First draft prepared by
Dr R. Fuchs
Ministry of Sciences
Republic of Croatia, Zagreb, Croatia
Spiramycin had previously been evaluated at the twelfth and
thirty-eighth meetings of the Committee (Annex 1, references 17 and
97). Based on the estimated concentration with no effect on the human
gut flora, a temporary ADI of 0-5 µg/kg bw was established at the
thirty-eighth meeting, with the requirement of additional in vivo
studies on the effect of spiramycin on the human intestinal flora.
This monograph addendum summarizes the data that have become
available since the previous evaluation (Annex 1, reference 98).
2. BIOLOGICAL DATA
2.1 Toxicological studies
2.1.1 Special studies on microbiological activity
A preliminary study of the effects of spiramycin on faecal
coliforms and enterococci of the human gastrointestinal flora was
carried out in mice. In this study, a dilution of pooled faecal flora
from healthy human volunteers was transferred anaerobically to twenty
6-week old female germ-free mice. A transfer of Bacteroides fragilis
was made prior to the human flora transfer. Seven days after the
human faecal flora transfer, the mice were divided into 4 groups of 5
mice each. Each mouse was caged individually. Group 1 (negative
control) received pure drinking-water. Group 2 (positive control)
received drinking-water containing spiramycin at a concentration of
200 ppm for 32 days. Groups 3 and 4 (test groups) received drinking-
water containing spiramycin at concentrations of 0.2 or 0.4 mg/l,
respectively (equivalent to 50 µg/kg bw/day and 100 µg/kg bw/day,
respectively), for 32 days.
Faecal samples were collected on day 0, on 10 occasions after day
9, and a final sample on day 32. Total counts for gram-negative
anaerobes, gram-positive anaerobes, coliforms and enterococci were
recorded on day 0. These same counts were performed on the 10 samples
taken from days 9 to 32. Additionally, an evaluation of the degree of
spiramycin resistance in coliforms and enterococci was performed on
these samples. For this purpose, coliforms were incubated and counted
on PCB-desoxycholate agar supplemented with 512 mg spiramycin/l.
Enterococci were incubated and counted on Bile Esculine agar
supplemented with 4 mg spiramycin/l.
No effect on coliform resistance to spiramycin was reported at
any dose level. The percentage of enterococci resistant to spiramycin
in the 50 µg/kg bw/day group was similar to negative controls. The
percent of enterococci resistant to spiramycin was increased in the
positive control and the high-dose group. In the negative control
group, large variations in the percentage of coliforms and enterococci
resistant to spiramycin were reported throughout the study. Values
ranged from 1.2 to 28% for coliforms and from 4.7 to 55% for
enterococci. Therefore, the significance of the increase in the
percentage of spiramycin resistant enterococci in test group was
questionable (Corpet, 1992).
A study was conducted to evaluate the effect of spiramycin on
chicks artificially infected with Salmonella typhimurium. Two
groups (A and C) of twelve 15-day old chicks received feed containing
no spiramycin and two groups (B and D) of twelve 15-day old chicks
consumed feed containing spiramycin embonate (equivalent to 20 mg
spiramycin base/kg feed). Groups A and B were inoculated with
Salmonella typhimurium, variety Copenhagen (strain 74-928,
resistant to nalidixic acid), 5 days after the initiation of
The following parameters were evaluated: the number of
salmonellae excreted per gram of faeces; the proportion of salmonellae
resistant to 10 antibiotics commonly used in human and veterinary
therapeutics; the degree of resistance; and the resistance spectrum.
These evaluations occurred at various times after the inoculation (2,
6, 8, 10, 13, 21, 28, 35, 42, and 49 days). At the beginning and end
of treatment, the proportion of faecal coliforms, staphylocci and
micrococci resistant to 10 commonly used antibiotics, the level of
resistance, and the resistance spectrum, were determined in the two
groups of noninfected chicks (groups C and D). Coliform counts were
conducted at the beginning and end of the trial on 100 strains (10
chicks selected per group, 5 strains per chick).
Spiramycin caused no significant increase in the relative number
of excreted salmonellae, nor did it produce an increase in the number
of chicks excreting salmonellae. The proportion of salmonellae
resistant to common antibiotics was not increased. At the end of the
trial, 83% of untreated chicks and 75% of treated chicks continued to
excrete salmonella in faeces. Twenty-seven percent of the E. coli
strains isolated before the beginning of treatment were found to be
resistant to 4 antibiotics (streptomycin, tetracycline,
chloramphenicol and sulfadiazine). At the end of the trial, the
proportion of resistant E. coli was similar in both groups (49% in
the untreated and 34% in the treated chicks). The salmonella
antibiotic resistance spectrum at the end of the trial was identical
to the one conducted at the beginning of the trial. Also, all
staphylococci and micrococci were sensitive to the antibiotics
studied. Thirty-five strains from 10 untreated chicks and 11 strains
from 10 treated chicks were isolated. Spiramycin intake was
determined to be 3000 µg/kg bw/day on day 20 and 1666 µg/kg bw/day on
day 70 (Benazet & Cartier, 1979).
An in vitro assessment of spiramycin MICs for 9 bacterial
species (10 or 20 strains of each species) from human gastrointestinal
flora was conducted. Dominant flora consisting of strictly anaerobic
bacteria were tested at a concentration of 109 bacteria/ml. These
included 10 strains of Bacteroides spp., Fusobacterium spp.,
Bifidobacterium spp., Eubacterium spp., Clostridium spp.,
Lactobacillus spp., and Peptostreptococcus spp. Sub-dominant
flora consisting of facultative aero-anaerobic and microaerophilic
bacteria, were also tested at a concentration of 107 bacteria/ml.
These included 20 strains each of Escherichia coli and
Enterococcus faecalis. In the total 110 strains tested, the MIC
value was >1 µg/ml. In 99 strains, the MIC value was >128 µg/ml
(Roques & Michel, 1993).
At the present meeting, the Committee considered data from new
in vivo and in vitro studies on the effect of spiramycin on
human gastrointestinal flora.
In an in vivo study in mice, a dilution of pooled faecal flora
from healthy human volunteers was transferred anaerobically to germ-
free mice. The animals were then treated with up to 200 mg
spiramycin/l of drinking-water for 32 days. Increases in resistant
microorganisms were observed at 0.2 mg/l of water, equivalent to 40
µg/kg bw/day. Although a quantitative endpoint was identified, there
were certain shortcomings in this study. There were large variations
in the number of resistant coliforms and resistant enterococci in the
non-treated control group and high populations of resistant organisms
in all groups before spiramycin treatment. Moreover, in the selective
medium used to determine the total and resistant coliforms and
enterococci in the pooled faeces of mice, only one concentration of
spiramycin was employed for each bacterial group.
The Committee also evaluated data from an in vivo study
performed in chickens in which the effects of the drug on Salmonella
typhimurium, Escherichia coli and several other microorganisms
were studied. The Committee concluded, however, that this study was
of little relevance for the microbiological evaluation of the effects
of spiramycin on human gastrointestinal flora because the micro-
organisms investigated in this study were not of human origin.
Studies to determine MIC values for spiramycin were conducted
using bacterial species isolated from healthy human volunteers.
Dominant flora tested consisted of strictly anaerobic bacteria (109
bacteria/ml), while the sub-dominant flora included facultative
aerobic and microaerophilic bacteria (107 bacteria/ml). In a total
of 110 strains tested, all the MIC values were >1 µg/ml. These
results confirmed those of the earlier studies evaluated at the
thirty-eighth meeting of the Committee, performed on a limited number
of strains. Taking into account the results of the studies already
evaluated at the previous meeting and new data from the in vitro
and in vivo studies, the Committee was reassured of the
microbiological safety of spiramycin.
At the thirty-eighth meeting of the Committee a temporary ADI of
5 µg/kg bw was calculated using the following formula:
Concentration without effect x Daily faecal
Upper limit of on human gut flora (1 µg/ml) bolus (g)
temporary ADI =
Fraction of x Safety x Human body
dose bioavailable factor weight
1 x 150
0.05 x 10 x 60
= 5 µg/kg bw
The safety factor of 10 was used to cover the variability between
individuals for all extrapolated parameters.
In view of the additional reassurance provided by the new data,
and as these studies covered a wide range of organisms, the Committee
reconsidered the magnitude of the safety factor and concluded that a
safety factor of 1 instead of 10, was appropriate. The other
parameters used in the previous evaluation provide a conservative
estimate. As a result the Committee established an ADI of 0-50 µg/kg
bw, using the above formula.
BENAZET, F. & CARTER, J.R. (1979). Influence of spiramycin (5 337
R.P.) on the implantation and excretion of Salmonella typhimurium
in artificially infected chicks and on the resistance of these
salmonenella, and faecal E. coli, Staphylococci and Micrococci
to common antibiotics. Unpublished report, Rhône Poulenc RP/RD/CNG
No. 20 193. Submitted to WHO by Rhône Mérieux, Toulouse, France.
CORPET, D.E. (1992). Effect of spiramycin residues on faecal
coliforms and enterococci in human flora associated mice. Unpublished
report from Institut National Recherche Agronomique, Toulouse, France.
Submitted to WHO by Rhône Mérieux, Toulouse, France.
ROQUES, C. & MICHEL, G. (1993). Determination of minimal inhibitory
concentrations (MIC) of spiramycin for bacterial species in the human
gut flora. Unpublished report Ph/93-152 from Faculté des Sciences
Pharmaceutiques, Toulouse, France. Submitted to WHO by Rhône Mérieux,