737. Spiramycin (WHO Food Additives Series 29)

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
assay^1,3 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
assay^1,3 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 (Vd_{ss 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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TIMMERMANS, L. (1974) Influence of antibiotics on spermatogenesis.
J. Urol., 112: 348-349.

VEIEN, N.K., HATTEL, T., JUSTESEN, O. & NORHOLM, A. (1980)
Occupational contact dermatitis due to spiramycin and/or tylosin
among farmers. Contact Derm., 6: 410-413.

VEIEN, N.K., HATTEL, T., JUSTESEN, O. & NORHOLM, A. (1983) Patch
testing with substances not included in the standard series.
Contact Derm., 9: 304-308.

WOEHRLE, R. (1968) The repartition in tissues and organic liquids of
antibiotics of natural origin. Thèse de Doctorat en Médecine, Lyon:
Quoted in: Rico A (1990). Monograph. Spiramycin. Submitted to WHO by
Rhône-Poulenc Santé, Toulouse, France.

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