doses          Results                                         Reference
                                                                                              

    Isolated         0.0009 to      No effect on non-stimulated organ.              Williams
    guinea-pig       90 g/ml       90 g/ml slightly inhibited acetylcholine-      et al.,
    ileum                           and angiotensin-induced contraction             1988
                                    and significantly inhibited electrically
                                    stimulated contraction

    Isolated         0.0009 to      No effect on non-stimulated tissue and          Williams
    rat uterus       90 g/ml       no change to oxytocin-induced                   et al.,
    (estrogen                       contractions                                    1988
    primed)

    Isolated rat     0.0009 to      No effect on non-stimulated tissue and          Williams
    vas deferens     90 g/ml       no change to noradrenaline-induced              et al.,
                                    contractions                                    1988

    Isolated         0.0009 to      In spontaneously beating tissue, > 90           Williams
    guinea-pig       900 g/ml      g/ml decreased force of contraction;           et al.,
    atria                           and 900 g/ml increased rate of                 1988;
                                    contraction. 90 g/ml inhibited                 Jordan
                                    isoprenaline-, noradrenaline- and Bay           et al.,
                                    K8644 (calcium agonist)-induced force           1990
                                    and rate of contraction, and
                                    histamine-induced rate of contraction.
                                                                                              

    Table 3.  Results of pharmacological assays with tilmicosin (cont'd).
                                                                                              

    Test system      Doses          Results                                         Reference
                                                                                              

    Conscious        0.25, 1, 2.5   Heart rate increased at > 1 mg/kg. Left         Jordan &
    dogs             or 5 mg/kg     ventricular function decreased at > 0.25        Sarazan,
                     iv             mg/kg. Aortic pulse pressure decreased          1991;
                                    at > 1 mg/kg. ECG revealed "Ventricular         Sarazan
                                    Alternans" (ventricular contraction fails       et al.,
                                    to achieve adequate pressure to open            1993
                                    aortic valve and cause an arterial pulse).

    Anaesthetised    0.5, 1 or 5    Heart rate increased, stroke volume,            Jordan
    dogs             mg/kg iv       stroke work index and cardiac output            et al.,
                                    decreased at > 1 mg/kg. At 5 mg/kg 2/3          1988
                                    dogs developed second degree heart block

    CD-1 mice        10, 100,       Piloerection and grasping loss at > 500         Jordan
                     500 or         mg/kg. Ptosis at 1000 mg/kg. No effect          et al.,
                     1000 mg/kg     on electroshock- or pentylenetetrazol-          1989d
                     oral           induced seizures or on body temperature.
                                    Hexobarbital-induced sleep time
                                    prolonged by 500 (x1) and 1000 (x3) mg/kg

    Sprague-         10, 30, 90     Urine volume and creatinine reduced             McGrath
    Dawley           or 270         at 270 mg/kg with increases in osmolality       et al.,
    rats             mg/kg oral     and electrolytes.                               1988b
                                                                                              
    
    2.3  Observations in humans

         A total of 241 human exposures to tilmicosin (Micotil) were
    reported to the Rocky Mountain Poison Center from May 1992 to May
    1993. Needlesticks and scrapes (n=112) and accidental injection
    (n=43) caused either no effect or local reactions including
    soreness, numbness, stinging, swelling, redness, burning and
    stiffness. Some injected subjects experienced anxiety, sweating,
    headache and lightheadedness. Skin exposures (n=50) resulted in
    redness and tingling of the skin, and eye exposures (n=11)
    resulted in stinging and swelling. Persons accidentally ingesting
    the drug (n=39) reported either no symptoms or bitter taste,
    nausea, numbness of lips and tongue, vomiting, thirst and
    headache (Montanio & Dart, 1993).

         Over a 30-month period, 36 cases of accidental exposure to
    tilmicosin (Micotil) were reported to the Ontario Regional Poison
    Information Centre. Percutaneous injection (n=26) always resulted
    in pain at the site and seven subjects mentioned a variety of
    local reactions consistent with an irritant action. One person
    showed "peaked T waves" 30 minutes following the injection of
    1 ml into his arm, but ECG changes were not noted in other
    subjects. The remaining subjects received splashes into the mouth
    or eyes or onto the skin. Unpleasant taste, a burning sensation
    on the hard palate and ocular irritation were each recorded in
    one person (McGuigan, 1994).

    3.  COMMENTS

         The Committee considered toxicological data on tilmicosin,
    including the results of studies on acute and short-term
    toxicity, pharmacokinetics, metabolism, reproductive toxicity,
    teratogenicity, genotoxicity, antimicrobial activity and
    pharmacology. The Committee also considered observations in
    humans accidentally exposed to tilmicosin.

         Tilmicosin is absorbed from the gastrointestinal tract as
    shown by the recovery of radiolabelled compound in urine and/or
    bile of orally dosed pigs (110 to 400 mg per animal), and the
    presence of dose-related serum concentrations of tilmicosin dosed
    orally in dogs for 3 months. However, the results from available
    studies did not allow any conclusion to be reached concerning the
    extent of absorption. The metabolites detected in a range of farm
    animals were also found in rats, which suggests that the rat is a
    suitable model for determining the potential toxicological
    hazards posed by tilmicosin.

         The LD50 in fasted rats was 800 to 850 mg/kg bw, but
    toxicity was substantially lower in non-fasted animals, among
    which there were no deaths following administration of a single
    dose of 2000 mg tilmicosin/kg bw. Studies in a range of
    pharmacological models identified depression of cardiac muscle
    contractility and reduced heart function as the main adverse
    effects.

         Rats were administered oral doses of 50, 250 or 1000 mg/kg
    bw per day for 3 months. At 1000 mg/kg bw per day, animals
    exhibited chromorhinorrhea, chromodacryorrhea, alopecia, poor
    grooming, reduced food consumption and they appeared thin.
    Body-weight gain was reduced and mortality was increased in
    females at 250 mg/kg bw per day and in both sexes at 1000 mg/kg
    bw per day. Toxic effects on the kidney and liver were indicated
    by increased organ weights, increased serum levels of alanine
    aminotransferase and blood urea nitrogen, and the presence of
    blood in the urine. However, pathological examination of these
    organs showed only slight nephrosis in two males in each of the
    groups given 250 or 1000 mg/kg bw per day. At the highest dose,
    hypertrophy of the zona fasciculata of the adrenal cortex was
    noted in most rats, and myocardial degeneration, necrosis of
    individual skeletal muscle fibres and lymphoid depletion in the
    spleen and thymus were seen in some rats. The NOEL was 50 mg/kg
    bw per day.

         Dogs were given oral doses of 6, 20 or 70 mg/kg bw per day
    for three months. Half of the animals given 70 mg/kg bw per day
    died during the first month. Heart rate was increased at 20 and
    70 mg/kg bw per day and at the higher dose there was depression
    of the ST segment of the electrocardiogram. Ophthalmoscopic
    examination of the eyes showed bilateral areas of subretinal
    fluid concentrated along arterioles in the tapetal region of two
    of the four surviving dogs at 70 mg/kg bw per day. The NOEL was
    6 mg/kg bw per day.

         Dogs were given oral doses of 4, 12 or 36 mg/kg bw per day
    for twelve months. Body weight gain was depressed at 12 and
    36 mg/kg bw per day. Heart rate was markedly increased at 36
    mg/kg bw per day, with occasional depression of the ST segment
    of the electrocardiogram. Hearts were enlarged at 36 mg/kg bw per
    day, but there were no associated pathological changes. The NOEL
    was 4 mg/kg bw per day.

         In a two-generation reproductive toxicity study, rats were
    administered oral doses of 10, 45 or 200 mg tilmicosin/kg bw per
    day. Body weight gain and food consumption were depressed in
    females given 45 or 200 mg/kg bw per day, but only during the
    pre-mating period. Fertility and reproduction parameters were
    unaffected at any dose level. At 200 mg/kg bw per day, mortality
    in pups was slightly increased in both first-generation litters
    up to post-partum day 4, but not in either second-generation
    litter. The NOEL was 10 mg/kg bw per day.

         Developmental toxicity studies were reported in rats and
    rabbits. Rats were dosed with 10, 70 or 500 mg tilmicosin/kg bw
    per day by gavage. Growth and development in rat fetuses were not
    affected by treatment, but increased salivation and reduced body
    weight gain of dams were observed at 70 and 500 mg/kg bw per day.
    The NOEL for maternal toxicity in rats was 10 mg/kg bw per day.
    In the study in rabbits, there were dose-related signs of
    toxicity in the does at all doses (8, 19 and 48 mg/kg bw per
    day). Severely affected members of the groups given 19 or
    48 mg/kg bw per day produced offspring that had open eyelids and
    low incidences of cleft palate or club foot. These fetuses had
    low body weights and were clearly retarded in development. The
    effects were attributed to treatment-related malnutrition in
    maternal animals, which is commonly observed in rabbits dosed
    with antibiotics, confirming that this species is an inappropriate
    model for studying the teratogenic potential of antibiotics.

         Tilmicosin has been tested for reverse mutation in bacteria,
    forward mutation in cultured mammalian cells, unscheduled DNA
    synthesis in primary cultures of rat hepatocytes, and in  in vivo
    cytogenetic assays. All results were negative, and the Committee
    concluded that tilmicosin has no genotoxic potential.

         The carcinogenic potential of tilmicosin has not been
    tested. However, a number of factors are relevant in assessing
    the carcinogenicity of this drug. Toxicity studies with
    tilmicosin have not resulted in lesions or proliferative changes
    suggestive of neoplasia, and tilmicosin was uniformly negative in
    a wide range of genotoxicity assays. Tilmicosin is a macrolide
    antibiotic, and this class of chemicals is not known to induce
    neoplasia despite widespread use in humans over many years. The
    closest structural analogue, tylosin, was reviewed at the
    thirty-eighth meeting of the Committee (Annex 1, reference 97).
    In a 2-year feeding study in rats, tylosin administration was
    associated with an increased incidence of pituitary adenomas in
    male animals when compared to concurrent controls. New evidence,
    not considered at the thirty-eighth meeting, indicates that
    spontaneous pituitary neoplasms occur in male rats at variable
    rates. Additionally, increased rates occur in animals with higher
    body weights. In tylosin-treated groups of male rats, the
    12-month body weights were somewhat higher than in the control
    groups and the incidences of pituitary adenomas were comparable
    to the upper limit of the historical control range. The lower
    incidence of pituitary neoplasms in the control animals may have
    been due to the earlier mortality in this group caused by
    respiratory disease. In light of this information the Committee
    considered that the concerns of the thirty-eighth meeting over
    the potential tumorigenicity of tylosin had been satisfactorily
    addressed and that there was no concern for the carcinogenic
    potential of tylosin. For the above reasons, the Committee
    considered that carcinogenicity studies would not be required for
    tilmicosin.

         In assessing the microbiological activity of tilmicosin, the
    results of a study using germ-free rats colonized with human
    intestinal microflora was considered the most relevant. At the
    highest dose administered (0.4 mg/kg bw per day for 5 days) there
    were no significant alterations in numbers of total anaerobes or
    enterobacteria in rat faeces.

         Reports of accidental human exposure to tilmicosin have
    identified local reactions indicative of an irritant action
    following dermal and ocular exposure. Humans accidentally
    injected with tilmicosin have reported anxiety, sweating,
    headache and lightheadedness. Changes in the electrocardiogram
    pattern were observed in only one person. Contact with the buccal
    mucosa or ingestion has resulted in a range of symptoms including
    nausea, vomiting, thirst, numbness or burning sensation in the
    mouth, and headache.

    4.  EVALUATION

         The NOEL from toxicological studies was 4 mg/kg bw per day
    in a 12-month study in dogs. Treatment of rats colonized with
    human intestinal microflora with 0.4 mg tilmicosin/kg bw per day
    produced no significant microbiological effect. The Committee
    established an ADI of 0-40 g/kg bw, based on the toxicological
    NOEL of 4 mg/kg bw per day and a safety factor of 100. An
    identical ADI would have been established using the data from the
    study on rats colonized with human intestinal microflora and a
    safety factor of 10 to account for variability among humans.

    5.  REFERENCE

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    Donoho, A.L. (1988). Comparative metabolism of 14C-tilmicosin
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    Donoho, A.L. & Thomson, T.D. (1988). 14C-Tilmicosin balance-
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    Donoho, A.L., Darby, J.M., Helton, S.L., Sweeney, D.J.,
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    Garriott, M.L., Rexroat, M.A., & Jordan, W.H. (1993). The effect
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    Giera, D.D. & Thomson, T.D. (1986). Preliminary 14C EL-870
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    Gries, C.L. & Young, S.S. (1982). Positive correlation of body
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    Jordan, W.H. (1988). The toxicity of tilmicosin given orally to
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    Jordan, W.H. & Higdon, G.L. (1988). A teratology study of
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    Jordan, W.H. & Ivett, J.L. (1989). Mutagenicity test on
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    Jordan, W.H. & Sarazan, R.D. (1991). An acute study of the
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    Jordan, W.H. & Shoufler, J.R. (1990). The acute toxicity of
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    Jordan, W.H., Hill, L.E., & Probst, G.S. (1985). The effect of
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    in primary cultures of adult rat hepatocytes. Unpublished studies
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    Jordan, W.H., McKinley, E.R., Brown, G.E., & Hawkins, D.R.
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    Jordan, W.H., McKinley, E.R., Brown, G.E., & Hawkins, D.R.
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    Jordan, W.H., McKinley, E.R., Brown, G.E., & Hawkins, D.R.
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    Jordan, W.H., Neal, S.B., & Probst, G.S. (1986d). The effect of
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    Jordan, W.H., Oberly, T.J., Bewsey, B.J., & Probst, G.S. (1986e).
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    Jordan, W.H., Rexroat, M.A., & Probst,G.S. (1986f). The effect of
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    Jordan, W.H., Richardson, K.K., & Oberly, T.J. (1989c). The
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    Scott, R.J.D., Morgan, J.M., Pether, J.V.S., & Gosling, P.
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    Williams, P.D., Jordan, W.H., & Colbert, W.E. (1988).  In vitro
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    Laboratories. Submitted to WHO by Lilly, Basingstoke, UK.
    


    See Also:
       Toxicological Abbreviations
       TILMICOSIN (JECFA Evaluation)