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    TYLOSIN

    First Draft prepared by
    Dr.F.X.R. van Leeuwen
    Toxicology Advisory Centre, National Institute of
    Public Health and Environmental Protection
    Bilthoven, The Netherlands

    1.  EXPLANATION

         Tylosin is a macrolide antibiotic produced by a strain of
     Streptomyces fradiae. The compound is active against most
    gram-positive bacteria, mycoplasma and certain gram-negative
    bacteria. The antibiotic is used in animal feed and veterinary
    medicine. The chemical structure of tylosin, and of certain of its
    metabolites, is shown at Figure I.

    FIGURE I

         Tylosin was evaluated at the 12th meeting of the Joint FAO/WHO
    Expert Committee on Food Additives in 1968 (Annex 1, reference 17).
    No ADI was established. It was concluded that tylosin used in animal
    feed or in veterinary medicine should not give rise to detectable
    residues in edible products of animal origin; when using the
    recommended methods of analysis it will be possible to ensure that
    residues in meat for human consumption not exceed 0.2 ppm. Since
    that time additional data have become available which are summarized
    and discussed in this monograph addendum (Annex 1, reference 17). 

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

         Fasted rats received a single oral dose of 50 mg/kg b.w.
    tylosin base or tylosin tartrate. Tylosin activity was assayed in
    serum after 15 and 30 minutes and 1, 2, 4, 5, 7, and 24 hours after
    treatment. Peak serum levels < 1.0 mg/l were seen after 1-2
    hours. Within 7 hours serum levels decreased to less than the limit
    of detection (i.e. 0.10 mg/l, microbiological assay). Four rats were
    given i.p. 100 mg/kg b.w. tylosin base. Bile samples were collected
    for 2 hours. The bile/serum concentration ratio ranged from 143-266
    (Anderson et al., 1966).

         Rabbits received i.m. 10 mg/kg b.w. tylosin base as a 5%
    aqueous solution acidified with hydrochloric acid to pH 5.5. Serum
    levels were determined after 1.5, 4, 7, and 24 hours. Peak serum
    levels ranging from 0.57 to 0.88 mg/l were observed after 1.5 hours.
    A similar study was carried out using tylosin tartrate in aqueous
    (25 mg/kg b.w.) as well as in PEG-200 (10 mg/kg b.w.) solutions.
    Peak serum levels at 1.0 hour were 4.7-7.2 and 0.96-1.25,
    respectively. Within 24 hours serum levels were below the limits of
    detection, which were 0.05 mg/l for tylosin hydrochloride and
    0.10 mg/l for tylosin tartrate.

         Two dogs given 25 mg/kg b.w. tylosin base orally in capsules
    excreted 2% of the dose in the urine within 5 hours. Serum
    concentrations were very low, < 0.05 and 3.3 mg/l at 2.5 hours
    (microbiological assay). In another study groups of 8 dogs received
    orally by capsule 1, 10, or 100 mg/kg b.w./day for 8 days. Blood
    levels determined 2 hours after the last dose ranged from
    < 0.15 mg/ml to 9.5 mg/ml mostly ranging with the dose. Two dogs
    received 25 or 100 mg/kg b.w. tylosin base orally by capsule daily
    during 29 days. Serum levels were determined 0, 1, 2, 3, 4, 5, 6,
    and 7 hours after the 1st, 15th, and 29th dose. At 25 mg/kg b.w/day
    peak serum levels (1.4-2.7 mg/l) were seen 2 hours after dosing and
    at 100 mg/kg b.w./day the highest levels (2.7-4.6 mg/l) were seen
    2-5 hours after dosing. No accumulation was observed. One dog given
    i.v. 10 mg/kg b.w. tylosin base (dissolved in a minimal amount of
    hydrochloric acid) excreted a total of 25.2% of the activity in the
    urine. During 5 hours after dosing 13.7% of the dose was recovered
    from bile. The bile/serum concentration ratio varied from 1230 to
    3780. Four dogs were administered 10 mg/kg b.w tylosin base i.v. (as
    an aqueous solution acidified with dilute hydrochloric acid). Blood
    t was calculated as 48 min. Urinary recovery was 18.8% of the
    dose during 6 hours after dosing (15.7% within 2 hours). Serum

    levels of tylosin in 4 dogs receiving 25 mg tylosin base
    intraduodenally at 0.25, 0.5, 1, 2, 3, 4, and 5 hours averaged 0.78,
    1.98, 1.77, 1.94, 0.56, 0.29, and 0.13 mg/l, respectively. Urinary
    recovery was 7.2% of the dose in 5 hours (Anderson, et al., 1966).

         Thirty mg/kg b.w. tylosin tartrate was administered orally by
    gavage or intravenously to groups of pigs (5/group, 30-days old). At
    38 days of age the treatments were crossed over. Tylosin activity
    was measured in blood samples taken at 10 intervals up to 24 hours
    after treatment. After oral administration tylosin activity was
    present in plasma 10 minutes after dosing with a peak concentration
    of 2.4 mg/l at about 1.5 hours. By comparing the areas under the
    curve of the tylosin concentration in blood following the 2 routes
    of administration a biological availability of 22.5% was determined
    (Shionogo & Co. Ltd., 1981).

         Tylosin at 110 mg/kg b.w. (as the granulated phosphate) was
    orally administered to 3 male and 3 female pigs. Tylosin activity
    was assayed in blood samples taken up to 24 hours after dosing.
    Serum activity peaked 1 hour after dosing (average 17.81 mg/l); 24
    hours after dosing tylosin was not detectable (< 0.1 mg/l) (Van
    Duyn & Kline, undated).

         Six pigs (weight 56 kg) were orally given 50 mg/kg b.w. tylosin
    phosphate in water. Blood and tissue samples were taken at intervals
    up to 24 hours. Tylosin levels were detected in serum from 10
    minutes to 8 hours after dosing and peaked at 1 hour (8.53 mg/l).
    Tylosin was widely distributed in the body with tissue
    concentrations in liver and kidney peaking at 1 hour. No activity
    was found in the brain or spinal cord. Highest activity was found in
    bile and urine (Nakamura et al., 1969).

         Young calves (weight range 44.4-59.0 kg) were injected with
    10 mg/kg Tylan 200 either subcutaneously or intramuscularly. The
    rate of tylosin absorption, time to peak concentration and decline
    of serum concentrations were very similar via both routes of
    administration (Thomson, undated a).

         Groups of calves (average weight 60 kg) received 10 mg
    tylosin/kg b.w. subcutaneously or intramuscularly {as formulations
    of tylosin tartrate in water, tylosin tartrate in propylene glycol
    and water and tylosin base in propylene glycol and water (Tylan 200
    injection)}. Another group of calves received the same dose (as
    Tylan 200 injection) intravenously. Tylosin activity in serum was
    measured in blood samples taken at various intervals after
    treatment. Absorption of the formulations containing tylosin
    tartrate was faster after subcutaneous and intramuscular
    administration than absorption of the formulation containing tylosin
    base (Thomson, undated b).

         Holstein calves (1-3 weeks, 38-56 kg) were given 1 or 1 to 5
    daily intramuscular injections of 17.6 mg tylosin/kg b.w. (as Tylan
    200). In all experiments tylosin levels in serum peaked 2 hours
    after administration (average 2.0 mg/l) decreasing to about 0.1 mg/l
    at 36 hours. Peak lung tissue levels were observed at 6 to 24 hours
    post injection varying from 12.6 to 15.7 mg/l. Lung tissue levels
    were greater than serum levels and still detectable (2.2 mg/l) at 48
    hours after administration (microbiological assay) (Van Duyn &
    Folkerts, 1979; Van Duyn & Johnson (undated a); Van Duyn & Handy
    (undated)).

         A serum half-life of 1.62 hours was established in cows given a
    single i.v. injection of 12.5 mg tylosin/kg b.w. (as Tylan 200). The
    apparent specific volume of distribution was 1.10 l/kg indicating no
    specific accumulation (Gingerich et al., 1977).

         Neonatal holstein calves with a natural occuring pneumonia were
    treated intramuscularly with 17.6 mg tylosin/kg b.w. (as Tylan 200)
    daily for 3 consecutive days. Healthy calves were subjected to the
    same treatment. Six hours following the last dose all the calves
    were sacrificed and tylosin activity was measured in the lungs.
    Tylosin distributed equally into both normal and pneumonic lung
    tissue (Thomson, undated a).

         Groups of neonatal healthy calves were fed a milk replacer
    containing 1.0 g tylosin (as tylosin tartrate) for 4, 7, and 10
    days. Serum samples were taken 4 hours after each dose; the animals
    were killed after the final sample had been taken and lung tissues
    were analyzed. Mean serum and lung tissue levels were 0.41, 0.37,
    and 0.42 mg/l, and 1.76, 3.16, and 3.17 mg/l for the 4, 7, and
    10-day treatment groups, respectively. Lung/serum tylosin ratios
    were 7.24, 9.36, and 14.01, respectively (Buck et al., undated).

         Groups of 4 calves (weight 250 kg) received intramuscular
    injections with 10 mg tylosin/kg b.w. (as Tylan 200) for 5 days. The
    calves were killed 2, 4, 6, 12, or 72 hours after the last
    injection. Tylosin activity was measured in serum and lungs. Tylosin
    activity in serum was highest at 4 hours after treatment (1.3 mg/l)
    and was no longer detectable after 72 hours using a microbiological
    assay whose detection limit was 0.05 mg/l. The mean tylosin activity
    in the lungs was 5.9, 5.0, 6.6, 4.4, and 0.6 mg/l at 2, 4, 6, 12,
    and 72 hours after treatment, respectively (Lilly, undated a).

         Four broiler chickens (weight 720 g) were given a single dose
    of 50 mg tylosin/bird (as tylosin tartrate) by stomach intubation.
    Tylosin activity was detected in serum after 0.5 hours and peak
    concentrations of 0.6-4.0 mg/l were found after 2 hours, declining
    to negligible after 24 hours. Repeated oral doses of 50 mg tylosin
    to chickens (weight 2 kg, dosed at 1, 2, and 3 hours) caused serum
    peak levels at 4 hours (about 0.28 mg/l) declining to negligible at
    24 hours (Lilly, undated b, undated c).

         Groups of 6 chickens (5-7 weeks old, surgically prepared)
    received orally 25, 100 or 250 mg tylosin/kg bw (as tartrate). Urine
    and faeces were collected during 72 hours. Peak tylosin levels in
    urine (< 100 mg/l at the 25 mg/kg dose and > 1400 mg/l at the
    250 mg/kg dose) occurred 2-4 hours after dosing and declined rapidly
    thereafter. Peak levels in faeces occurred at 8 hours and varied
    from 300 to 2000 g/g with the dose (Lilly, undated d)

    2.1.2  Biotransformation

         Four male rats, preconditioned on unlabelled tylosin (10 mg/kg
    b.w.) for 3 days, received daily during 5 days by gavage 2 ml of a
    solution containing 14C-labelled (in lactone ring) tylosin base.
    The rats were killed 4 hours after the last dose; 99% of the
    radioactivity was excreted in the faeces and 1% in the urine. The
    greatest part of the excreted residues was found to be tylosin
    factor A, tylosin factor D and dihydrodesmycosin. Less than
    0.25 mg/kg total 14C-residue was found in liver and kidney (Sieck
    et al., 1978b).

         A male pig, preconditioned on feed containing 110 mg/kg
    unlabelled tylosin for 2 weeks, received for 3 days feed with
    110 mg/kg of feed tylosin base 14C-labelled in the lactone ring.
    Four hours after the last dose the pig was killed. The radioactivity
    was excreted 99% in the faeces and 1% in the urine. The majority of
    the excreted residues (15% of the radioactivity in faeces was not
    extractable) was found to be tylosin factor D (33%), tylosin factor
    A (6%) and dihydrodesmycosin. At least 10 minor metabolites (< 5%
    of activity) were present in the excreta. In liver and kidneys
    < 0.25 mg/kg tylosin was found. At least 4 different metabolites
    (of which one was detected as dihydrodesmycosin) were detected in
    liver and kidneys (Sieck et al., 1978b).

         Three pigs received twice daily for 4 days a ration containing
    110 mg tylosin base/kg of feed 14C-labelled in the lactone ring. A
    control pig received the basal diet throughout the experiment. Pigs
    were sacrificed within 4 hours after the last dose. Total
    14C-residues in liver and kidney were < 0.28 mg/kg and in
    muscle and < 0.04 mg/kg in fat. Investigations by TLC of liver
    residues revealed 5 or 6 metabolites. Two of the metabolites could
    be identified as tylosin factor A and dihydrodesmycosin, each
    representing about 5% of the total extractable liver residue. Mass
    spectrometer analysis of the faeces identified 3 metabolites as
    tylosin factor A, tylosin factor D and dihydrodesmycosin (Sieck
    et al., 1978a; Sieck et al., 1980).

    2.2.  Toxicological studies

    2.2.1  Acute toxicity

         The acute toxicity of tylosin formulations and of tylosin are
    given in Tables 1 and 2, respectively.

        Table 1.  The acute toxicity of tylosin formulations

                                                                                                 
    Species     Sex        Route          LD50            LC50           Reference
                                        (mg/kg b.w)       (mg/l)
                                                                                                 

    rat         M&F        oral          > 5001                          Gries et al.,
                                                                         1985a
                M&F        oral          > 0.52           > 1.054        Gries et al.,
                                                          1 hr > 0.65    1985b
                M&F        inhal                                         Gries et al.
                M&F        inhal                                         1985c

    rabbit      M&F        dermal       > 20001                          Gries et al.,
                                                                         1985a
                M&F        dermal       > 20003                          Gries et al.,
                                                                         1985c
                M&F        dermal        > 2.02                          Gries et al.,
                                                                         1985b
                                                                                                 

    1.   administered as granulated tylosin concentrate, a formulation
         containing 26.7% of tylosin base activity as the phosphate salt
    2.   ml/kg b.w. administered as undiluted tylan 200 injection
    3.   administered as tylan soluble, a dry granular formulation, also
         known as tylosin tartrate
    4.   liquid droplet aerosol of tylan 200/injection formulation at
         1.05 mg/litre for 1 hour
    5.   solid particulate aerosol of tylan soluble at 0.6 mg/litre.

    Table 2. The acute toxicity of tylosin
                                                                                  

    Species   Sex        Route        LD50                Reference
                                    (mg/kg b.w.)
                                                                                  

    mouse     F          oral       > 62001               Anderson & Worth,
              -          oral       > 50002               undated
              -          oral       > 62005
              -          i.p.          492.51
              -          i.p.          594.12
              -          s.c.         13545
              -          s.c.          784.11
              -          s.c.       > 25002               Gries et al.,
              -          i.v.          385.71             1983
              -          i.v.          581.73
              -          i.v.          588.84
              -          i.v.          588.95
              F          i.v.       approx 3216

    rat       M          oral       > 62001               Anderson & Worth,
              -          oral       > 50002               undated
              -          oral       > 62005
              -          i.p.         10011
              -          i.p.       > 25005
              -          i.v.          6955               Gries et al.,
              -          s.c.         40831               1985c
              -          s.c.       > 30005
              M&F        oral       > 5005

    dog       M&F        oral       > 8002                Anderson & Worth,
                                                          undated
                                                                                  

    1.   administered as tylosin phosphate
    2.   administered as tylosin base
    3.   administered as tylosin hydrochloride
    4.   administered as tylosin lactate.
    5.   administered as tylosin tartrate.
    6.   administered as 20 mg tylosin/ml of acidified sterile water for
         injection, USP (2.0%)
    

         After oral administration no deaths were recorded at the
    highest dose used; dogs vomited at 800 mg/kg b.w. but not at
    400 mg/kg b.w.; at both these doses the dogs salivated and
    defecated. Intravenous and intraperitoneal administration caused
    depression, prostration, convulsions and death or recovery within 24
    hours.

    2.2.2  Short-term studies

    2.2.2.1  Rats

         Groups of rats (Harlan strain, 5 females/group) received daily
    s.c. injections of 10, 20, 50 or 100 mg/kg b.w. tylosin base as a
    suspension in 5% acacia gum for 1 month. No effects were seen on
    food intake, body weight gain, adrenal weight and terminal blood
    cell counts. Macroscopy and microscopy did not show abnormalities
    (Anderson et al., 1966; study R2-58). Remark: Summary only.

         Groups of rats (Harlan strain, 6/sex/group) received daily s.c.
    injections of 100, 250, 500, or 1000 mg/kg b.w. tylosin tartrate or
    2.5 ml/kg b.w. saline for 1 month. At doses > 250 mg/kg b.w.
    diarrhoea was seen during the first week, regressing to soft stools
    (occasionally seen at 100 mg/kg b.w. too) during the remainder of
    the study. At doses > 250 mg/kg b.w. scarring and scabbing at the
    injection site was seen (occasionally at 100 mg/kg b.w.). No effects
    were observed on growth, haematology, organ weight, macroscopy and
    microscopy (Anderson et al., 1966; study R19-59). Remark: Summary
    only.

         Groups of rats (Harlan Wistar, 15/sex/group, F1a offspring
    from parents fed the same amount of tylosin base for about 10 weeks
    prior to mating and during gestation and lactation) were fed diets
    containing 0, 0.1, 0.5 or 1.0% tylosin base for 1 year. No
    treatment-related effects were observed on mortality, growth, food
    consumption, ophthalmoscopy, organ weights, macroscopy and
    histopathology. Treated rats appeared moderately hyperirritable and
    hyperactive after 7 to 12 months of treatment. An increase in
    lymphocytes and a corresponding reduction in neutrophils were
    observed in both sexes (significantly in females) at 0.5 and 1.0%. A
    trend towards a slightly more alkaline urine occurred in females at
    0.5% and 1.0%. The authors concluded that the NOEL was 1.0% tylosin
    base, equivalent to 500 mg/kg b.w. However, the Committee concluded
    that the NOEL in this study was 0.1% tylosin base, equivalent to
    50 mg/kg b.w. (Broddle et al., 1978a).

    2.2.2.2  Dogs

         Groups of 8 dogs (2/sex mongrel dogs and 2/sex beagle dogs)
    received orally by gelatin capsule during 2 years 0, 1, 10 or 100 mg
    tylosin base/kg b.w./day. Groups of mongrel dogs (2/sex/group) given
    200 or 400 mg tylosin base/kg b.w./day in capsules for 2 years or
    longer were subsequently added. Salivation, vomiting and diarrhoea
    were observed at 200 and 400 mg/kg b.w./day (at 100 mg/kg b.w./day 1
    dog vomited once). Haematology, urinalysis and relative organ
    weights did not reveal abnormalities. No changes were observed in
    faecal microbiological flora. Liver and kidney function tests
    revealed two dogs at 100 mg/kg b.w./day and 1 dog at 400 mg/kg

    b.w./day with a transient increased BSP retention time. Macroscopy
    and microscopy did not show compound-related changes except for mild
    pyelonephritis seen in 1/4 dogs at 200 mg/kg b.w./day and bilateral
    nephrosis, mild chronic pyelonephritis and mild cystitis seen in 1/4
    dogs at 400 mg/kg b.w./day. Terminal bone-marrow counts were normal
    (only measured in normal study). At dose levels > 10 mg/kg
    b.w./day serum tylosin levels in blood could be detected. The NOEL
    in this study was 100 mg/kg b.w./day (Anderson et al., 1966; study
    D4-59). Remark: The limited details provided and the poor reporting
    of the study made proper interpretation difficult.

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Rats

         In a limited study rats (3/sex/group) were fed diets containing
    0, 0.1, 0.3, or 1.0% tylosin base for 17 months. In the 0.3% group 1
    female died due to malnutrition. No effects on growth or on terminal
    haematological parameters were seen. Relative organ weights revealed
    changes in weights of ovaries and uteri due to thickening in uteri
    and a decrease in size of the ovaries in 1/3, 3/3, 2/3, and 2/3
    female rats at the 0, 0.1, 0.3, and 1.0% levels, respectively.
    Macroscopy and microscopic examination revealed squamous metaplasia
    of the uterine glands in 2 female rats at the highest dose (Anderson
    et al., 1966; Study R9-58). Remark: Incomplete report.

         Groups of about 25 male and female Harlan rats (total 213)
    received 0, 0.001, 0.01 or 0.1 % tylosin base in their diet for 2
    years. Survival was better in tylosin treated groups than in control
    rats (54% and 30% respectively). No effects were seen on growth,
    haematology, or relative organ weights. Macroscopy and microscopy
    revealed an increased number of animals with fatty changes in livers
    and kidney at all dose groups and a slight increased incidence of
    bile duct proliferation at the 0.1 and 0.01% levels, but neither was
    dose-related (Anderson et al., 1966; Study R10-58). Remark:
    Incomplete reports of growth and haematology; limited
    histopathology.

         In another 2-year study groups of Harlan rats (30/sex/group)
    were fed diets containg 0, 0.01, or 1.0% tylosin base. Survival was
    better in rats fed tylosin than in control rats (57% and 29%
    survival in high dose and control rats, respectively). No effects
    were seen on growth, haematology, urinalysis, organ weights,
    macroscopy and microscopy. A dose-unrelated increase of fatty
    changes in liver and kidney was observed. (Anderson et al., 1966;
    R3-59). Remark: Incomplete reports of growth, haematology and
    urinalysis.

         In a very limited study groups of 10 male and 10 female rats
    were fed diets containing 0, 2, 5, 10, or 20% tylosin base for up to
    2 years. No effects on food consumption, growth and haematology were
    observed on rats at 2% and 5%. At the 10% level, growth and food
    consumption were slightly reduced. At the highest dose food
    consumption and growth were markedly reduced followed by death
    (Anderson et al., 1966; R6-60). Remark: Incomplete reports.

         Two replicate 2-year studies were carried out with Wistar rats
    (40/sex/group in each study derived from F1a offspring from
    parents fed diets containing tylosin from 10 weeks prior to mating
    up to the time of weaning). The control groups (60/sex/group in each
    replicate study) were derived from parents fed untreated diet.
    Treated groups were fed diets containing 0.1, 0.5, or 1.0% tylosin
    base. Observations included clinical signs, mortality, food
    consumption, food efficiency, body weight, terminal haematology and
    biochemistry, urinalysis, organ weights, macroscopy, and
    histopathology. There were trends towards improved survival in
    males, although overall survival was low (20% and 28% for the
    control and treated groups, respectively), and increased food
    consumption and body weight gain in both males and females in all
    treated groups. At histopathology an increased incidence of
    pituitary adenomas in males (but not in females) was observed. The
    combined incidence of pituitary adenomas in males for both
    replicates was 6/120 (5%) in the control group, 9/80 (11%) in the
    low dose (0.1% tylosin) group, 18/80 (22.5%) in the mid dose (0.5%)
    group, and 20/80 (25%) in the high dose (1.0%) group. The authors
    concluded that the increase in pituitary tumours was an indirect
    result of the ability of tylosin to increase survival and weight
    gain. The incidence of malignant tumours was unaffected in males or
    in females (Gries, 1980)

    2.2.4  Reproduction studies

    2.2.4.1  Mice

         Groups of 7-8 male and 14-17 female ICR mice were fed diets
    containing 0, 0.1, or 1.0% tylosin (composition unknown) for two
    succesive generations with 2 litters/generation. Some of the mice
    were maintained on an ordinary diet, but all the mice received the
    experimental diet prior to delivery of the offspring. No
    treatment-related effects were observed on reproductive performance
    (sexual maturation, number of pups or weaning of pups) (Tsubura
    et al., undated). Remark: Only a few summary tables were available.

    2.2.4.2  Rats

         A 3-generation reproduction study was performed with 2 groups
    of 5 male and 10 female Harlan rats/group receiving 0 or 1.0%
    tylosin base in their diet. Fertility, viability, gestation and
    lactation indices in the F0 generation rats did not reveal any

    abnormality. Growth was equal in all generations for both control
    and treated rats. In each succeeding generation the capability for
    reproduction and perpetuation was unaffected (Anderson et al., 1966;
    R3-59). Remark: Incomplete report.

         In a special study weanling Wistar rats (25/sex/group, 6-7
    weeks old) were fed diets containing 0.1, 0.5, or 1.0% tylosin base
    for 10 weeks prior to mating and thereafter for about 6 months
    total. A control group consisted of 35 rats/sex. Only one litter was
    bred. No effects were observed on parental body weight, food
    consumption, reproductive indices (male or female fertility,
    gestation length, number of live fetuses, mean litter weight or
    offspring survival) or biochemistry. High dose male rats revealed
    significantly decreased white blood cells at termination. Sera
    collected from parental rats (approx. 150 days on experimental
    diets) did not contain detectable levels of tylosin (< 0.1 mg/l).
    Offspring were physically normal and were assigned to the one year
    toxicity study with tylosin (see Section 2.2.2.1) (Broddle, et al.,
    1978b). Remark: Only summarized data given on reproductive indices.

    2.2.5  Special studies on embryotoxicity and teratogenicity

    2.2.5.1.  Mice

         Groups of 10 pregnant mice (2 strains, CBA and A/Jax) received
    orally 100, 500, or 1000 mg/kg b.w./day tylosin (composition not
    given) in 0.1 ml water from days 7-12 of gestation. Two control
    groups of 3 and 5 mice received saline or remained untreated,
    respectively. Females were killed on day 18 of pregnancy. No effects
    were observed on number of corpora lutea, number of implantations,
    number of early and late deaths, number of embryos alive, or fetal
    development (Tsuchikawa & Akabori, undated).

         Four groups of mice given 0 or 500 mg/kg b.w. and a further 2
    pregnant females (A/Jax x male CBA) per group, receiving orally 0 or
    1000 mg tylosin/kg b.w./day during days 7-12 of gestation, were
    allowed to deliver and rear their young for 4 weeks. No effects were
    observed in growth, survival, or genital system of all mice born
    determined up to 9 weeks. At 8 weeks after birth rearing ability,
    hearing ability and kinetic functions were not effected. At the 9th
    week the mice were killed and visceral and skeletal examinations
    were performed. No abnormalities were seen (Tsuchikawa & Akabori,
    undated).

    2.2.5.2  Rats

         Groups of 15 Wistar rats were fed diets containing 0.1, 1.0, or
    10.0% tylosin (composition not given; equal to 60.5, 725, or
    4800 mg/kg b.w./day, respectively) in their diet during days 1-20 of
    gestation. A control group consisted of 10 rats. Females were killed

    on day 20 of gestation. Observations included number of resorptions
    and live and dead fetuses, sex ratio, fetal weight and external,
    visceral and skeletal abnormalities. Fetus weight at the highest
    dose was slightly decreased (Terashima, undated).

         In another study 3 groups of 15 Wistar rats received 0, 1.0, or
    10.0% tylosin (composition not given, equal to 0, 725, or 4800 mg/kg
    b.w.) during days 1-20 of gestation. Normal delivery was allowed.
    Number of fetuses, sex ratio, external abnormalities and growth
    during the weaning period (3 weeks) were determined. Motor functions
    and senses were examined. The weanlings were killed and visceral and
    skeletal examinations were performed. A slightly reduced growth was
    observed in weanlings at the highest dose (Terashima, undated).

    2.2.6  Special studies on genotoxicity

         Tylosin was tested for genotoxicity in an  in vitro
    chromosomal assay with Chinese hamster ovary cells, a mouse lymphoma
    assay and an  in vivo assay for cytogenetic damage. The results are
    summarized in Table 3.

    2.2.7  Special studies on microbiological activity

         The anti-microbial activity of tylosin has been described in
    the published literature. Tylosin is markedly active  in vitro
    against gram-positive bacteria, certain gram-negative bacteria and
    mycobacteria; it is inactive against Enterobacteriaceae (McGuire
    et al., 1961).

        Table 3: Results of genotoxicity assays on Tylosin
                                                                                               

    Test system       Test          Concentration     Purity     Results      Reference
                      object        of substance
                                    tested
                                                                                               

    Chromosome        Chinese       500-1000          99.3         -          Gries,
    aberration        hamster       g/ml2,                                   1990a
    assay1            ovary         250-750
                      cells         g/ml3, both
                                    in DMSO

    Lymphoma          Mouse         10-1000 g/ml2    99.3         +5         Gries,
    assay             L5178Y        10-1000 g/ml3                            1990b
                      TK+/-         (1000 g/ml
                      cells         toxic) both in
                                    DMSO

    Micronucleus      ICR           2 daily doses     966          -          Gries,
                      mice          of 1250, 2500                             1990c
                                    or 5000 mg/kg4
                                                                                               

    1.   Mitomycin C and cyclophosphamide, used as positive controls, yielded
         positive results
    2.   without metabolic activation
    3.   with metabolic activation
    4.   the positive control cyclophosphamide yielded positive results.
    5.   positive at cytotoxic dose
    6.   administered as tylosin base
    
         In recent studies the minimal inhibitory concentration (MIC) of
    tylosin has been determined for bacterial pathogens isolated from
    target animal species of European and North American origin since
    1984. Additional information on sensitivity of bacterial isolates to
    tylosin was obtained from literature published since 1980. Tylosin
    was active against most Gram-positive bacteria and mycoplasmas
    tested  in vitro. Activity against Gram-negative bacteria was
    generally lower. Tylosin was also found to be active against
    isolates of  Chlamydia psittaci. MIC values for streptocci,
    enterococci and staphylococci are given in Table 4 (Herd, 1990).

        Table 4. MIC1 values for tylosin against some Gram-positive bacteria
                                                                                  

    Organism                         MIC range            Total No.
                                     (mg/1)               of isolates
                                                                                  

    Streptococcus pyogenes           0-1  -  0.2             5
    Streptococcus pneumoniae         0.2  -  0.4             4
    Streptococcus dysgalactiae       0.06 -  128            50
    Streptococcus agalactiae         0.125 -  0.5           51
    Streptococcus suis               0.125 - > 128          42
    Streptococcus uberis             0.125 - > 128          53
    Enterococcus faecalis            0.25 - > 128           31
    Staphylococcus aureus2           0.125 - > 128          98
    Staphylococcus Spp.3             0.78 -  1.0             7
                                                                                  

    1.   Minimum inhibitory concentration
    2.   Coagulase - positive
    3.   Coagulase - negative
    
    2.2.8  Special studies on neurotoxicity

         Three cats received daily for 90 days subcutaneously 200 mg/kg
    b.w. tylosin tartrate as a 20% solution divided in 2 doses during
    the first 37 days and thereafter as a single dose (with addition of
    sodium citrate for buffering). An untreated control group consisted
    of 3 cats. Slight reduction (25-35%) was observed in the
    post-rotatory nystagmus response, but the auditory response appeared
    normal. All cats landed on their four feet when dropped from 1
    metre. No ataxia was seen (Anderson, 1966; C2-59)

    2.2.9   Special studies on skin and eye irritation

         Granulated tylosin concentrate (2 g/kg b.w.) was not irritating
    when applied to the rabbit skin but when the formulation (52 mg
    (0.1 ml)) was instilled to the rabbit eye corneal dullness, slight
    corneal opacity, slight to marked irititis and moderate
    conjunctivitis were observed. The effects resolved within 14 days of
    treatment (Gries et al., 1985a).

         The formulation Tylan 200 injection (2 ml/kg b.w.) caused very
    slight erythema to the rabbit skin and the formulation (0.1 ml)
    caused slight conjunctival hyperemia to the rabbit eye both within
    24 hours after application. No skin and eye irritation were observed
    after 48 hours (Gries, et al., 1985b).

         The formulation Tylan soluble (2 g/kg b.w.) caused very slight
    erythema and slight desquamation to the rabbit skin. Slight to
    moderate corneal opacity, marked iritis and moderate conjunctivitis
    were observed following the instillation of 58 mg of the formulation
    into the rabbit eye; these effects had cleared within a week (Gries
    et al., 1985c).

    2.2.10  Special studies on skin sensitization

         Tylosin tartrate was used as a positive control in a
    sensitization test by the method of Landsteiner and Jacobs conducted
    for tilmicosin. Guinea-pigs (12) were administered 10 intracutaneous
    injections of 50 mg/ml tylosin tartrate; a challenge with a further
    dose was given after 14 days. At challenge a mild positive response
    was observed (Jordan et al., 1989).

    2.3.  Observations in humans

         Trials in human subjects have been conducted to investigate
    antibiotic-resistant bacteria. In one trial 12 or 11 human
    volunteers were given 20 mg tylosin per day or a placebo,
    respectively, for up to 6 months. There was no significant change in
    the total number of tylosin-resistant staphylococci and lactobacilli
    in weekly faecal samples or in coliform or yeast forms whereas a
    significant increase was observed in total number of resistant
    streptococci. In another trial no tylosin resistant organisms were
    detected in specimens (including faeces samples) from hospitalized
    patients who had never had any exposure to the nonmedical antibiotic
    tylosin. In both studies no regular pattern was observed in
    cross-resistance to related antibiotics (Malin & Silliker, 1966)

         In a Japanese trial 2 human volunteers were given 2 or 5 mg
    tylosin (as 1 mg tablets; composition not given) per day for 3
    months. Faeces were inspected for  E. coli and Enterococci and
    Staphylococci at intervals of 1 to 2 weeks from 2 months prior to
    administration and 3 months from the start of administration. No
    tendency towards increased resistance was noted (Kuwabara, undated).

         Tylosin resistance was examined in human cultures of
     Staphylococcus aureus, Streptococcus pyogenes and  Campylobacter
     spp. Of 3812 human cultures isolated between 1985 and 1987 only
    1.0% were resistant to tylosin. There was no evidence for a
    significant animal source of these resistant cultures (Lacey, 1988).

    3.  COMMENTS

         The Committee considered toxicological data on tylosin,
    including the results of studies on biochemical aspects,
    mutagenicity, and microbiological activity. The Committee noted that
    most of the toxicity studies had been carried out about 20 years
    ago, had not been conducted according to current protocols, and were
    poorly reported.

         Following administration of tylosin by various routes, peak
    serum levels in rats, dogs, pigs, and cattle were observed within
    1-2 hours, and then declined rapidly. In pigs, about 22% was
    bioavailable after oral administration. Excretion of tylosin was
    rapid and largely in the bile.

         After oral administration of radiolabelled tylosin to rats and
    pigs 99% of the radioactivity was excreted via the faeces. Major
    products identified in faeces were tylosin (factor A), macrosin
    (factor C), relomycin (factor D), and dihydrodesmycosin. In pig
    liver and kidney, only very small amounts of tylosin and
    dihydrodesmycosin could be found.

         Several short- and long-term studies in rats and dogs were
    performed. In a 1-year study in rats, tylosin base was administered
    in the diet at concentrations up to 10 g/kg of feed. A NOEL of
    1 g/kg of feed, equivalent to 50 mg/kg b.w./day, was established,
    based on haematological and urinary pH changes. In a study in dogs,
    tylosin base was administered orally at dose levels up to 400 mg/kg
    b.w./day for 2 years. At the two highest dose levels, salivation,
    vomiting, and diarrhoea as well as mild pyelonephritis were
    observed. The NOEL was 100 mg/kg b.w./day.

         In two replicate, but not independent, carcinogenicity studies
    in rats, tylosin base was administered in the diet for 2 years at
    levels up to 10 g/kg of feed. Food consumption and body-weight gain
    were increased in both males and females in all treated groups. In
    male rats, a dose-related increase in pituitary adenomas was
    observed, from 5% in the control to 25% in the highest-dose group.
    There was no such increase in female rats. The authors of the report
    concluded that the increase in pituitary tumours was an indirect
    result of the ability of tylosin to increase survival and weight
    gain. However, this hypothesis was neither tested experimentally nor
    verifiable in detail by the Committee because individual body
    weights at about 12 months of age were not available.

         No effects on reproduction performance were observed in a
    two-generation study in mice and in one- and three-generation
    studies in rats. No malformations were observed in mice or rats, but
    the Committee noted that these studies were poorly reported.

         Tylosin was not mutagenic in an  in vitro test for chromosomal
    aberrations and in an  in vivo micronucleus test. In a mouse
    lymphoma assay, no activity was found with metabolic activation;
    however a weak, but significant activity was observed in the absence
    of such activation.

         In studies in human volunteers, there was no evidence of the
    emergence of cross-resistance to therapeutically important
    antibiotics, but volunteers given oral doses of 20 mg of tylosin
    daily for 6 months showed an increase in the number of resistant
    streptococci. The Committee concluded that additional studies
    showing no microbiological effects in two individuals at doses up to
    5 mg/person/day were inadequate to establish a NOEL. In addition, no
    suitable  in vitro data were available to establish a NOEL with
    respect to the microbiological risk for humans.

    4.  EVALUATION

         Because of the deficiencies in the toxicological and
    microbiological data, the Committee was not able to establish an
    ADI.

    5.  REFERENCES

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    Study numbered 893//FAANIM/AM/14-26. Submitted to WHO by Lilly
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    ANDERSON, R.C., HARRIS, P.N., LEE, C.C., MAZE, N., SMALL, R.M. &
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    ANDERSON, R.C. & WORTH, H.M. (undated) The acute toxicity of tylosin
    phosphate. Unpublished report from the Lilly Research Laboratories,
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    BRODDLE, W.D., GOSSETT, F.O., ADAMS, E.R., HOFFMAN, D.G., GRIES,
    C.L., GIBSON, W.R. & MORTON, D.M. (1978a) Chronic toxicity of
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    BRODDLE, W.D., GOSSETT, F.O., ADAMS, E.R., HOFFMAN, D.G., KITCHEN,
    D.N., GIBSON, W.R. & MORTON, D.M. (1978b) A study of a parental
    population of rats bred to produce offspring assigned to one- and
    two-year dietary studies of tylosin. Unpublished Report Study No.
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    BUCK, A.M., COOPER, T.R. & THOMSON, T.D. (undated) Serum and lung
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    tartrate B.I.D. in milk replacer. Unpublished Report T1Y768501 from
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    GINGERICH, D.A., BAGGOT, J.D. & KOWALSKI, J.J. (1977) Tylosin
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     Jour., 18(4), 96-100.

    GRIES, C.L. (1980) The toxicological evaluation of tylosin (compound
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    GRIES, C.L., McKINLEY, E.R. & QUARLES, J.P. (1983) Acute comparative
    intravenous toxicity testing of tylosin, desmycosin and macrocin in
    the ICR mouse. Unpublished report dated August 1983 with studies
    M-V-46-83, M-V-45-83, and M-V-44-83 from Toxicology Division, Lilly
    Research Laboratories, Greenfield, Indiana. Submitted to WHO by
    Lilly Research Centre Ltd., Windlesham, Surrey, England.

    GRIES, C.L., DOWNS, O.S. & NEGILSKI, D.S. (1985a) The acute oral,
    dermal and ocular toxicity of granulated tylosin concentrate.
    Unpublished report dated April, 1985 with studies R-O-365-79,
    R-366-79, B-D-109-79, and B-E-94-79 from Toxicology Division, Lilly
    Research Laboratories, Greenfield, Indiana. Submitted to WHO by
    Lilly Research Centre Ltd., Windlesham, Surrey, England.

    GRIES, C.L., DOWNS, O.S. & NEGILSKI, D.S. (1985b) The acute oral,
    dermal, ocular and inhalation toxicity of tylan 200 injection.
    Unpublished report dated September 1985 with studies R-O-344-79,
    R-O-343-79, B-D-103-79, B-E-87-79, and R-H-39-79 from Toxicology
    Division, Lilly Research Laboratories, Greenfield, Indiana.
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    GRIES, C.L., NEGILSKI, D.S. & DOWNS, O.S. (1985c) The acute oral,
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    report dated September 1985 with studies R-O-367-79, R-O-368-70,
    B-D-94-79, B-E-90-79, and R-H-40-79 from Toxicology Division, Lilly
    Research Laboratories, Greenfield, Indiana. Submitted to WHO by
    Lilly Research Centre Ltd., Windlesham, Surrey, England.

    GRIES, C.L. (1990a) The effect of tylosin on the  in vitro
    induction of chromosome aberrations in chinese hamster ovary cells.
    Unpublished report with studies 891109CTX3279, 891129CAB3279, and
    891220CAB3279 from Lilly Research Laboratories, Toxicology Division,
    Greenfield, Indiana.

    GRIES, C.L. (1990b) The effect of tylosin on the induction of
    forward mutation at the thymidine kinase locus of L5178Y mouse
    lymphoma cells. Unpublished report with studies 891011MLT3279,
    891017MLA3279, and 891114MLA3279 from Lilly Research Laboratories,
    Toxicology Division, Greenfield, Indiana.

    GRIES, C.L. (1990c) The effect of tylosin (compound 027892) on the
     in vivo induction of micronuclei in bone marrow of ICR mice.
    Unpublished report, study 891212MNT3279 from Lilly Research
    Laboratories, Toxicology Division, Greenfield, Indiana.

    HERD, R.M. (1990) The antimicrobial activity of tylosin  in vitro
    1980-1989. Unpublished Report dated 27-06-90 from Lilly Research
    Centre Ltd. Submitted to WHO by Lilly, Windlesham, Surrey U.K.

    JORDAN, W.H., GARDNER, J.B. & WEAVER, D.E. (1989) An intracutaneous
    sensitization stidy in albino guinea pigs with tilmicosin.
    Unpublished toxicology report No. 32 from Toxicology divison, Lilly
    Research Laboratories, Greenfield, Indiana. Submitted to WHO by
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    KUWABARA, S. (undated) A study on the effect of continuous and
    minute amount of tylosin on human intestinal flora. Medical
    Department Toho University. Submitted to WHO by Lilly Research
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    LACEY, R.W. (1988) Rarity of tylosin resistance in human pathogenic
    bacteria. Report from Department of Microbiology, University of
    Leeds, Leeds, England. Submitted to WHO by Lilly Research Centre
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    LILLY (undated a) The determination of tylosin levels in lung and
    serum following administration of tylan 200 by intramuscular
    injection to weaned calves. Unpublished Report T1Z769001.
    Supplementary information submitted to WHO under
    893/IJB200/FDISTR/AM/26-35 by Lilly Research Centre, Windlesham,
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    LILLY (undated b) Tylosin absorption studies in chickens.
    Unpublished Report VPR-17-418 submitted under 893//FAANIM/AM/1-9 to
    WHO by Lilly Research Centre, Windlesham, Surrey, England.

    LILLY (undated c) Tylosin blood titres in chickens. Unpublished
    Report VPR-21-418 submitted to WHO under 893//FBLDLV/AM//1-3 by
    Lilly Research Centre, Windlesham, Surrey, England.

    LILLY (undated d) Tylosin excretion studies in chickens. Unpublished
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    Lilly Research Centre, Windlesham, Surrey, England.

    MALIN, B. & SILLIKER, J.H. (1966) Low level tylosin and the
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    McGUIRE, J.M., BONIECE, W.S., HIGGENS, C.E., HOEHN, M.M., STARK,
    W.M., WESTHEAD, J. & WOLFE, R.N. (1961) Tylosin, a new antibiotic: I
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    320-327.

    NAKAMURA, H. ET AL. (1969) Studies on distribution of antibiotics
    inblood and tissue. XIX. Blood level and tissue concentration of
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     Proc. Jap. Soc. Vet. Science, 67th meeting, No. 128.

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    SIECK, R.F., GRAPER, L.K., GIERA, D. D., HERBERG, R.J. & HAMILL,
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    Unpublished Report dated November 1978 from Agricultural
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    SIECK, R.F., GRAPER, L.K., GIERA, D. D., HERBERG, R.J. & HAMILL,
    R.L. (1978b) Metabolism of tylosin in swine and rat. Unpublished
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    TERASHIMA, H. (undated) The effect of tylosin on a fetus and a
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    THOMSON, T.D. (undated a) Relative bioavailability of TYLAN 200
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    THOMSON, T.D. (undated b). The relative absorption rates of tylosin
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    THOMSON, T.D. (undated c). Tylosin distribution in pneumonic calf
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    VAN DUYN, R.L. & JOHNSON, W.S. (undated a) Tylosin serum and lung
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    Windlesham, Surrey, England.


    See Also:
       Toxicological Abbreviations
       TYLOSIN (JECFA Evaluation)