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    SPIRAMYCIN

    First Draft prepared by
    Dr. K.N. Woodward
    Veterinary Medicines Directorate
    Weybridge, Surrey, England

    1.  EXPLANATION

         Spiramycin is a macrolide antibiotic used for the treatment and
    control of a number of bacterial and mycoplasmal infections in
    animals. It is available as a spiramycin embonate for use in animal
    feed, and as the adipate, a more soluble form, for administration by
    other routes. Spiramycin was previously evaluated at the twelfth
    meeting of the Joint FAO/WHO Expert Committee on Food Additives
    (Annex 1, reference 17).

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

         Spiramycin appeared to be relatively well absorbed in the rat
    after oral doses of 150 or 400 mg/kg b.w./day for 6 days with high
    levels being found in bone (40 and 50 µg/g bone). Bone spiramycin
    slowly decreased over a 24-day period (Woehrle, 1968).

         After intramuscular injection of 50 mg/kg b.w. to the
    guinea-pig, relatively high levels (23-70 µg/g) were found in blood,
    heart, liver, lung, spleen in kidney one day after administration.
    Tissue levels were still relatively high (20-58 µg/g) four days
    after administration (Pellerat & Maillard, 1959).

         When given as the adipate in water to pigs at 50 or 100 mg/kg
    b.w., high levels were noted in the liver, bile and kidney (15-40
    and 45-320 µg/g at 500 and 100 mg/kg b.w., respectively). Lower
    levels were found in lung, intestine and muscle. Similar levels were
    found after subcutaneous injection of 25-50 mg/kg b.w. (Ferriot &
    Videau, 1971).

         Several studies with spiramycin embonate in the feed of pigs
    revealed highest levels in the liver and kidney after doses
    equivalent to 8-128 mg/kg b.w. for periods of 6-18 days (Genin &
    Pascal, 1981; Genin, 1983 a,b; Genin, 1984). Dietary administration
    to pigs at levels equivalent to 16 mg/kg b.w./day for 7 days
    resulted in liver and kidney concentrations of 4-7.5 and 7-12 µg/g,
    respectively, 12 hours after administration. Levels in muscle and
    fat were low (0.12 and < 0.1 µg/g, respectively). By 3 days after
    administration, levels in kidney and liver had fallen markedly
    (1-2 µg/g) and were below 0.1 µg/g in muscle and fat. At day 10
    levels in kidney and liver were less than 0.3 and 0.15 µg/g,
    respectively. Very similar results were noted with 25 mg/kg b.w./day
    given in the same way (Pascal et al., 1990).

         There are no extensive data available on excretion. Studies in
    the pig suggest biliary and subsequent faecal excretion may be
    involved (Ferriot & Videau, 1971). Studies in cattle indicate that
    spiramycin is excreted in milk after intramuscular and subcutaneous
    administration (Moulin, 1989).

    2.1.2  Biotransformation

         There were no data available from studies in laboratory
    animals. In cattle, the metabolite neospiramycin, the demycarosyl
    derivative, is formed. Concentrations of neospiramycin in muscle and
    kidney were marginally higher than those of spiramycin 14-28 days
    after dosing; in muscle, levels of neospiramycin and spiramycin were
    approximately equal (Sanders, 1990).

    2.1.3  Effects on enzymes and other biochemical parameters

         Spiramycin (12.5 and 25 mg/kg b.w.), administered twice a day
    for 3 days to inbred Balb/c mice, had no effect on
    pentobarbital-induced sleeping time indicating no effect on drug
    metabolizing enzymes. Triacetylolandeomycin and josamycin
    significantly increased the sleeping time (Descotes & Evreux, 1983).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         Acute toxicity studies are summarized in Table 1. Spiramycin
    adipate and embonate were of low acute toxicity in the rat and mouse
    after oral administration. Similarly, the adipate had low toxicity
    in the guinea-pig, rabbit and dog. After subcutaneous administration
    the adipate was of low toxicity to the rat but more toxic to cats
    and turkeys. It was significantly more toxic to rats and mice after
    intravenous administration.

         After oral dosing signs of toxicity at high doses included
    anorexia, diarrhoea and lassitude in rats and dogs (Boyd, 1958).
    Restlessness, incoordination, convulsions, diarrhoea, phonation and
    ataxia occurred in mice given high doses. In rats, hepatic necrosis
    occurred whereas in dogs, vacuolar degeneration of hepatocytes was
    observed. Necrosis of the distal convoluted tubules occurred in both
    species (Boyd, 1968; Boyd & Price-Jones, 1960).

         Mice given intravenous spiramycin adipate developed
    hyperactivity, ataxia, tremor, dyspnoea, aggressiveness, convulsions
    and diarrhoea. Similar signs were noted in rats given intravenous
    spiramycin (Cordier et al., 1984a,b). Dogs given intravenous
    spiramycin at relatively high doses developed facial oedema, ear
    oedema and erythema. Hypersalivation occurred. Animals which died
    had hepatic, renal and pulmonary congestion (Cordier et al., 1985a).

        Table 1: Acute toxicity
                                                                                               

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

    Adipate

    Mouse                M                    oral          3130       Boyd & Price-Jones,
                                                                       1960
    Mouse                M&F                  iv             220       Cordier et al. 1984a
    Rat                  M                    oral          9400       Boyd, 1958
    Rat                  M                    oral          4850       Boyd & Price-Jones
                                                                       1960
    Rat                  M                    s.c.          3500       Boyd, 1957/58
    Rat                  M&F                  iv             350       Cordier et al., 1984b
    Guinea-pig           M                    oral         3-4000      Boyd & Price-Jones,
                                                                       1960
    Rabbit               M                    oral          4330       Boyd & Price-Jones,
                                                                       1960
    Cat                  M&F                  s.c.           950       Boyd & Price-Jones,
                                                                       1959
    Dog                  F                    oral          5200       Boyd, 1958
    Turkey               Unspecified          s.c.           850       Cook & Inglis, 1963

    Embonate

    Mouse                M&F                  oral          >5000      Delongeas et al.
                                                                       1989b
    Rat                  M&F                  oral          >4350      Delongeas et al.
                                                                       1989a
                                                                                               
    
         In oral studies with the embonate in mice and rats, no clinical
    signs were noted at the highest dose in rats (4350 mg/kg b.w.) but
    mice became ataxic and dyspnoeic at doses of 2500 and 5000 mg/kg
    b.w.. These signs disappeared within 2 days (Delongeas, 1989 a,b).

    2.2.2  Short-term studies

    2.2.2.1  Rats

         Groups of 6 male and female rats of unspecified strain were
    given oral doses of spiramycin adipate at 0.25 or 1 g/kg b.w. daily
    for 4 weeks. There were no effects on urinalyses or haematology and
    no signs of toxicity were noted (Dubost et al., 1956).

         Groups of 20 male and 20 female Crl : CD(SD)BR rats were given
    diets containing 0, 2000, 10 000, or 50 000 ppm spiramycin embonate
    for 13 weeks. At the end of this time, 10 rats/sex/group were killed
    and the remainder maintained on the treatment diets for a further
    week and then allowed a 4-week recovery period (i.e., up to
    week 18). The equivalent doses varied over the test period, being
    219, 1204, and 5858 mg/kg b.w./day for 2000, 10 000, and 50 000 ppm
    males and 214, 1019, and 5233 mg/kg b.w./day for 2000, 10 000, and
    50 000 ppm females in week 1 of the study while at week 13 the
    respective values were 89, 473, and 2488 mg/kg b.w./day and 116,
    581, and 2779 mg/kg b.w./day. These were equivalent to mean doses of
    140, 718, and 3695 mg/kg b.w./day for males and 162, 785, and
    3911 mg/kg b.w./day for females.

         During the study clinical observations were made daily, body
    weights measured weekly and food consumption weekly. Ophthalmology
    was conducted at weeks 6 and 13 in high level and control animals.
    Haematological and biochemical tests and urinalyses were carried out
    at weeks 6 and 13, and 16 and 18 in recovery groups. At necropsy,
    all major organs were weighed and the animals were subjected to full
    histopathological examination.

         There was no compound-related mortality during the study and no
    major clinical signs attributable to spiramycin intake. Significant
    reductions in body weight gains were noted in high dietary level
    animals as were significant reductions in food consumption in high
    level males.

         Haematological examination showed reductions in neutrophils in
    mid- and high-dietary level animals at week 6 in males and females
    although the effect in the latter group was not statistically
    significant. At week 16 of the recovery period, reductions in both
    neutrophil and lymphocyte counts were noted in mid- and high-dietary
    level males. There were no significant biochemical effects but
    urinalyses showed reductions in urinary protein at weeks 6 and 13 in
    high level males, although the effect was not statistically
    significant at the latter time point. 

         At termination, there were reductions in mean relative liver 
    weights. Bone marrow examination showed reductions in lymphocytes in 
    high level males. The authors considered the effects on circulating 
    lymphocytes to be unrelated to compound intake as they were not seen 
    during the treatment period. The only other major effect noted on 
    histopathological examination was dilatation of the caecum. Testicular 
    degeneration was not seen. The no-observed effect level was 
    approximately 140 mg/kg b.w./day (Powell et al., 1990).

         Groups of 20 male and 20 female albino rats (strain
    unspecified) were fed diets containing 0, 800, 2400, and 7200 ppm
    spiramycin adipate; these levels were calculated by the authors to
    be equivalent to 0, 80, 240, and 720 mg/kg b.w./day, for a period of
    one year.

         By week 14, no adverse effects on body weights were apparent.
    An interim kill of 5 males and 5 females was conducted at this time.
    There were no effects on organ weights except for a slight increase
    in relative spleen and kidney weights in females given 2400 and 7200
    ppm spiramycin adipate.

         At the end of the study the animals were sacrificed. Prior to
    this, there were no adverse effects noted on body weights in treated
    males nor in females given the low and intermediate dietary levels.
    Females given the highest level showed a significant reduction in
    body weight gain. Food intake in all treated groups was similar to
    control values. There were no haematological abnormalities noted.

         At necropsy, the only changes seen were increases in relative
    liver, kidney and adrenal weights in animals given the highest
    dietary level. Treated groups but not control animals showed
    hepatocytic glycogen depletion. No other changes were noted
    (Johnson, 1962a; Johnson, 1962b).

         Spiramycin adipate was administered subcutaneously at doses of
    0, 200, and 600 mg/kg b.w./day for 8-10 weeks to groups of male
    Wistar rats (numbers unspecified). At 200 mg/kg b.w./day,
    hyperreflexia and convulsions appeared within one hour of dosing.
    Necrosis developed at the injection site. Body weights and food
    intake fell below those of controls at 7 days, and blood haemoglobin
    decreased at 4 weeks. Water intake and urine volume increased.
    Urinalyses were normal although the specific gravity was less than
    in controls. Pallor of visceral organs was noted at necropsy and the
    adrenals were enlarged. Animals given 600 mg/kg b.w./day showed
    signs similar to those seen in those given the lower dose. At
    necropsy, oedema in the kidney was evident, with necrosis of cells
    of the convoluted tubules. Degenerative changes were noted in the
    gastrointestinal tract and dilatation of the intestinal crypts was
    seen. Vacuolation of cells in the adrenal cortex occurred and no
    sperm was evident in the testes (Boyd & Brown, 1958).

         Groups of 10 male and 10 female CD rats were given daily
    intravenous injections of 0, 90 000, 180 000, and 270 000 iu/kg
    b.w./day spiramycin adipate in 0.9% saline (doses equivalent to 0,
    2.8, 5.6, and 84.4 mg/kg b.w./day), twice daily, 7 days per week for
    32 days. Groups of 5 males and 10 female rats in a reversibility
    study were treated in a similar manner.

         Salivation and tremor were noted in intermediate and high dose
    animals during dosing. A slight reduction in appetite was noted in
    high dose males as was a reduction in weight gain in these animals.
    Low alanine aminotransferase activities were noted in high dose
    females. There were no effects on urinalyses. High liver weights
    were found in high-dose males and these persisted for the 15-day

    reversibility period. No macroscopic abnormalities were seen at
    necropsy. Histological examination showed foamy macrophages in the
    spleens of all high dose rats, in some intermediate dose animals and
    in one rat given the low dose. These effects were not seen in
    animals allowed the 15-day reversibility period.

    2.2.2.2  Dogs

         Two dogs of unspecified breed were given oral doses of 200 or
    500 mg/kg b.w./day spiramycin adipate each day for 4 weeks. No signs
    of toxicity occurred and there were no effects on liver or kidney
    histology, nor on haematology (Dubost et al., 1956).

         A group of 14 male and 6 female adult mongrel dogs was given
    oral doses of 500 mg/kg b.w./day spiramycin adipate for 56 days or
    until death occurred; 10 dogs served as controls. All but two of the
    dogs receiving the dose died. Varying degrees of anorexia,
    salivation, vomiting, diarrhoea and irritability occurred. Prior to
    death, apathy and pallor were seen. Impaired vision was evident in 3
    dogs. Prostration, dark faeces, rigor and anal incontinence occurred
    immediately prior to death. Reduction of food intake was seen after
    1-2 weeks of treatment and in the days preceding death this was
    marked. A pronounced anaemia developed in the treated dogs with
    haematocrit, erythrocytes and haemoglobin progressively falling
    throughout the study. There were also declines in plasma lipids and
    cholesterol. Albumin and bilirubin appeared in the urine during the
    weeks preceding death. Half the dogs survived to 4 weeks but by the
    end of the study only two of the animals remained alive.

         Gross examination at necropsy showed the gastrointestinal tract
    to be empty except for green fluid in the small bowel and a
    green-black fluid in the colon. The spleen was pale, enlarged, and
    soft; the pancreas and liver were also pale. The adrenals were
    enlarged. Testicular atrophy occurred. Histological examination
    showed no major abnormalities of the gastrointestinal tract,
    however, there was a reduction in spermatogenesis, and cells lining
    the liver sinusoids were swollen while the central veins appeared
    empty. Hepatocytes appeared compressed by the swelling of the
    sinusoidal cells. "Considerable damage" was seen in the kidney
    particularly in the loop of Henle. Necrotic changes were apparent in
    several areas of the kidney (Boyd et al., 1958).

         Four groups of 3 male and 3 female purebred beagles were given
    0, 60, 120, and 240 mg/kg b.w./day spiramycin adipate orally as a
    capsule formulation, 6 days a week for 28 weeks.

         Diminished activity was noted in dogs given the highest dose.
    Some of these dogs produced mucous in the faeces for the first few
    days of the test, and during this time of these animals also
    appeared to have vomited during the night.

         There were no pronounced effects on body weight gain,
    haematology and qualitative urinalysis. Blood urea nitrogen values
    were elevated in intermediate and high dose dogs at 13, 19, and 26
    weeks but no other effects on blood biochemistry were seen.

         Animals given the highest daily dose had splenomegaly. 
    Thyroids and kidneys appeared pale. Degenerative liver and kidney
    changes were noted in mid- and high-dose dogs. There was no evidence
    of testicular atrophy. The no-effect level in this study was
    60 mg/kg b.w./day (Johnson, 1962a).

         Groups of 4 male and 4 female purebred beagles were fed diets
    containing 0, 3000, 4000, 5000, and 6000 ppm, spiramycin adipate,
    equivalent to 0, 75, 100, 125, and 150 mg/kg b.w./day, for 2 years.
    Two additional dogs (1 male, 1 female) were placed on the highest
    dietary level at week 70 and returned to basal diet after 45 weeks.

         No deaths occurred during the study and all dogs appeared
    normal. Body weights, haematological and clinical chemistry data
    were within normal limits and there were no effects on qualitative
    urinalyses, blood pressure or heart rate.

         Ophthalmological changes were noted at examination after 28
    weeks of study. These changes were also evident at weeks 43, 82, and
    105, and they occurred at all levels except the 3000 ppm level. They
    consisted of a flecking of the pigment of the  tapetum lucidum with
    degrees of absence of this structure. The 2 dogs entered on the high
    level diet at week 70 showed tapetal changes after 15 days of
    treatment and after 45 days these effects were as severe as the
    cases noted in the main study. When returned to the control diet,
    the condition resolved within 14 days. Vision did not appear to be
    impaired. However, dogs given 5000 and 6000 ppm spiramycin adipate
    showed incomplete accommodation and light sensitivity around week
    65, and there were bluish areas on the tongues of animals in these
    groups. No other effects were seen.

         At necropsy, absolute and relative weights of the heart, liver,
    kidney, spleen, adrenal and pancreas were increased in dogs given
    5000 and 6000 ppm spiramycin adipate. Histopathological examination
    showed degenerative changes in several organs including the liver,
    spleen, prostate, pancreas, lymph nodes, stomach, gall bladder, bile
    duct, adrenal, and small intestine. These were prominent in dogs
    given 5000 and 6000 ppm and minimal in those given 4000 ppm. The
    changes consisted largely of cell vacuolation. The thickness of the
    walls of small arteries was increased in sections of heart from dogs
    given 5000 and 6000 ppm spiramycin adipate.

         Retinal vacuolation and atrophic changes occurred in dogs given
    5000 and 6000 ppm. The two dogs allowed the recovery period showed
    no evidence of retinal damage. The no-effect level for retinal
    damage in this study was 3000 ppm, equivalent to 75 mg/kg b.w./day
    spiramycin adipate (Thompson et al., 1967).

         Groups of 3 male and 3 female beagle dogs were given daily
    intravenous doses of 240 000, 360 000, and 540 000 iu/kg b.w./day
    spiramycin adipate (doses equivalent to 7.5, 112.5, and 168.8 mg/kg
    b.w./day) daily as two injections, 7 days per week for 4 weeks. Ear
    erythema and oedema of the ears and face occurred during the
    injections. Body weights remained normal. There were no effects on
    food consumption, ECG, ophthalmology, blood chemistry, haematology
    and urinalyses.

         At necropsy, no gross pathology was evident. Absolute and
    relative spleen weights were increased in male and female high dose
    animals and in some dogs given the intermediate dose. At the high
    and intermediate doses, hypertrophy of the spleen occurred with
    disseminated or focal macrophages in the peripheral areas of
    Malgipi's follicles. All high dose animals also showed slight
    hypertrophy and clear appearance of the juxtaglomerular system and
    of the cells of mesangial cells of the glomeruli (Cordier et al.,
    1985b).

         Four purebred beagles (2 male, 2 female) were given intravenous
    doses of 50 mg/kg b.w./day spiramycin adipate, 5 days a week for 4
    weeks. No controls were used. After the first dose, prostration,
    convulsions, emesis, salivation, cyanosis, and ataxia occurred.
    Subsequent doses produced head shaking and salivation. Blood
    biochemistry and haematology were normal. At necropsy, gross
    examination revealed no adverse effects. There were no effects on
    absolute or relative organ weights. Histopathological examination
    revealed no significant changes (Johnson, 1962a).

    2.2.2.3  Monkeys

         Groups of 2 male and 2 female monkeys (Macaca fascicularis)
    were given daily intravenous injections of 0, 240 000, 360 000, and
    540 000 iu/kg b.w./day spiramycin adipate (doses equivalent to 0,
    75, 112, and 169 mg/kg b.w./day) for 5 days. Hypersalivation
    occurred during injection in all dose groups. Muscle hypotonia and
    nauseous spasticity occurred in several high dose monkeys and in one
    given the low dose. No abnormalities of body weights occurred but
    food consumption was reduced in all treated animals. A slight
    decrease in haemoglobin, red cell numbers and haematocrit was noted
    in high dose animals. No abnormalities in blood chemistry urinalyses
    occurred. There were no drug-related changes at gross examination;
    histological examinations were not conducted (Courdier et al, 1985).

         In these short-term studies oral spiramycin at doses of up to
    4000 mg/kg b.w./day for 12 days, 1000 mg/kg b.w./day for 28 days and
    500 mg/kg b.w./day for 13 weeks had no apparent effects on the rat.
    When given orally at doses of up to 720 mg/kg b.w./day for 52 weeks,
    spiramycin produced a reduction in body weights only at the highest
    dose level in females; the no-effect level was 240 mg/kg b.w./day.

         There was no evidence of testicular atrophy in this study as
    determined by testicular weights but it was not clear if specific
    subclinical investigations were made although all organs were
    subject to histopathological examination (see Sections 2.2.2.2 and
    2.2.4). Intravenous doses of up to 84.4 mg/kg b.w./day for 32 days
    produced various clinical signs including salivation, reductions in
    weight in high-dose males and elevated liver weights; the no-effect
    level was 2.8 mg/kg b.w./day. Rats given 200 or 600 mg/kg b.w./day
    subcutaneously for 8-10 weeks displayed hyperreflexia and
    convulsions at both doses. Similarly, pallor of visceral organs and
    adrenal enlargement were also noted at both doses; a no-effect level
    was not identified.

         Dogs given oral spiramycin at doses of 200 and 500 mg/kg
    b.w./day for 28 weeks showed no major effects but only kidney and
    liver histology were studied. In another investigation animals given
    500 mg/kg b.w./day for 56 days developed reductions in
    spermatogenesis, testicular atrophy (see Section 2.2.4) and other
    clinical and histopathological effects. As only one dose level was
    used, a no-effect level could not be identified. However, in a
    28-day study with oral doses of up to 240 mg/kg b.w./day, no
    testicular effects were seen. However, in the latter study,
    spiramycin adipate produced reversible retinal damage with effects
    on the  tapetum lucidum. The no-effect level was 75 mg/kg b.w./day.
    Similar effects were noted in another dog study. The relevance of
    these findings to non-tapetal mammals including humans in unclear.
    Intravenous doses of up to 168 mg/kg b.w./day spiramycin adipate
    produced a range of effects in dogs in 4-week studies, including ear
    erythema and oedema during injection, and splenomegaly. The
    no-effect levels could not be identified in the study using a
    50 mg/kg b.w./day dose level but in the multi-dose study it was in
    the region of 7.5 mg/kg b.w./day. An intravenous study in the monkey
    with doses of up to 169 mg/kg b.w./day spiramycin adipate for 5 days
    revealed hypotonia and spasticity as the major clinical signs in
    monkeys at all dose levels.

         Decreases in haemoglobin occurred in high-dose animals.
    Although the clinical signs occurred at all dose levels, these were
    minimal at the low dose (75 mg/kg b.w./day) and occurred in only one
    animal. It seems likely therefore that the intravenous no-effect
    level was in the range of 70 mg/kg b.w./day in this species.

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Rats

         Groups of 50 male and 50 female OFA (Sprague-Dawley) rats were
    given diets containing 0, 1500, 3000, and 6000 ppm spiramycin
    adipate, equivalent to oral doses of 0, 75, 150, and 300 mg/kg
    b.w./day for 2 years.

         No adverse clinical signs were noted during the study but a
    small reduction in the rate of weight gain (around 17% of control
    values at the end of the study) was seen in rats given the highest
    dietary level. There was no excess mortality in spiramycin-treated
    rats. Haematology, blood biochemistry and urinalyses were
    essentially normal. There were slight increases in renal and uterine
    weights in rats given the highest level.

         Histological examination revealed no compound-related effects;
    there was no increase in the incidence of any tumour type. The
    no-effect level in this study was 300 mg/kg b.w./day (Coquet, 1975).

    2.2.4  Reproduction Studies

         No data were available from conventional fertility studies.

    2.2.4.1  Rats

         In a study where male rats were given doses of 30 mg/kg
    b.w./day for 8 days by an unspecified route, mitotic and meiotic
    abnormalities in spermatogonia were noted. Nuclear degeneration in
    spermatocytes was evident and the activities of some enzymes in the
    testes (glucose-6-phosphate dehydrogenase, succinic dehydrogenase
    and lactate dehydrogenase) appeared to be lowered in germinal cells.
    It is likely that such changes would to some extent reduce male
    fertility, but the degree of any reduction is impossible to assess
    from this study (Timmermans, 1974).

    2.2.5  Special studies on genotoxicity

         Results are shown in Table 2. Spiramycin has been tested for
    its ability to induce forward mutation in mammalian cells  in vitro,
    and  in vitro cytogenetics assays, and in the micronucleus test in
    the mouse, both as the adipate and the embonate. Negative results
    were obtained in all these tests.

    2.2.6  Special studies on teratogenicity

    2.2.6.1  Mice

         Groups of approximately 30 pregnant CD-1 mice were given gavage
    doses of 0, 100, 200, and 400 mg/kg b.w./day spiramycin adipate on
    days 5 to 15 of gestation. Mice were allowed to give birth normally
    on day 21 but those which did not were sacrificed and the uterine
    contents examined. At day 21, the young were examined and returned
    to the dams for 30 days. At this point the young were sacrificed and
    necropsied.

         No adverse effects were noted in dams given 100 or 200 mg/kg
    b.w./day but those given the highest dose showed slight decreases in
    food intake whilst on days 15 and 19 the body weights were
    significantly lower than control values.

         There was no evidence of embryotoxicity in treated animals and
    no effects on birth weight. Spiramycin treatment had no effects on
    post-natal development nor on post-natal mortality. There were no
    effects on body weights of pups from treated dams in the 30 day
    post-natal period. There was no evidence of any teratogenic effects
    (Pasquet, 1971a).

    
    Table 2: Results of genotoxicity assays on spiramycin
                                                                                               

    Test            Test Object      Concentration         Results        References
    system
                                                                                               

    Forward         Chinese          0-40000 iu/ml         negative2      Bonneau &
    mutation        hamster          (0-2,5 mg/ml)                        Cordier, 1986
    assay1          ovary cells      0-20000 iu/ml
                    (HGPRT           0-6.25 mg/ml)
                    locus)

    Forward         Chinese          0-1250 µg/ml          negative4      Diot et al., 1989
    mutation        hamster
    assay1,3        ovary cells      0-1250 µg/ml          negative4
                    (HGPRT
                    locus)

    In vitro        Chinese          0-5800 iu/ml          negative       Fournier et al.,
    cytogenetics    hamster          (0-1.8 mg/ml)                        1986
    assay1          ovary cells      0-600 iu/ml           negative
                                     (0-0.19 mg/ml)

    In vitro        Chinese          0-1000 µg/ml          negative       Melcion &
    cytogenics      hamster                                               Fournier, 1989
    assay1,3        ovary cells

    Micro           CD1 mouse        0,0.21, 0.42          negative       Cordier &
    nucleus test                     and 0.63M                            Fournier, 1989
                                     iu/kg b.w.,
                                     once or twice,
                                     i.v. (0, 65.6,
                                     131.3, 196.9
                                     mg/kg b.w.)

    Micro           CD1 mouse        0, 1250, 2500         negative       Fournier &
    nucleus test3                    and 5000 mg/kg                       Cordier, 1986
                                     b.w., once or
                                     twice, oral
                                                                                               

    1.   ± S9
    2.   Significant cytototoxicity at 10 000 and 10 000 iu/ml (3.13 and
         6.25 mg/ml)
    3.   Embonate; other tests conducted with adipate
    4.   Cytotoxic without metabolic activation at concentrations above
         500 µg/ml
    
    2.2.6.2  Rats

         Groups of 20 pregnant CD rats were treated intravenously on
    days 6-15 of gestation with doses of 0, 90 000, 180 000, and
    270 000 iu/kg b.w./day, equivalent to 0, 28, 56, and 84 mg/kg
    b.w./day spiramycin adipate. On day 21, females were killed by
    carbon dioxide inhalation and the uterine contents examined.

         The highest dose given produced brief (5 minutes) ataxia and
    tremors immediately after dosing, with occasional salivation. No
    other signs of maternal toxicity were noted. There were no effects
    on food consumption or body weight, and no adverse macroscopic
    findings were made at termination.

         There were no adverse effects on the numbers of corpora lutea,
    implantations or viable young, and the numbers of resorptions were
    not affected. A slight but significant reduction in fetal weight
    occurred at the intermediate dose but all values were within
    historical control ranges.

         There were no increased incidences of any foetal anomaly noted
    in this study (Tesh et al., 1985).

    2.2.6.3  Rabbits

         Groups of approximately 20 pregnant Fauve de Bourgogne rabbits
    were given 0, 100, 200, and 400 mg/kg b.w./day spiramycin adipate on
    days 6-16 of gestation. Animals were killed by cervical dislocation
    on day 28 of gestation and the uterine contents examined. Fetuses
    were investigated for gross defects, sex, and skeletal anomalies.

         A slight decrease in food intake was noted at the two highest
    doses. In animals which remained pregnant during treatment, there
    were no effects on body weights, but in those where pregnancy failed
    to continue, there was a fall in body weight compared with controls.
    No deaths occurred in any of the groups. Necropsy revealed marked
    caecal enlargement in females given the 200 and 400 mg/kg b.w./day
    doses. A small study conducted by the authors showed that oral
    spiramycin at 200 and 400 mg/kg b.w./day for 12 days produced
    approximately 43% and 70% increases in caecal weights indicating a
    substantial effect on microflora.

         The numbers of implantation sites was similar in treated and
    control animals. At the lowest dose there were no embryotoxic
    effects noted and the resorption rate was lower and the mean numbers
    of live fetuses higher than in controls. However, the 200 and
    400 mg/kg b.w./day doses produced pronounced embryotoxic effects.
    The numbers of animals which did not remain pregnant until
    termination was significantly decreased although the number of live

    litters per animal was unaffected in females which remained
    pregnant. The numbers of resorptions were much higher in these
    animals. The 100 and 200 mg/kg b.w./day doses had no effect on fetal
    weights or degree of ossification. However, the 400 mg/kg b.w./day
    dose produced growth retardation in the form of significantly
    reduced fetal weights. Ossification was normal. No teratogenic
    effects were noted (Pasquet, 1971b).

         Groups of 14 pregnant New Zealand white rabbits were given
    intravenous doses of 0, 90 000, 180 000, and 270 000 iu/kg b.w./day
    spiramycin adipate on days 6-19 of gestation, equivalent to 0, 28,
    56, and 84 mg/kg b.w./day. On day 29, the animals were killed by
    intravenous injection of pentobarbitone sodium and the uterine
    contents examined.

         During the study, 4 controls and two high dose rabbits died or
    were killed  in extremis. Necropsies were conducted but no effects
    attributable to treatment were found. Involuntary mastication,
    salivation and increased respiratory rate occurred in all groups
    following treatment but these effects were more pronounced in
    animals given spiramycin; the effects were not dose-related. Body
    weights were not affected by compound-treatment and food intake was
    unaffected. No adverse effects were detected at necropsy.

         The numbers of implantations, viable young, post and
    pre-implantation losses, fetal and placental weights showed no
    compound-related effects. There were no increased incidences of any
    fetal anomalies in this study (Tesh et al., 1986).

    2.2.7  Special studies on sensitization

         Spiramycin was tested for its skin sensitizing potential in the
    Dunkin-Hartley albino guinea-pig using the Magnusson-Kligman
    maximization test. Induction was carried out using the intradermal
    and topical routes followed by respective intradermal and topical
    challenges. There was no evidence of sensitization on challenge
    (Guillot, 1987).

    2.2.8  Special studies on microbiological activity

         Studies in animals were not available.

         The effects of orally administered spiramycin on the faecal and
    oral bacterial flora of humans were studied in healthy volunteers.
    Six subjects were given 1 g spiramycin twice a day for 5 days.
    Faecal and saliva samples were collected for examination before
    treatment with the drug, during the treatment, and 7 and 21 days
    after.

         There was no evidence of an increased colonization of the oral
    cavity by enterobacteria, enterococci, staphylococci or fungi as a
    result of treatment with spiramycin. The mean anaerobe counts of
    faeces were not affected by spiramycin intake; there were no effects
    on enterobacteria and enterocci counts. Similarly, there were no
    increases in fungi, staphylococci or  Pseudomonas aeruginosa. There
    was a shift in the numbers of enterobacteria resistant to the
    effects of high concentrations of spiramycin with counts increasing
    during treatment. Similarly, increases in Minimum Inhibitory
    Concentration (MIC) values for anaerobes and enterococci occurred
    during treatment. These changes were considered by the authors
    merely to reflect a selective pressure in the treatment period and
    they concluded that spiramycin had a limited effect on the
    intestinal flora of healthy volunteers at 2 g per day (approximately
    33 mg/kg/day) (Andremont et al., 1989).

         Spiramycin is known to be inactive against bateria of the
    family Enterobacteriacae. MICs were available for eight strains
    representative of four species of the anaerobic dominant flora of
    the large intestine  (Bacteroides, Eubacterium, Clostridium and
     Peptostreptococcus species) (Rico, 1990).

         The data indicated that the MICs varied from 0.25 to
    2 µg/ml in pure culture at 106 bacteria/ml. At 109 bacteria/ml
    the observed MIC values for the strains used varied from 2 to
    >128 µg/ml. In mixed culture the MIC values were 16 µg/ml at 106
    bacteria/ml and >128 µg/ml at 109 bacteria/ml. A model MIC of
    0.5 µg/ml was estimated (Roques, 1989, as cited in Rico, 1990).

    2.3  Observations in humans

         Spiramycin is well absorbed in humans after oral
    administration. Oral administration of 15-30 mg/kg b.w. to healthy
    young male adults resulted in peak plasma levels in 3-4 hours and
    plasma concentrations of 0.96-1.65 mg/l. After intravenous dosing
    (7.25 mg/kg b.w.) a large volume of distribution (Vdss 5.6 l/kg)
    was observed indicating extensive tissue distribution.
    Biotransformation did not appear to be important. Biliary excretion
    was the main route of excretion; only 7-20% of an oral dose was
    excreted in the urine. Spiramycin is known to achieve high
    tissue:serum concentrations in pulmonary and prostatic tissues, and
    in skin (Borgogne-Frydam et al., 1988; Levrat et al., 1964;
    Borgogne-Berezin, 1988; MacFarlane et al., 1968).

         Spiramycin appears to have no effects on the activities of drug
    metabolizing enzymes in humans. It did not affect the metabolism of
    theophylline, antipyrine or cephalosporin in man (Descotes et al.,
    1988; Pessayre et al., 1985; Debruyne et al., 1986; Descotes et al.,
    1986; Guillemain et al., 1989). It does not bind to cytochrome P-450
    and, as supporting evidence, it did not affect barbiturate-induced
    sleeping time in mice (see Section 2.1.3).

         Adverse reaction reporting data from France suggests a low
    incidence of effects in humans (Artiges, 1987). Mild
    gastrointestinal disorders have been noted after spiramycin therapy
    (Descotes et al., 1988). A 500 mg dose (approximately 7.5 mg/kg
    b.w.), four times daily for 5 days, resulted in severe abdominal
    cramps with bloody diarrhoea. The condition resolved within 24 hours
    of cessation of therapy (Decaux and Devroede, 1978). Studies in dogs
    suggest, however, that spiramycin has no effect on gut motility (Qin
    et al., 1987; Pilot & Qin, 1988) and the mechanism of action of the
    gastrointestinal tract in humans in unknown. There is a single case
    reported where spiramycin led to ulceration of the oesophagus in man
    (Perreard & Klotz, 1989).

         There are no cases of hepatitis reported which could be
    attributed to spiramycin therapy (Descotes et al., 1988).

         Despite the negative results obtained in animal sensitization
    studies, spiramycin has induced contact dermatitis in farmers and
    veterinarians handling the substance (Veien et al., 1980; Hjorth &
    Weismann, 1973; Foussereau et al., 1982) and the effects have been
    confirmed by patch testing (Veien et al., 1980; Veien et al., 1983;
    Hjorth & Weismann, 1973). Bronchial asthma has also been reported in
    workers exposed to spiramycin. Symptoms were produced following
    spiramycin diagnostic challenge (Moscato et al., 1984; Malo &
    Cartier, 1988). A single case of allergic toxic dermal vasculitis
    has been reported after spiramycin treatment (Galland et al., 1987).

    3.  COMMENTS

         The Committee considered animal pharmacokinetic data, the
    results of short-term studies in rats, dogs, and monkeys, a
    carcinogenicity study in rats, teratogenicity studies in mice, rats,
    and rabbits, and genotoxicity data. It also considered information
    on pharmacokinetics, adverse reactions, and microbiological effects
    in humans and minimum inhibitory concentration (MIC)1 data.

         Limited data suggested that orally administered spiramycin was
    well absorbed in rats; the same was true in humans. Studies in
    animals and humans suggested extensive tissue distribution. In pigs,
    the highest levels of spiramycin were found in the liver and kidney
    after dietary administration.

         In a short-term dietary study in which rats were given the
    equivalent of up to 3900 mg/kg b.w./day for 13 weeks, the only major
    effects noted were a reduction in neutrophil counts in some mid- and
    high-dose animals, and dilatation of the caecum; the latter was
    attributed to antibiotic effects on the rodent gut flora. The NOEL
    was equivalent to 140 mg/kg b.w./day. In another dietary study in
    the rat, animals were given up to the equivalent of 720 mg/kg
    b.w./day for 1 year. The only notable effects were reductions in the
    body weights of females receiving the higher doses, and increases in
    relative liver, kidney, and adrenal weights, at high-dose level
    animals in both sexes. Hepatic glycogen depletion occurred at all
    dose levels but not in controls. However, the significance of this
    was unknown.

         Oral doses of 200 and 500 mg/kg b.w./day of spiramycin given to
    dogs for 28 days produced no adverse effects. However, in a second
    study, when mongrel dogs were given 500 mg/kg b.w./day for up to 56
    days, reductions in spermatogenesis and testicular atrophy occurred.
    Kidney damage was also seen. All but two of the dogs (out of a total
    of 20) died before the end of this latter study. A NOEL could not be
    established, as only a single dose level was used. When beagles were
    given dietary spiramycin at up to the equivalent of 150 mg/kg
    b.w./day for 2 years, testicular damage was not seen, although
    degenerative changes occurred in other organs. The NOEL in this
    study was 75 mg/kg b.w./day. No adequate reproduction studies were
    available to the Committee.

    _______________

    1  The minimum inhibitory concentration is defined as the minimum
    concentration of an antimicrobial drug giving complete inhibition of
    growth of a particular microorganism, as judged by the naked eye
    after a given period of incubation (WHO Technical Report Series, No.
    610, 1977).

         In teratogenicity studies in mice, oral doses of spiramycin of
    up to 400 mg/kg b.w./day given over days 5-15 of gestation had no
    effects on the outcome of pregnancy. Intravenous doses of up to
    84 mg/kg b.w./day given on days 6-15 of gestation to rats and days
    6-19 to rabbits had no effect on development, but oral doses of 200
    and 400 mg/kg b.w./day in rabbits produced caecal enlargement in
    mothers and significant embryotoxicity. It is unlikely that these
    findings in rabbits are of significance for human hazard assessment,
    because this species is known to be particularly susceptible to the
    effects of antibiotics on the gut microflora. The embryotoxicity was
    probably related to maternal toxicity as neither was evident at
    100 mg/kg b.w./day.

         The genotoxic potential of spiramycin was investigated in a
    range of studies. Negative results were obtained with spiramycin
    adipate and embonate in a forward mutation test in mammalian cells
     in vitro, in an  in vitro cytogenetic assay and in the mouse
    micronucleus test.

         There was no evidence of carcinogenicity in the rat when
    spiramycin was tested in a dietary study at levels equivalent to up
    to 300 mg/kg b.w./day over a 2-year period.

         Adverse reactions in humans following spiramycin treatment are
    uncommon but, when encountered, the most frequently reported are
    mild gastrointestinal disturbances.

         In assessing the microbiological effects of spiramycin, the
    Committee considered the results of a study in human volunteers. Six
    subjects were given 1 g of spiramycin twice a day for 5 days and the
    effects on microorganisms of the oral cavity investigated. There was
    no evidence of increased colonization by the microorganisms
    investigated. Similarly, there were no major changes in the
    populations of the faecal microorganisms studied. However, MIC
    values for faecal anaerobes and enterococci increased during
    treatment and a NOEL could therefore not be established.

    4.  EVALUATION

      In view of the results of the toxicological studies of spiramycin
    and those of the human volunteer study on the effects of the drug on
    the gut flora, the Committee concluded that, of the data available,
    the results of  in vitro MIC investigations were the most
    appropriate for use in safety assessment. Data previously cited
    under 2.2.8, special studies on microbiological activity, were used
    in the Committee's evaluation. Data from recent work indicated that
    the MICs varied from 0.25 to 2 µg/ml in pure culture at 106
    bacteria/ml. With increasing density of the bacteria, however, the
    MIC values also increased significantly. At 109 bacteria/ml, the
    observed MICs for the same strains varied from 2 to > 128 µg/ml and
    were between 8 and 128 times as high as that at 106 bacteria/ml.
    In mixed culture the MIC of spiramycin for the mixed population was
    16 µg/ml at 106 bacteria/ml and greater than 128 µg/ml at 109
    bacteria/ml.

         In order to estimate the approximate concentration without
    antimicrobial effect on the intestinal flora, the following
    calculations were made (see Annex 5 of the report - Annex 1,
    reference 97). The modal value of the above  in vitro
    determinations was used as the initial value in subsequent
    calculations. This modal MIC1 was reported as 0.5 µg/ml at 106
    bacteria/ml. In order to cover adequately the range of MICs of
    sensitive bacteria, this value was divided by a factor of 10. In the
    light of data on the influence of bacterial density on MICs, the
    conditions of co-culture and anaerobiosis, and the unfavourable pH
    in significant parts of the intestine for the activity of
    spiramycin, the resulting value was multiplied by 20. This rather
    conservative factor seemed appropriate in view of the uncertainties
    involved in the extrapolation of data on the inhibition of bacterial
    growth from standardized  in vitro conditions to the conditions of
    growth in the gut. As a result of such extrapolation, a
    concentration without effect on the human intestinal flora of
    0.5 x 20/10 = 1 µg/ml (equivalent to 1 µg/g) was estimated.

         In human volunteers who had received two oral doses of 1 g of
    spiramycin daily on five consecutive days, a concentration of
    689 ± 48 µg (SD) of spiramycin in faeces was found on the fifth day
    of treatment. On the basis of a daily faecal bolus of about 150 g,
    it was possible to estimate the fraction of spiramycin that was
    bioavailable in active form to the bacteria of the intestine after

    ________________

    1  For the purpose of evaluation, the modal MIC means the most
    frequently observed MIC out of a frequency distribution of MICs
    determined for strains of the relevant species that were tested.

    oral administration. In subsequent calculations, this fraction was
    assumed to be 5% of the orally ingested dose. In view of the
    conservative margin of safety already provided by the estimated
    no-effect concentration, an additional safety factor of 10 was used
    to cover fully the variability between people of all extrapolated
    parameters. A temporary ADI was therefore calculated as follows:


                        Concentration
                        without effect  x   Daily faecal bolus (g)
                        on human gut
    Upper limit of      flora (µg/ml)
    temporary ADI  =    ___________________________________________
    (µg/kg of body
    weight)

                        Fraction of
                        oral dose       x   Safety factor x Weight of
                        bioavailable                        human (60 kg)


                   =    1 x 150
                        _______________

                        0.05 x 10 x 60

                   =    0-5 µg per kg of body weight

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    See Also:
       Toxicological Abbreviations
       Spiramycin (WHO Food Additives Series 34)
       SPIRAMYCIN (JECFA Evaluation)