INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
Toxicological evaluation of certain veterinary drug
residues in food
WHO FOOD ADDITIVES SERIES 39
Prepared by:
The forty-eighth meeting of the Joint FAO/WHO Expert
Committee on Food Additives (JECFA)
World Health Organization, Geneva 1997
ENROFLOXACIN (addendum)
First draft prepared by Dr P.L. Chamberlain
Center for Veterinary Medicine
Food and Drug Administration,
Rockville, Maryland, USA
1. Explanation
2. Biological data
2.1 Biochemical aspects
2.1.1 Absorption, distribution, excretion
2.2 Toxicological studies
2.2.1 Special studies on microbiological effects
2.2.1.1 Tests in vitro
2.2.1.2 Observations in humans
3. Comments
4. Evaluation
5. References
1. EXPLANATION
Enrofloxacin is a fluroquinolone antibiotic that was evaluated at
the forty-third meeting of the Committee (Annex 1, reference 114). A
temporary ADI of 0-0.6 µg/kg bw was established at that time on the
basis of limited microbiological data. The Committee requested the
following information for evaluation in 1997: (i) detailed reports of
the investigations of MIC values in vitro that were submitted for
evaluation and (ii) information on the effects of enrofloxacin and
ciprofloxacin on specific genera of microorganisms obtained from the
human intestine. In addition, the Committee required that the results
of studies to determine the antimicrobial activity of the residues
other than enrofloxacin and ciprofloxacin be submitted for review as
soon as they became available.
This monograph addendum summarizes new data on the
microbiological activity of enrofloxacin and ciprofloxacin and the
pharmacokinetic properties of ciprofloxacin in humans that have become
available since the previous evaluation (Annex 1, reference 113).
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion
Ciprofloxacin has been identified as a major metabolite of
enrofloxacin in some food-producing animals. Because it is used
routinely in human medicine, its pharmacokinetic properties in humans
have been well studied.
The time to peak serum concentration in healthy subjects given a
single oral dose of 400 mg ciprofloxacin is 1-2 h. The peak serum
concentration is reported to be 1.5 µg/ml. The beta half-life of
elimination is 3.5 h. The area under the curve for serum concentration
versus time is about 5.5 mg.h/litre. The absolute oral bioavailability
is 63-69%. Administration with food does not cause clinically
important alterations in oral bioavailability. Elimination occurs by
both renal and non-renal routes, and metabolism accounts for < 20% of
elimination. The apparent volume of distribution exceeds 70 litres
(total body water). Only 14-25% of ciprofloxacin binds to serum
proteins, which facilitates tissue penetration and access to target
sites. The peak concentrations in urine are 25 to many hundred times
the serum concentrations. The concentrations in renal tissue tend to
be two to 10 times higher than the serum concentrations. The faecal
concentrations in the gastrointestinal tract are very high, and the
concentrations in bile tend to be two to 10 times greater than the
serum concentrations. The peak concentrations in saliva and bronchial
secretions are usually 0.2-1.0 of the serum concentrations, but the
concentrations in lung tissue are 1.6 to four times greater.
The main sites of absorption of ciprofloxacin are the duodenum
and jejunum. Radiolabelled ciprofloxacin given intravenously to rats
is found in the lower gastro-intestinal tract too soon after injection
to be accounted for by biliary excretion, suggesting transintestinal
secretion; 11% of ciprofloxacin administered intravenously to healthy
subjects was secreted across the bowel wall into the intestinal lumen.
Transintestinal elimination has been suggested elsewhere as the route
of elimination by the gastrointestinal tract of humans (Wolfson &
Hooper, 1991).
2.2 Toxicological studies
2.2.1 Special studies on microbiological effects
2.2.1.1 Tests in vitro
The minimum concentration of enrofloxacin that inhibited 50%
(MIC50) of colonies of 100 bacterial strains (10 isolates of 10
genera) of human intestinal origin was determined at three inoculum
levels: 109, 107, and 105 colony forming units (cfu) per ml.
Isolates were obtained before antibiotic therapy. The spectrum of
bacteria included gram-positive and gram-negative organisms and
facultative, moderate, and stringent anaerobes. This spectrum was
considered to be representative of the typical overall human
intestinal flora with respect to oxygen tolerance and taxonomy. The
study was conducted in full accordance with the principles of GLP. The
results are presented in Table 1 (Marshall, 1996).
Table 1. Mean minimum concentrations of enrofloxacin resulting in
inhibition of 50% of colonies (MIC50) of 100 bacterial strains of
human intestinal origin at three inoculum levels
Genus No. of strains Mean MIC50 (µg/ml)
109 107 105
Escherichia coli 10 0.062 0.031 0.031
Enterococcus spp. 10 1 1 0.25
Lactobacillus spp. 10 2a 0.5 0.5
Proteus vulgaris 10 0.25 0.125 0.125
Bacteroides spp. 10 2 1 2
Bifidobacterium spp. 10 2b 0.5 0.125
Fusobacterium spp. 10 0.25 0.125 0.125
Eubacterium spp. 10 0.5 0.25 0.25
Peptostreptococcus spp. 10 0.5b 0.25 0.25
Clostridium spp. 10 4 0.5 0.062
a Nominal concentration of 109 not achieved for three species
b Nominal concentration of 109 not achieved for one specie
In another study in vitro, the MIC50 levels of enrofloxacin
and nine of its metabolites were tested against 164 aerobic bacterial
strains of human origin (23 isolates of each of five genera) at a
single inoculum level (1 × 104 cfu/inoculation site, except for
Proteus spp. which were inoculated at 1 × 105 cfu/inoculation
well). Strains of the genera Escherichia, Enterococcus,
Staphylococcus, and Proteus were isolated from clinical sources
during the period 1992-95. The strains of E. coli, Staphylococcus,
and Proteus tested in this study were selected after classification
as 'untreated' with fluoroquinolones. Because of their low natural
susceptibility to fluoroquinolones, strains of the genera
Enterococcus and Lactobacillus were not selected. Lactobacilli
were isolated from the oral cavities of healthy people who had not
received antibiotics within four weeks before sampling.
The metabolites of enrofloxacin tested were ciprofloxacin,
oxoenrofloxacin, oxociprofloxacin, N-formyl ciprofloxacin,
desethylene enrofloxacin, desethylene ciprofloxacin, ring-opened
oxociprofloxacin, 7-aminoacetic fluoroquinolonic acid, and
7-aminofluoroquinolonic acid. The study was conducted in full
accordance with the Standard Laboratory Protocol, the principles of
the National Committee of Clinical Laboratory Standard, and the
guidelines of the Deutsches Institut fur Normung e.V. The
susceptibility of the tested strains to enrofloxacin and its
metabolites is summarized in Table 2. The most active substances
tested were enrofloxacin and ciprofloxacin, and the most susceptible
bacterial species was E. coli (Pirro, 1996).
The MIC50 of ciprofloxacin was determined against 100 bacterial
strains of human intestinal origin (10 isolates of each of 10 genera)
at a single inoculum level (107 cfu/ml). The isolates were obtained
from clinical cases before the initiation of antibiotic therapy. The
spectrum of bacteria included gram-positive and gram-negative
organisms and facultative, moderate, and stringent anaerobes. This
spectrum was considered to be representative of the typical overall
human gut flora with respect to oxygen tolerance and taxonomy. The
study was conducted in accordance with the principles of GLP
(Pridmore, 1996a). The results are given in Table 3.
In another study, the effect of pH on the MIC50 of enrofloxacin
against 36 bacterial isolates of human intestinal origin in vitro
was evaluated. The pH levels were chosen to encompass the range of
conditions likely to occur in the lower regions of the human
intestinal tract, while avoiding extremes likely to inhibit bacterial
growth in vitro. The isolates were obtained from human clinical
cases before initiation of antibiotic therapy. The spectrum of
bacteria included Gram-positive and gram-negative organisms and
facultative, moderate, and stringent anaerobes. This spectrum was
considered to be representative of the overall typical human
intestinal flora with respect to oxygen tolerance and taxonomy. The
strains were considered as representative of their respective genera
if they exhibited MIC50 values against enrofloxacin that were in the
region of the geometric mean for their genus in the study of Marshall
(1996), summarized above. A larger number of E. coli isolates was
selected on the basis of reference data generated by the sponsor. A
0.5 McFarland standardized inoculum was produced from each culture;
however, the inocula were not enumerated. The study was conducted in
full accordance with the principles of GLP. The results of this study
are presented in Table 4. The investigator reported that reduction of
fluoroquinolone activity at low pH has been demonstrated by other
investigators (Pridmore, 1996b).
Table 2. Susceptibility of aerobic human bacterial strains to enrofloxacin and nine of its metabolites
Compound Mean MIC50(µg/ml) against:
E. coli Proteus Lactobacillus Enterococcus Staphylococcus
(33 strains) (49 strains) (32 strains) (23 strains) (25 strains)
Enrofloxacin 0.03 0.125 8 1 0.125
Ciprofloxacin 0.015 0.03 16 1 0.25
Oxoxiprofloxacin 0.25 1 4 2 0.25
Desethylene 2 4 > 128 > 128 32
ciprofloxacin
Ring-opened 16 32 > 128 > 128 16
oxociprofloxacin
7-Aminoacetic 16 8 > 128 > 128 32
fluoroquinolonic
acid
Desethylene 2 8 > 128 16
enrofloxacin
N-Formyl cipro- 0.06 0.125 16 2 0.25
floxacin
7-Aminofluoro- 0.5 0.25 > 128 128 8
quinolonic acid
Oxoenrofloxacin Not tested 1 8 2 0.125
Table 3. Mean minimum concentrations of
ciprofloxacin resulting in inhibition of 50% of
colonies (MIC50) of 10 strains of each of 10
bacteria of human intestinal origin at one
inoculum level of 107 cfu/ml
Genus Mean MIC50
(µg/ml)
Escherichia coli 0.016
Enterococcus spp. 1
Lactobacillus spp. 0.5
Proteus vulgaris 0.031
Bacteroides spp. 8
Bifidobacterium spp. 0.031
Fusobacterium spp. 0.125
Eubacterium spp. 0.5
Peptostreptococcus spp. 0.125
Clostridium spp. 0.25a
a This result is questionable owing to low
inoculum counts and ranges (103-107 cfu/ml)
for the strains tested.
Table 4. Minimum inhibitory concentrations of enrofloxacin or concentrations that
inhibit 50% of colonies of bacterial isolates of human intestinal origin
in culture at three pH levels
Genus No. of Mean MIC50 (µg/ml)a or
isolates geometric mean MIC (µg/ml)
pH 7 pH 6 pH 5.2
Escherichia coli 9 0.031 0.062 0.25
Proteus spp. 3 0.125 0.315 2.52
Lactobacillus spp. 3 0.400 0.500 2.50
Enterococcus spp. 3 0.63 1.00 4.00
Bacteroides spp. 3 1.26 2.00 Not calculatedb
Fusobacterium spp. 3 0.315 0.315 Not calculatedb
Peptostreptococcus spp. 3 0.198 0.250 0.200
Clostridium spp. 3 0.157 0.198 Not calculatedb
Eubacterium spp. 3 0.198 0.198 0.125
Bifidobacterium spp. 3 0.397 1.00 5.04
a Mean MIC50 calculated only for E. coli, because of small numbers of isolates of
other genera tested
b Insufficient growth in the three isolates
The effects of differences in vitro and in vivo on the
microbiological activity of enrofloxacin against selected bacterial
strains of human intestinal origin were investigated using a test
system in which enrofloxacin and the test strains were exposed to
conditions simulating those found in the human intestinal tract. The
study was conducted in full compliance with the principles of GLP. The
MIC50 values of enrofloxacin against the cultures used in this study
had been determined previously in the study of Marshall (1996). Two
isolates of each of five genera obtained from patients before
antibiotic therapy were selected. The spectrum of bacteria included
gram-positive and gram-negative organisms and facultative, moderate,
and stringent anaerobes. This spectrum was considered to be
representative of the overall typical human intestinal flora with
respect to oxygen tolerance and taxonomy. The procedure used was as
follows:
* Enrofloxacin was added at two concentrations to cooked meat
medium:
- at a concentration similar to the geometric mean MIC50
determined for the respective microbial groups and
- at a concentration likely to be achieved in the gut after
consumption of typical residue levels.
* The enrofloxacin/cooked meat medium mixture was allowed to
equilibrate for 1 h, then supplemented with physiological levels
of pepsin, adjusted to pH 2, and incubated anaerobically at 37°C
for 1 h. The mixture was supple-mented with physiological levels
of bile salts and pancreatin, then adjusted to pH 7.
* The culture was inoculated into the mixture and incubated at 37°C
for 18 h.
* Viable counts were performed to determine bacterial survival, and
the results were compared with those for a control medium
inoculated with the test culture but no bacteria.
The results of this study are summarized in Table 5. The investigator
pointed out that the viable counts of all 10 bacterial strains exposed
to enrofloxacin in this intestinal model experiment increased during
the 18-h incubation period. These results suggest that the
concentrations of enrofloxacin tested, i.e. those that could
potentially occur in the human intestine, are unlikely to have a
significant effect on the complex intestinal ecosystem of the human
intestine (Watson, 1996).
Table 5. Effect of enrofloxacin on human intestinal isolates after incubation in a simple intestinal model in vitro
Strain Enrofloxacin Inoculum Total viable count Enrofloxacin
concentration density (cfu/ml test system) MIC (µg/ml)
(µg/ml) (cfu/ml)
On inoculation After 18 h
Enterococcus faecalis 0 1.6 × 109 4.9 × 106 1.3 × 108 1
0.56 5.6 × 106 5.6 × 107
0.90 6.4 × 106 6.1 × 107
Enterococcus spp. 0 3.1 × 108 1.3 × 106 9.4 × 107 1
0.56 1.2 × 106 6.1 × 107
0.90 1.4 × 106 6.9 × 107
Escherichia coli 0 9.1 × 108 7.0 × 106 9.6 × 107 0.062
0.56 7.4 × 106 5.2 × 107
0.04 8.9 × 106 8.0 × 107
Escherichia coli 0 2.3x 109 4.7 × 106 8.9 × 107 0.062
0.56 3.9 × 106 2.9 × 107
0.04 5.0 × 106 7.5 × 107
Clostridium 0 1.1 × 108 1.3 × 106 > 1.0 × 108 2
sporogenes 0.56 1.5 × 106 > 1.0 × 108
0.90 1.2 × 106 > 1.0 × 108
Clostridium 0 7.1 × 108 4.1 × 106 > 1.0 × 108 0.25
perfringens 0.56 3.8 × 106 > 1.0 × 108
0.90 4.7 × 106 > 1.0 × 108
Bifidobacterium 0 2.7 × 107 8.9 × 103 2.1 × 105 0.25
adolescentis 0.56 5.4 × 102 5.1 × 104
0.4 3.0 × 102 5.5 × 104
Bifidobacterium spp. 0 7.8 × 109 6.0 × 104 2.0 × 105 0.5
0.56 5.8 × 104 2.6 × 105
0.4 1.1 × 105 2.9 × 105
Bacteroides 0 4.4 × 106 1.7 × 104 1.1 × 109 2
thetaiotaomicron 0.56 1.7 × 104 1.6 × 109
1.4 1.8 × 104 1.6 × 109
Bacteroides vulgatus 0 6.9 × 106 7.8 × 104 1.7 × 109 2
0.56 7.6 × 104 1.8 × 109
1.4 6.9 × 104 1.6 × 109
2.2.1.2 Observations in humans
Fluroquinolones as a class have a broad spectrum of activity
against aerobic gram-negative bacteria, and their primary human
clinical use is for selective decontamination of the gastrointestinal
tract by decreasing the intestinal reservoir of potential aerobic and
facultative anaerobic pathogens while preserving the predominant
anaerobic bacteral flora. These conditions are clinically useful in
the treatment of travellers' diarrhoea, therapy for immunocompromised
patients, selective decontamination before colorectal surgery, and
therapy for burn victims and leukaemia patients. Therapeutic oral
doses of fluroquinolones to humans do not alter the intestinal
bacterial ecology or weaken the barrier effect. In addition, anaerobic
bacteria such as Bifidobacterium, Bacteroides, Eubacterium,
Fusobacterium, and Peptostreptococcus spp. are the main components
of the human gastrointestinal tract and are affected only slightly or
not at all by fluroquinolones. Although E. coli is extremely
sensitive to fluroquinolones in general, this species is a minor
component of the flora in the gastrointestinal tract (Goldstein
et al., 1987; Midtvedt, 1990; Vollaard et al., 1990; Lewin
et al., 1991; Boisseau, 1993; Nord, 1993; Hill, 1995; Nord, 1995).
A study of the effects of ciprofloxacin on human intestinal flora
in vivo was conducted in 12 healthy male subjects aged 19-40 years,
who were given an oral dose of 500 mg of ciprofloxacin every 12 h for
seven days. Faecal specimens were collected on the day before the
first dose was taken, on the last day of treatment, and one week
later. Bacterial counts were made, and ciprofloxacin concentrations
were assayed by a microbiological assay (sensitivity not stated). No
antibacterial activity was found in specimens taken before treatment
or on day 14. On day 7, the concentration of ciprofloxacin in the
faeces was 185-2220 µg/g (mean, 891; SD, 624). The authors attributed
these high concentrations to poor gastrointestinal absorption and not
to biliary excretion. They remarked that, although biliary excretion
is known to be significant in humans, it cannot account for the amount
of drug that is recovered from faeces (unpublished data). The effects
of ciprofloxacin on the faecal flora of the volunteers is shown in
Table 6. In addition, although most (75%) of the anaerobic strains
were sensitive to ciprofloxacin before treatment, only 12% were
sensitive after treatment. This difference was statistically
significant (p < 0.001) (Brumfitt et al., 1984).
The effects of therapeutic oral doses of ciprofloxacin on human
intestinal flora were studied in 12 patients with acute leukaemia who
were given a prophylactic oral dose of 500 mg ciprofloxacin every 12 h
during treatment to induce remission. The mean duration of treatment
was 42 days. The colon enterobacteria were eliminated within three to
five days. Bacteroides and Clostridium species were not affected,
but the numbers of anaerobic cocci were decreased. Pseudomonas and
Acinetobacter species were recovered, but these did not colonize or
cause infection (Rosenberg-Arska et al., 1985).
Table 6. Mean log10 counts of faecal flora per gram of
faeces of 12 healthy male volunteers treated orally with
ciprofloxacin
Flora Pretreatment Day 7 Day 14
Coliforms 7.24 < 2 6.25
Faecal streptococci 6.3 3.31a 6.93
Other streptococcib 5.61 2.72a 4.22
Staphylococci 4.86 2.14a 3.07a
Yeasts 2.75 3.45 3.27
Anaerobes 9.64 8.54 9.49
a Difference statistically significant from pretreatment value
(p < 0.05)
b Usually Lancefield group C
The effects of therapeutic oral doses of ciprofloxacin on human
intestinal flora were also studied in 12 volunteers (six male, six
female, 21-30 years old) given 400 mg ciprofloxacin orally every 12 h
for seven days. Faecal samples were obtained one to two days before
and two, five, eight, 10, and 15 days after ingestion of
ciprofloxacin. Qualitative and quantitative analysis of microflora
were performed. The results are shown in Table 7. The authors noted
that anaerobic species that are considered to be the main stabilizing
components of the bowel flora ( Bacteroides and Bifidus species)
were not affected by treatment; furthermore, no selection for
C. difficile was observed. This study thus shows that treatment with
ciprofloxacin can eliminate major components of the aerobic
gram-negative flora with little or no influence on anaerobic organisms
(Enzensberger et al., 1985).
The effect of repeated oral doses of ciprofloxacin (500 mg every
12 h for five consecutive days) on the oropharyngeal and colon
microflora was investigated in 12 healthy volunteers. The peak serum
concentration of ciprofloxacin was reported to be 2.8 mg/litre. Of the
oral microflora, only the aerobic gram-negative cocci, e.g.
Neisseria spp., were affected. In the colon, marked decreases in
the numbers of enterobacteria and enterococci were observed; changes
in the anaerobic microflora of the colon were considered minor. The
oral and colonic microflora returned to normal 14 days after the last
treatment. No new colonization of ciprofloxacin-resistant bacteria
(MIC50 > 1 mg/l) was observed. Neither C. difficile nor its
cytotoxin was detected (Bergan et al., 1986).
Table 7. Effects of oral doses of ciprofloxacin on faecal flora (mean colony forming units per gram
faeces (log10))
Species Day of treatment
0 2 5 8 10 15
Escherichia coli 7.6 0 0 0 5.8 (2) 6.8 (8)
Proteus vulgaris 0 0 0 0 4.0 (1) 7.6 (1)
Providencia stuartii 0 0 0 0 7.6 (1) 0
Citrobacter freundii 0 0 6.9 (1) 5.3 (1) 8.5 (1) 5.4 (3)
Streptococcus faecalis 7.8 5.7 (9) 5.5 (7) 5.8 (8) 6.2 (7) 6.1 (9)
Streptococcus faecium 7.4 (4) 5.7 (3) 5.3 (3) 4.2 (5) 6.2 (6) 6.1 (10)
Bacteroides spp. 9.3 8.7 9.2 9.5 9.5 9.8
Bifidobacterium spp. 9.1 8.3 8.6 9.3 9.6 9.1
Candida spp. 5.3 (3) 4.1 (6) 4.6 (6) 5.6 (5) 5.0 (5) 4.9 (2)
Geotrichum spp. 6.6 (1) 4.5 (4) 4.3 (4) 3.6 (1) 4.8 (4) 4.3 (2)
In parentheses, number of volunteers if fewer than 12
The effect of ciprofloxacin on the colonization resistance of
colonic microflora was investigated in 12 healthy volunteers given
50 mg ciprofloxacin every 6 h for six days. After two or three days of
treatment, Enterobacteriaceae strains had been eliminated from faecal
samples of all volunteers. A slight effect was observed on
enterococci, and the number of Candida spp. showed a minor increase.
No ciprofloxacin-resistant bacteria were isolated. The flora was
considered normal one week after the last treatment (van Saene
et al., 1986).
Six volunteers were given 500 mg ciprofloxacin orally once a day
for five days. Faecal samples were collected before, during, and after
treatment and analysed qualitatively and quantitatively for aerobic
and anaerobic microorganisms. In all volunteers, a marked reduction in
Enterobacteriaceae strains was observed during the treatment period
(from 106-107 to < 104 cfu/g faeces). In two volunteers,
colonization by resistant coagulase-negative staphylococci or
Corynebacterium spp. was observed. These strains were not detected
by the eighth day of the study. In one volunteer, the anaerobic
bacteria count decreased from 1012 to 109 cfu/g faeces during the
study (Holt et al., 1986).
Fourteen patients with liver cirrhosis were treated for urinary
or respiratory tract infections with 250 mg ciprofloxacin twice daily
or 500 mg ciprofloxacin once daily for five to 10 days. A marked
decrease in the Enterobacteriaceae count was observed during the first
few days at both doses; they disappeared between days 3 and 6 of
therapy but returned to normal two weeks after the last treatment. The
concentrations of Bacteroides spp. decreased during therapy. Two
patients, one receiving 250 mg and the other receiving 500 mg, had
Candida albicans in their faeces during therapy, and these were
still detectable 14 days after the last dose (Esposito et al.,
1987).
Studies of the effect of fluoroquinolones on the intestinal
microflora have been reviewed, including a summary of the results of
studies with ciprofloxacin, as shown in Table 8 (Korten & Murray,
1993).
Ten healthy volunteers were given single oral doses of 750 mg
ciprofloxacin, and blood samples were collected 1 h later. Faecal
samples were collected once before treatment then daily after
treatment for eight consecutive days. The mean serum concentration of
ciprofloxacin in the 10 volunteers 1 h after administration of the
drug was about 3 mg/litre. Faecal concentrations exceeded 3 mg/g in at
least one sample from each volunteer on days 2-5 after treatment. The
author did not propose an explanation for the sustained high
concentrations of the drug in faeces but attributed them to failure of
complete absorption and to gastrointestinal secretion of
ciprofloxacin. The total counts of anaerobes, streptococci,
staphylococci, and yeasts were not significantly modified by
treatment. No overgrowth by P. aeruginosa and no colonization by
C. difficile was observed, except in one subject who was apparently
a healthy carrier of C. difficile. A 2 log10 decrease in total
aerobe counts was attributed to a significant reduction in the total
count of Enterobacteriaceae (8 log10 cfu/g faeces down to 6 log10
cfu/g faeces; p < 0.01). A slight but significant increase in the
counts of Enterobacteriaceae resistant to nalidixic acid was also
observed (from 2 to 4 cfu/g faeces; p < 0.01). This resistance showed
a return to pretreatment levels on days 5-8 after treatment (Pecquet
et al., 1990).
Table 8. Representative studies of the effect of ciprofloxacin on human intestinal flora
Dose No. of Length of No. of Effect on Colonization Emergence Reference
(mg) doses/day treatment patients with yeasts of resistance
(days) Enterobacteria Enterococci Anaerobes
500 2 7 12 +++ ++ - - + (anaerobes) Brumfitt et al.
(1984)
400 2 7 12 +++ + - - - Enzensberger
et al. (1985)
500 2 42 15 +++ + + - - Rosenberg-Arska
et al. (1985)
500 2 5 12 +++ ++ + - - Bergan et al.
(1986)
500 1 5 6 +++ + + - - Holt et al.
(1986)
250 2 5-10 7 +++ - + Minor - Esposito et al.
(1987)
500 1 5-10 7 +++ - + Minor -
500 2 10 23 +++ ND ND - - Daikos et al.
(1987)
50 2a de Vries-Hospers
et al. (1987)
100 2 5 10 +++ + - Yesb -
200 2
500 2 10 12 +++ - ++(Veillonella) - - Shah et al.
(1987)
500 2 1-42 11 +++ - ND - - Scully et al.
(1987)
/750 3
50 4 7 15 +++ + - Yesb ND van Saene
et al. (1988a)
200 1 6 6 +++ + - Minor - van Saene
et al. (1988b)
Table 8. (continued)
Dose No. of Length of No. of Effect on Colonization Emergence Reference
(mg) doses/day treatment patients with yeasts of resistance
(days) Enterobacteria Enterococci Anaerobes
500 1 7 25 +++ - ND +c - Rademaker
et al. (1989)
750 2 2 21 +++ ++ +++ - - Brismar et al.
(1990)
/400 i.v. 2
500 2 5 14 ++ ++ + - ND Ljungberg
et al. (1990)
+++, strong suppression (> 4 log10/g faeces) or elimination of Enterobacteriaceae; ++, moderate suppression (approximately 3 log10/g
faeces); +, mild suppression (< 2 log10/g faeces); -, no significant effect; ND, not determined
a A mean faecal concentration of 80 mg/kg was measured.
b All persons became colonized with yeasts during treatment.
c Colonization with yeasts, but not different from placebo group
3. COMMENTS
Because ciprofloxacin has been shown to be a major metabolite of
enrofloxacin in some food-producing animals, the Committee considered
new information on the pharmacokinetics and effects of ciprofloxacin
on human intestinal microflora. New studies of the microbiological
effects of enrofloxacin and ciprofloxacin in vitro were also
evaluated by the Committee. All of these studies were conducted in
accordance with appropriate standards for study protocol and conduct.
The oral bioavailability of ciprofloxacin in humans is 63-69%,
and this value is not significantly altered by administration with
food. The main sites of absorption are the duodenum and jejunum. The
recovery of high concentrations of ciprofloxacin in faeces is
attributed primarily to secretion into the intestine.
The Committee noted that fluoroquinolones as a class have a broad
spectrum of activity against aerobic gram-negative bacteria. The
primary human clinical use of the fluoroquinolones is for selective
elimination of potential aerobic and facultative anaerobic pathogens
from the gastrointestinal tract while preserving the predominant
anaerobic bacterial flora. These properties of the drug are clinically
useful in the treatment of travellers' diarrhoea, therapy for
immunocompromised patients, selective decontamination prior to
colorectal surgery, and therapy associated with burns and leukaemia.
The Committee also noted that therapeutic oral doses of
fluoroquinolones to humans have been shown to have no appreciable
effect on the intestinal bacterial ecology and do not weaken the
barrier effect. In addition, anaerobic bacteria such as
Bifidobacterium, Bacteroides, Eubacterium, Fusobacterium, and
Peptostreptococcus spp., the main components of the flora of the
human gastrointestinal tract, are largely unaffected by these
compounds. Finally, the Committee noted that, although Escherichia
coli is extremely sensitive to fluoroquinolones in general, this
species is a minor component of the flora in the gastrointestinal
tract. These concepts guided the Committee in its evaluation and
interpretation of the results of studies of the effects of
enrofloxacin and ciprofloxacin on bacteria of human intestinal origin
in vitro and in vivo.
The minimum concentration of enrofloxacin causing 50% inhibition
(MIC50) was determined for 100 bacterial strains comprising 10
isolates from 10 bacterial genera, many of which are typically found
in the human gastrointestinal tract. These included Enterococcus
spp., E. coli, Lactobacillus spp., Proteus spp., Bacteroides
spp., Bifidobacterium spp., Fusobacterium spp., Eubacterium
spp., Peptostreptococcus spp., and Clostridium spp. E. coli was
found to be the most sensitive to enrofloxacin, with a mean MIC50
value of 0.03 µg/ml at an inoculum density of 107 colony forming units
per ml. The mean MIC50 value for the 10 strains of the most
sensitive relevant genus isolated from the human gastrointestinal
tract was 0.125 µg/ml for Fusobacterium spp. at an inoculum density
of 107 colony forming units per ml.
A study of identical design was conducted to determine the
MIC50 of ciprofloxacin against the same 100 bacterial strains.
E. coli was the most sensitive species, with a mean MIC50 value of
0.016 µg/ml at an inoculum density of 107 colony forming units per
ml. The mean MIC50 value for the 10 strains of the most sensitive
relevant genus isolated from the human gastrointestinal flora was
0.031 µg/ml for Bifidobacterium spp. at an inoculum density of 107
colony forming units per ml.
A study to determine the microbiological activity of enrofloxacin
metabolites against aerobic bacteria was reviewed by the Committee. In
this study, the MIC50 values of enrofloxacin and nine of its
metabolites were determined against 164 aerobic bacterial strains
isolated from the human intestinal microflora. Enrofloxacin and
ciprofloxacin were the most active compounds. E. coli was the most
sensitive species tested, the mean MIC50 values for enrofloxacin and
ciprofloxacin against 33 strains of E. coli being 0.030 and 0.015
µg/ml, respectively, at an inoculum density of 107 colony forming
units per ml.
The effect of pH on the MIC50 value of enrofloxacin against 36
bacterial isolates representative of the human intestinal flora was
considered. The pH levels tested (7.2, 6.2, and 5.2) encompassed the
range of conditions likely to occur in the lower regions of the human
intestinal tract, while avoiding extremes likely to inhibit bacterial
growth in vitro. The results were in agreement with those of
previous studies with fluoroquinolones, which showed that
antimicrobial activity decreased as the pH was lowered.
The effect of enrofloxacin on the growth of 10 bacterial strains
isolated from the human gastrointestinal tract was evaluated after
incubation in a simple in-vitro model. Enrofloxacin was added to the
test system at a concentration similar to the geometric mean MIC50
previously determined for each microbial group and at 0.56 µg/ml, the
concentration estimated to occur in the intestine after consumption of
residues. The viable counts of all 10 strains exposed to enrofloxacin
increased during the 18-h incubation period. The growth of eight of
the 10 strains was comparable to that of controls.
Numerous reports on the effects of oral doses of ciprofloxacin on
the intestinal flora of volunteers were available. The doses ranged
from 50 mg twice daily to 750 mg three times daily. In general, the
anaerobic microflora were, at most, mildly suppressed. The aerobic
microflora were the most sensitive to all doses of ciprofloxacin
tested.
Three studies of the levels of ciprofloxacin in faecal material
from volunteers who received oral doses of the compound enabled the
Committee to obtain a more direct estimate of the concentration of the
antimicrobial agent present in the colon than from the commonly used
indirect approach of subtracting the value for bioavailability from 1.
In these studies, volunteers received daily oral doses of
ciprofloxacin ranging from 100 to 1000 mg for up to seven days. The
concentrations of ciprofloxacin in faecal samples varied widely among
individuals. Two of the studies resulted in mean data that were used
to estimate that approximately 20% of an oral dose of ciprofloxacin is
present in the colon, assuming a colonic content of 220 g. The
Committee used this figure in establishing the upper limit of the ADI
for the antimicrobial activity of enrofloxacin. This is in keeping
with the decision taken at the forty-third meeting of the Committee to
use data on excretion from studies with ciprofloxacin in humans for
the purpose of calculating the upper limit of the ADI for the
antimicrobial activity of enrofloxacin.
The upper limit of the ADI based on the antimicrobial activity of
enrofloxacin was calculated on the basis of the formula described on
p. 12, as follows:
0.125 µg/ga × 220 g
Upper limit =
of ADI 0.20b × 1c × 60 kg
= 2.3 µg/kg bw
a For the purpose of this evaluation, the MIC50 value is the
mean MIC50 for enrofloxacin against the 10 strains of the
sensitive relevant genus isolated from the human intestinal
tract, in this case Fusobacterium spp.
b The fraction of an oral dose available to act upon
microorganisms in the colon was determined on the basis of a
study in volunteers in which about 20% of an oral dose of
ciprofloxacin was present in the colon.
c A safety factor of 1 was used because extensive, relevant
microbiological data were available.
4. EVALUATION
The Committee noted that the antimicrobial activity of
ciprofloxacin against the relevant human intestinal microflora was
about four times greater than that of enrofloxacin and that consumers
may be exposed to residues of ciprofloxacin in some species of
food-producing animals. The Committee considered that the
approximately fourfold greater microbiological activity of
ciprofloxacin should be taken into account in recommending MRLs for
this metabolite.
The forty-third Committee established a temporary ADI of 0-0.6
µg/kg bw on the basis of the limited summary data from microbiological
tests on ciprofloxacin. The present Committee considered the
toxicological data on enrofloxacin and the microbiological effects of
enrofloxacin and ciprofloxacin and concluded that the microbiological
effects in vitro were the most sensitive end-point on which to
establish an ADI. Therefore, an ADI of 0-2 µg/kg bw was established on
the basis of data on the antimicrobial activity of enrofloxacin
against 10 strains of the most sensitive relevant genus isolated from
the human gastrointestinal tract. The Committee noted that this ADI
provides an adequate margin of safety in relation to the NOEL of 1.2
mg/kg bw per day for testicular toxicity in dogs described in the
report of the forty-third Committee.
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See Also:
Toxicological Abbreviations
Enrofloxacin (WHO Food Additives Series 34)
ENROFLOXACIN (JECFA Evaluation)