CHLORAMPHENICOL

1. EXPLANATION

2. BIOLOGICAL DATA

2.1 Biochemical aspects

2.2 Toxicological studies

2.3 Observations in humans

3. PROBLEMS IN TRADE CAUSED BY RESIDUES IN FOODS

From Food Standards Agency of Ireland (2002/2003).

4. ANALYTICAL METHODS

4.1 Preparation of the primary extract of the sample

4.2 Purification of the primary extract

4.3 Detection and quantification of residues of chloramphenicol

APCI, atmospheric pressure chemical ionization; ECD, electron capture detection; ESI, ion electrospray; ESI(-), negative ion electrospray; GC, gas chromatography; HRMS, high-resolution mass spectrometry; LC, liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MS, mass spectrometry; NICI, negative ion chemical ionization; RP, reverse phase; UV, ultraviolet.

^a (µg/l).

Chloramphenicol was first described as a new antibiotic produced by cultures of an actinomycete isolated from soil by Ehrlich et al. (1947). The soil samples from which the strains were isolated were collected from a mulched field near Caracas, Venezuela (strain ATCC 10712) and from a compost soil on the horticultural farm of the Illinois Agricultural Experiment Station at Urbana (strain ATCC 10595), respectively. It was demonstrated by Ehrlich et al. (1948) that this actinomycete was a new species. The dynamics of the synthesis of chloramphenicol were studied under laboratory conditions by Legator & Gottlieb (1953), who showed that the peak concentration of chloramphenicol in the culture medium was reached hours after the growth peak of the microorganisms. The antibiotic was not accumulated intracellularly. Addition of chloramphenicol to the culture medium inhibited the synthesis of the antibiotic.

Chloramphenicol was also isolated from the soil actinomycete Streptosporangium viridogriseum var. kofuense by Tamura et al. (1971) and from the marine snail Lunatia heros (moon snail) by Price et al. (1981).

The biosynthetic route of chloramphenicol starts with the general shikimate pathway for assembling aromatic structures. It then branches at chorismic acid to generate p­amino-phenylalanine, which serves as an advanced precursor of the p­nitrophenylserinol moiety of chloramphenicol (He et al., 2001; Lewis et al., 2003). 3’-O-Acetyl-chloramphenicol, which is commonly formed from chloramphenicol by many resistant bacteria, has also been isolated from the antibiotic-producing organism. It has been suggested that it is a protected intermediate in chloramphenicol biosynthesis, implicating acetylation as a self-resistance mechanism in the producing organism (Gross et al., 2002). 3’-O-Acetyl-chloramphenicol esterase activity was detected in cell-free extracts of strains of Streptomyces venezuela, other Streptomyces spp. and Streptosporangium viridogriseum var. kofuense, which produced chloramphenicol (Nakano et al., 1977).

Gottlieb & Siminoff (1952) studied the adsorption, stability, and rate of production of chloramphenicol in soil under different laboratory conditions. Chloramphenicol was poorly adsorbed to soil. When the drug was added to sterilized soil at a concentration of 50 mg/kg, approximately 80% could be recovered over the whole observation period of 14 days. When the same experiment was carried out using non-sterile soil, the antibiotic was slowly degraded. When sterilized soil was infested with S. venezuelae and was incubated for long periods, the authors were able to show the presence in soil of chloramphenicol formed by the microorganism following a lag phase of several weeks. The highest concentration measured was 1.12 mg/kg. Table 3 summarizes some of the results obtained in experiments designed to study the interaction of S. venezuelae and chloramphenicol-sensitive Bacillus subtilis. The table gives the results of the control experiments in which S. venezuelae was the only microorganism added.

Table 3. Production of chloramphenicol in sterilized soil infested with S. venezuelae

When organic substrates were added to the soil before sterilization, the production of chloramphenicol increased after the addition of S. venezuelae. Under the most favourable conditions of growth (in the presence of tryptone), chloramphenicol accumulated in the soil at a concentration of 25.0-27.8 mg/kg during 18-31 days of incubation. Addition of 1% of more "natural" substrates like alfalfa, corn stover and soybean straw also increased the production of chloramphenicol. However, only in the presence of alfalfa were significant quantities (concentration, 1.4mg/kg) observed.

These results should not be interpreted to mean that S. venezuelae produces chloramphenicol in soil in appreciable amounts under natural conditions. Natural soil is not usually a good substrate for production of antibiotic (Gottlieb, 1976). Ehrlich's group has investigated soils from 91 cultivated and grassland sites in nine states of the USA and from 13 other countries and found that soil samples were either infested with Streptomyces venezuelae or were not infested. No chloramphenicol was identified in extracts from either of these soils. Initially, the LOD of chloramphenicol was 0.3 mg/kg using a test based on the antimicrobial activity of chloramphenicol. In other experiments with LOD of 0.05 mg/kg in a chemical assay selective for the nitro group in the chloramphenicol molecule, these negative results were confirmed. If the soils were sterilized before seeding, chloramphenicol was found and identified in the infested soils (Ehrlich et al., 1952a, 1952b).

In their soil studies, Ehrlich et al. also investigated the recovery of smaller amounts of chloramphenicol added to non-sterilized soil. When chloramphenicol was incubated in sterilized soil at a concentration of 4.6 mg/kg for 92 days at 23-27 °C, recoveries were approximately 40% throughout the observation period. When the same incubation was performed with non-sterile samples of soil, the concentrations of chloramphenicol declined as a function of the incubation time as shown in Table 4. A graph of the same data (see Figure 1) suggests a biphasic curve for the degradation of chloramphenicol in soil. However, the second phase could be the result of the closeness to the LOQ of the observed concentrations. For the values ranging sufficiently above the LOQ (days 0-17 of the experiment), a half-life in the order of 3-4 days was estimated.

Whether antibiotics are produced in soil in appreciable amounts by indigenous soil organisms has remained a scientific dispute for several decades. Only recently, it has been demonstrated that an antibiotic can be synthesized in detectable amounts in soil. Using biosensor methods with very low LODs, Hansen et al. (2001) have demonstrated the presence of oxytetracycline produced by Streptomyces rimosus in untreated soil. However, similar studies with chloramphenicol were not found.

Table 4. Recovery of chloramphenicol from non-sterile soil after the addition of chloramphenicol at 4.6 mg/kg

Figure 1. Stability of chloramphenicol in non-sterile soil

The Food and Drug Administration of the USA has published an environmental assessment of chloramphenicol in the context of a proposal made in March 1985 to withdraw approval of new animal drug applications (NADAs) using chloramphenicoloral solution (Food and Drug Administration, 1985). Further information was obtained from the Hazardous Substances Databank, a database of the National Library of Medicine's TOXNET system (Hazardous Substances Databank, 2003). The information available from these sources (most of the original literature cited was not available for this review) is summarized as follows. The solubility of chloramphenicol in water at 25°C is 2.5 g/l over a wide range of pH. Chloramphenicol is not adsorbed to clay or soil to any significant degree and therefore has very high mobility in soil. Adsorption to sediment and bioconcentration in aquatic organisms should not be important processes. Chloramphenicol is degraded by biological, chemical, and photolytic means and undergoes oxidation, reduction and condensation reactions upon exposure to light in aqueous solution. Photochemical decomposition of chloramphenicol in vitro by ultraviolet-A (UV-A) light leads to the formation of p­nitrobenzaldehyde (pNB), p­nitrobenzoic acid (pNBA) and p­nitrosobenzoic acid (pNOBA); the latter comprises up to 45% by molarity of the starting amount of chloramphenicol (de Vries et al., 1994).

The half-life of chloramphenicol in soil at 25°C is 4.5 days; in pond water the half-life is 10.3 days at 25 °C and pH 8, and 20.8 days at 37°C and pH 6.

The log K_ow for chloramphenicol is 1.14. With regard to sorption coefficients to soil solids (K_d,solid), the range of values for chloramphenicol is in the same group as, for example, olaquindox, sulfamethazine, sulfathiazole, and metronidazole, which also appear to have little sorption affinity to soil particles, as is evidenced by their low values of K_d,solid (0.2-2 l/kg) (Tolls, 2001; Rabolle & Spliid, 2000).

Lai et al. (1995) estimated K_d,solid values of 0.4 l/kg for a freshwater sediment (salinity, 0 g/kg ; pH 7.7; sulfate, 4.8 mmol/l) from an eel pond in Taiwan, China, and of 0.2 l/kg for a marine sediment (salinity, 33 g/kg; pH 8.2; sulfate, 25.6 mmol/l) from a shrimp farm in Taiwan, China. The authors used top sediment (0-5cm). The typical concentration of chloramphenicol was 60-70 mg/l throughout all experiments. However, possible effects of the concentration of chloramphenicol were also studied using concentrations of 100, 200, and 400 mg/l. The average rates of chloramphenicol transformation were much higher under anaerobic conditions than under aerobic conditions. Some selected results are summarized in Table 5. Chloramphenicol also degraded very slowly in sterilized slurries.

Table 5. Rate of degradation (mg/l per day) of chloramphenicol in slurries of aquaculture pond soils

Although the physicochemical mechanism of sorption interactions is not known, it is possible that chloramphenicol might form charge-transfer complexes with soil constituents (Haderlein & Schwarzenbach, 1993).

Lai et al. (1997) added sodium chloride to brackish water top sediment (0-5cm) slurries (10% slurry; salinity, 24 g/kg; pH 7.9) of a shrimp pond to obtain salinities of 30 and 36 g/kg and conducted sorption and transformation experiments at final concentrations of chloramphenicol of 60-70 mg/l and temperatures of 20-25 °C. The experiments were carried out under either anaerobic or aerobic conditions. While sorption to soil of chloramphenicol was not influenced by changes in salinities and by aerobic or anaerobic conditions, the compound was much more stable under aerobic conditions. Increasing salinities also slowed down the degradation process under aerobic conditions but not under anaerobic conditions. Selected results from this study are summarized in Table 6.

Table 6. Influence of salinity and oxygen on sorption and stability of chloramphenicol in slurries of pond soils (brackish water)

From Lai et al. (1997).
^a k is the first order rate constant of the degradation of chloramphenicol. Its dimension is 1/day.

Chien et al. (1999) studied the degradation of chloramphenicol in aquaculture pond sediment. Freshwater (salinity, 0 g/kg) eel pond sediment slurries (10% w/v) were treated with sodium chloride to obtain salinities of 12, 24 and 36 g/kg. There were no significant differences in sorption rate either between aerobic and anaerobic conditions or among various salinities. Degradation of chloramphenicol fitted well to an exponential curve. The degradation rates under anaerobic conditions were significantly greater than those under aerobic conditions. As salinity increased, the degradation rates decreased under both aerobic and anaerobic conditions in this experiment. Selected results are summarized in Table 7.

Table 7. Influence of salinity and oxygen on sorption and stability of chloramphenicol in slurries of pond soils (eel pond)

From Chien et al. (1999).
^a k is the first order rate constant of the degradation of chloramphenicol. Its dimension is 1/day.

These studies demonstrate that chloramphenicol can be quite stable under suitable aerobic and ionic conditions and at normal pH.

Hirsch et al. (1999) have estimated that 20.1 million daily doses of chloramphenicol were prescribed in 1995 in Germany for human medical use (indications and doses not given). They estimate that 5-10% of the doses were excreted unchanged and 70-90% were excreted as the glucuronide. When they analysed 10 samples of sewage treatment plant effluents with a LOQ of 0.02 µg/l, they found one sample containing 0.56 µg/l of chloramphenicol. Of 52 samples of surface water, four contained chloramphenicol at concentrations greater than the LOQ, with a maximum of 0.06 µg/l in one sample. As a general rule, concentrations of antibiotic residues in sewage treatment plant effluents were approximately one order of magnitude higher than the concentrations found in surface water. In 59 samples of ground water, no chloramphenicol residues at concentrations greater than the LOQ were found. Chloramphenicol residues were not found in samples taken during a study conducted in the USA (Kolpin et al., 2002). It was also not found in samples of groundwater, surface water and drinking-water analysed in a recent study conducted in the Netherlands (Stolker et al., 2003) and using sensitive analytical methods with a LOQ for chloramphenicol of 0.005 µg/l (Versteegh et al., 2003).

Hamscher et al. (2003) studied sedimentation dust collected between 1981 and 2000 in a pig finishing unit (350-420 animals). Each year, 10-15 samples were collected over periods of 14-30 days. One randomly selected sample was analysed for each year. Chloramphenicol was detected in three out of 20 samples (representing the years of sampling 1989, 1991, and 1992) at concentrations of 1.96, 0.07, and 5.49 mg/kg, respectively. The samples had been stored for more than 10 years before analysis.

The mere isolation of chloramphenicol-resistant microorganisms from the environment, including soil, can probably not be used as an argument for the presence of the drug. The phenomenon of resistance is too complex and its occurrence does not need to be related to any history of the use of chloramphenicol itself. For example, Kardavy et al. (2000) isolated chloramphenicol-resistant species of Providencia rettgeri from the gut of larvae of the oil fly inhabiting the 40 000-year-old asphalt seeps of Rancho La Brea in California. They found a correlation between antibiotic resistance and organic solvent tolerance, which could be explained by the presence of an active efflux pump maintained by the constant selective pressure of the solvent-rich environment. These efflux pumps expel a broad range of comparatively hydrophobic antibiotics (chloramphenicol, erythromycin, nitrofurantoin, novobiocin, rifampin, spectinomycin, and vancomycin), most of which contain aromatic ring systems. Providencia spp. are also known as agents of nosocomial infections. Efflux pumps play also an important role in resistant strains of other bacterial species (Malléa et al., 2003).

Resistance to chloramphenicol in Salmonella enterica serovar typhimurium isolated from cattle in the USA has drastically increased over the years (Davis et al., 1999) owing to sharply increased occurrence of isolates displaying DT104-linked resistance. Although the use of chloramphenicol is prohibited in the USA, it was never authorized for use in food-producing animals and monitoring results do not suggest widespread illegal use. A gene conferring cross-resistance to florfenicol and chloramphenicol has been isolated from Salmonella enterica serovar typhimurium (S. typhimurium) DT104 (Bolton et al., 1999). A conjugative plasmid, pOLA52, conferring resistance to the antibiotic growth promoter olaquindox has been isolated from Escherichia coli from swine manure. It also confers resistance to ampicillin and chloramphenicol and has a high frequency of transfer between strains of E. coli (Sørensen et al., 2003).

Petersen et al. (2002) have studied the development of antibiotic resistance in integrated fish farms in Asia. The farms used antimicrobial agents and animal manure was shed directly into fish ponds as fertilizer in these farms. Three of the farms were using chloramphenicol in ducks and pigs. The impact of the use of antibiotics on the development of antimicrobial resistance among the indicator microorganisms was greatest at the beginning of a fish production cycle. However, the most significant increase in resistance to chloramphenicol occurred on a farm where this antibiotic was not used (amoxicillin, enrofloxacin, norfloxacin, tylosin were used on this farm).

For such reasons, the many reports dealing with resistance phenomena discovered in the environment have not been used here as an argument for the presence of chloramphenicol in the environment.

Table 8 provides an example and short summary of the relationship between age, live weight, feed intake and daily live-weight gain in pigs raised in the western hemisphere (Peer et al., 2001).

Table 8. Typical feed intakes and live-weight gain in pigs raised in the western hemisphere

From Peer et al. (2001).

A review of recent research papers indicates that the situation in south-east Asia could differ significantly from that observed in, for example, certain agricultural areas of the USA. Figure 2 summarizes the results of this review. More details are given in the Appendix I at the end of this document. From these details it can be seen that the typical daily dry matter intake of the animals is in the same order in the different hemispheres. However, the growth rates of the pigs used in the studies in south-east Asia were comparatively lower.

The data for the pigs in Iowa in Figure 2 were retrieved from the Internet (The pig site, 2003). The data for pigs in south-east Asia were taken from the studies summarized in the tables given in Appendix I at the end of this monograph. These data were collected from published results of research projects carried out in southeast Asia. They may give an incomplete picture and real conditions may vary largely from country to country, and between regions and provinces of a given country, and may also be subject to rapid changes. However, a complete review of animal feeding practices is outside the scope of this monograph. The information provided in the Appendix I is limited to the minimum necessary to derive suitable relationships between live weight, live-weight gain and feed intake since this information is needed to estimate soil ingestion as function of growth and intake of dry matter of the animals, and to relate hypothetical environmental doses of chloramphenicol to estimated soil ingestion.

Figure 2. Comparison of growth rates of pigs raised in different regions of the world

Body weight is the mean of the initial and the final body weight
of a feeding period. Average daily gain is the average over the whole feeding period.

As a result of the data discussed in this section, one can assume for simple model calculations that pigs generally eat approximately 35 g of dry matter per kg bw per day. Using the data in Figure 2, the average daily live-weight gain is estimated as a function of body weight (within the limits of 20 g and 75 kg body weight) according to the formula:

Average daily live-weight gain (g) = 0.164 g + 0.00693 (g/kg) × Body weight (kg)

It is not within the scope of this monograph to describe the range of different production systems. An example of data that have been gathered in the western hemisphere can be found in tabular form in a publication of the National Academy of Sciences of the USA (National Academy of Sciences, 1994). However, under the hypothesis that soil ingestion could be the cause of chloramphenicol residues in tissues and edible products, extensive systems—as they still exist, for example, in south-east Asia, with free-ranging scavenging chickens receiving supplementary feeds—are of particular interest. Local breeds may play an important role, at least regionally (Nguyen Dang Vang & Le Viet Ly, 2000). Some of these breeds have small bodies and growth rates and feed conversion may vary largely (Tran Thi Mai Phuong et al., 2003). A limited amount of data from south-east Asia were available from published results of research projects. These results, which are not necessarily representative, are summarized in more detail in Appendix I. Selected data are presented in Figure 3. The study of Duong Duy Dong (2003) provided useful data for model calculations.

Figure 3. Growth rate of chickens under different feeding conditions

As a result of the evaluation of the data discussed in this section, the daily live-weight gain of chickens with a body weight of between 150 and 1000 g was calculated according to the formula:

Average daily live-weight gain (g) = 3.599 g + 0.01623 (g/kg) × Body weight (kg)

The intake of dry matter is approximately 3 g per g of average daily live-weight gain.

Chloramphenicol is not highly systemically bioavailable after per-oral administration to ruminating cattle. The drug is largely degraded by microflora in the rumen. Corresponding calculations have not, therefore, been performed for cattle.

Soil ingestion varies seasonally and according to farm management. Using the titanium content of faeces as a stable indicator of soil ingestion, Thornton & Abrahams (1983) found that grazing cattle involuntarily ingest from 1% to nearly 18% of their dry matter intake as soil; sheep may ingest up to 30%. Abrahams et al. (2003) studied rates of soil ingestion by sheep grazing on metal-enriched flood-plain soils and found very high rates of soil intake during the winter/spring season with maximum rates during March, when soil ingestion exceeded 30% of the dry matter intake at two of the 11 sites investigated. No detailed quantitative data on soil ingestion of chickens and pigs were available for evaluation, although the phenomenon of soil ingestion in (food) animals is well recognized and frequently described in the literature.

Fries et al. (1982) investigated soil ingestion by dairy cattle using quantitative analysis of titanium in faecal samples and in soils to which the animals had access. Selected results are summarized in Table 9.

Table 9. Soil ingestion by dairy cattle

From Fries et al. (1982).

7.2.1 Pigs

Using the information collected in section 6, hypothetical intakes of chloramphenicol were calculated as a function of intake of dry matter and corresponding soil intake of the animals. It was assumed that soil represented 2% of dry matter intake. The concentration of chloramphenicol in soil was set at one of the following concentrations:

• <0.05 mg/kg, which corresponds to the LOD of the methods used by researchers from Parke-Davis who were unable to detect chloramphenicol above this limit when they analysed a large number of soil samples from different countries; or
• 1 mg/kg, which roughly corresponds to the highest concentration produced in soil samples to which "natural" organic matter was added, and which were then sterilized and infested with S. venezuelae; or
• 25 mg/kg, which roughly corresponds to the highest concentration found in laboratory experiments with soils treated with tryptone, and under the most favourable conditions.

The following steps were performed in the calculations¹:

• Calculation of body weight for every day of growth from 20 kg to 75 kg, using the formula for the average daily live-weight gain developed in section 6.1;
• Calculation of daily dry matter intake as a function of body weight;
• Calculation of soil intake as 2% of dry matter intake;
• Multiplication of the soil intake with the assumed chloramphenicol concentrations in soil;
• Summing up of the individual results obtained for days 1-119 of the theoretical period of growth of the animals, from 20 to 75 kg of body weight.

The hypothetical cumulative intake of chloramphenicol by a single animal whose live weight increases from 20 kg to 75 kg during 119 days (the average time required to achieve this body-weight gain) varies from <183 µg to 91.3 mg under these conditions. The results are summarized in Table 10. The cumulative intake overestimates the amount of chloramphenicol in the body of the animal. This value is much smaller since—even if the drug were 100% bioavailable—every daily dose is partly eliminated before the next dose is ingested.

Table 10. Hypothetical cumulative intake through ingestion of soil containing chloramphenicol, in pigs

In order to correctly estimate the amount of chloramphenicol in the body, bioavailability and elimination rate must be known. Unfortunately, the pharmacokinetic behaviour of low doses of chloramphenicol in pigs is not known. The elimination half-life for high doses is not well understood because quantitative determinations of chloramphenicol in plasma have usually not been extended to sufficiently long time periods after the administration of the dose. Boertz (1984) and Boertz et al. (1985) have carried out a residue study using 24 pigs each with a body weight of approximately 100 kg. The animals were given a single subcutaneous injection of chloramphenicol of 30 mg/kg bw. Two animals were slaughtered at each of 12 time-points between 4 h and 30 days after dosing. The kinetics in plasma and in all tissues examined suggest that there are at least two elimination phases, the first one characterized by a half-life of 6-10 h and a second one with a half-life of up to 100 h. There was excellent agreement between the results obtained with radioimmunoassay and with GC-ECD. However, the number of data points covering the terminal phase was too small to estimate the parameters of this phase with sufficient accuracy. A study using the same dose was carried out later in the same laboratory with the aim of producing reference material (Balizs & Arnold, 1989). Blood samples from five pigs were taken to predict the appropriate time of slaughter in order to obtain a muscle sample with a concentration of chloramphenicol of approximately 10 µg/kg. The seven time-points used covered the period between 1 h and 107 h. The following half lives were found under these conditions in the five animals: 10.6, 7.4, 6.5, 10.9 and 14.2 h, respectively. The data obtained in these two studies are summarized in Figure 4.

Figure 4. Plasma kinetics of parent chloramphenicol in pigs given a single subcutaneous dose at 30 mg/kg bw

The following assumptions were made and corresponding calculations were performed in order to obtain a crude estimate of the hypothetical amounts in the bodies of the animals corresponding to the cumulative intake shown in Table 10:

• Oral bioavailability was assumed to be 100%.
• The conditions defined in section 6, which are summarized in Appendix I, result in an oral dose of <0.035 or 0.7 or 17.5 µg/kg bw per day in pigs, depending on the hypothetical concentration of chloramphenicol in the soil.
• Two different models concerning the elimination half-life were then tested. In the first model, a single exponential term was used represented by half-lives of 2, 4, 6, 8, 10, or 100 h. In the second model, it was assumed that 95% of a daily dose is eliminated with a half-life of 10 h, and 5% is eliminated with a half-life of 100 h.

The results are summarized in Table 11.

Table 11. Estimated amounts of chloramphenicol in the body resulting from soil ingestion, in pigs^a

The apparent volume of distribution for chloramphenicol in pigs is given as 1.4 l/kg (Kroker, 1994). The studies of Boertz et al. (1984) have shown that concentrations of chloramphenicol in muscle were directly proportional to the concentrations found in plasma. Assuming 100% bioavailability of the ingested chloramphenicol, a crude estimate of maximum residue concentrations expected in pig muscle would result in the values presented in Table 12.

Table 12. Estimated muscle concentrations of chloramphenicol derived from soil ingestion, in pigs^a

From Boertz et al. (1984).

* Results are given as the range between the minimum amounts (calculated for the time-point immediately before the last dose) and the maximum amounts (calculated for the time-point immediately after the last dose).

For a given dose and all other conditions remaining constant the values of the minima and maxima and the differences between the extreme values depend only on the half-lives of elimination. This is illustrated in Figure 5 for constant multiple doses of 17.5 µg/kg bw per day and hypothetical elimination half-lives of 4, 10 and 100 h, respectively.

Figure 5. Effect of elimination half-life on steady-state concentrations

The uncertainties of the calculations are:

• The values used for development of body weight and intake of dry matter are likely to have a narrow range of variability.
• The amount of soil ingested by pigs is not known, but the estimate used here (2% of dry matter intake) is probably realistic or somewhat too low for free-ranging animals.
• The most influential factor is the production and resulting hypothetical (steady-state) concentration of chloramphenicol in soil. There is no evidence to assume that it is greater than 0.05 µg/kg in non-amended soil, and the possibility that it is much lower cannot be excluded. However, the effect of organic matter (straw, manure, compost, etc.) reaching the soil is totally unpredictable.
• The pharmacokinetics of low oral doses of chloramphenicol in pigs are not known. Should it be justified to use parameters derived from the kinetics of high doses—as has been done here—then the maximum hypothetical concentration in muscle tissue could be in the order of 0.04 µg/kg for chloramphenicol concentrations of <0.05 µg/kg in soil (see Table 12).
• Only assuming approximately 100% bioavailability and an extremely long elimination half-life of very small, ingested amounts of chloramphenicol, could residues exceed concentrations of 0.1 µg/kg for chloramphenicol concentrations of <0.05 µg/kg in soil (see Table 12).
• It is not known whether the addition of organic material (straw, bedding material, manure, slurry, etc. could enhance production of chloramphenicol by soil bacteria. If this were the case, as has been shown under laboratory conditions, residues in the above calculations could reach or even exceed concentrations of 1 µg/kg in muscle tissue according to Table 12.

7.2.2 Chickens

The calculations performed for chickens are similar to those described in section 6.1 for pigs. Therefore the description of the individual steps of the calculations already explained is not repeated here. The formulae describing live-weight gain and dry matter intake were developed in section 4.2. The basic assumptions and the results are summarized in Table 12. Only the maximum amounts and concentrations calculated for the time-point immediately after the last dose are given in Table 13.

Table 13. Hypothetical muscle concentrations of chloramphenicol derived from ingestion of soil, in chickens

Using a half-life of 7 h and a bioavailability of 35%.

A half-life of 7 h and a bioavailability of 35% were used for the above calculations on the basis of data published by Anadon et al. (1994). These authors have studied bioavailability, pharmacokinetics and residues of chloramphenicol in chickens. The pharmacokinetic properties (on the basis of a two-compartment open model) of chloramphenicol were determined in broiler chickens after intravenous and after oral administration. After oral administration at a dose of 30 or 50 mg/kg bw, chloramphenicol was absorbed rapidly (time to maximal concentration, 0.72 or 0.60 h, respectively) and eliminated with a mean half-life (t_1/2 beta) of 6.87 or 7.41 h, respectively. The bioavailability was 29% at a concentration of chloramphenicol of 30 mg/kg bw and 38% at 50 mg/kg bw.

The uncertainties of the calculations are similar to those described for the model in pigs:

• Again, the major uncertainty is associated with the estimates of likely concentrations of chloramphenicol in the soil.
• The well-established pharmacokinetic parameters given in the publication of Anadon et al. might not adequately describe the behaviour of chloramphenicol at very low doses. As has been noted for pigs, the kinetics of chloramphenicol at low doses is not known.

This section discusses the hypothetical possibility that residues resulting from past treatments of (humans and) animals could persist in the environment. Chloramphenicol was widely used in food producing animals in nearly all regions of the world until, within about the past 10 years, many countries and regions, including the European Union, imposed a complete ban on all uses in such animals in order to protect the health of consumers. A wide range of formulations, dosages and routes of administration, which cannot be reviewed here, were in use in terrestrial animals. Table 14 provides some additional examples of historical uses in aquaculture in Asia (Arthur et al., 2000). In Asia, chloramphenicol was widely used as a veterinarian antibiotic in aquaculture, according to a contemporary view (Somjetlertcharoen, 2002).

Table 14. Some historical uses of chloramphenicol in aquaculture in Asia

From Arthur et al. (2000).

Environmental residues of chloramphenicol in farm animals are most likely to originate from excreta of treated animals and a significant risk of secondary contamination could be assumed for farming systems in which manure is used as fertilizer.

Integrated farming of fish or shrimp and livestock, for example, combines aquaculture with production of pigs, ducks, chicken, and/or other livestock animals. Farming of such animals produces manure that may be used as soil fertilizer; however, it is also possible to make use of the nutrients contained in this manure in fish or shrimp ponds. These nutrients are taken up by bacteria and microalgae, which themselves are food for filtrating organisms, mostly zooplankton. Some of these organisms are then consumed by fish or shrimp.

According to the literature (Vincke, 1991), the number of pigs required per 10 000 m² of pond area varies from 40 to 300. Typically, about 100 weaned piglets (aged 2 months; average body weight, 12-15 kg) may be used per 10 000 m² (ponds are frequently much smaller than 10 000 m²). The pigs reach a body weight of 70-85 kg after 6-7 months. The pigsty may be constructed near the fish pond in traditional systems. Manure is then washed down to the pond.

If production of duck and fish is combined, ponds provide living and foraging space for both. A variety of strains of duck are used, each having a different fattening period. About 750-4000 ducklings may be stocked per 10 000 m², these animals reaching slaughter weight within 7-9 weeks. Both ducks and chickens (broilers or layers, 1000-6000 animals per 10 000 m²) are traditionally reared in pens beside or over the ponds.

If, in such systems, the terrestrial animals were to be treated with chloramphenicol, drug residues contained in their excreta would constantly be added to and diluted by the pond water, and would disappear according to the rate of degradation discussed in previous sections (half-life, 10-20 days in pond water; see section 5.4), and also according to the extent to which the water of the pond is exchanged.

However, it is also possible that fresh manure may be either directly applied to soil or that it remains in the housing or in a specific storage area before further use. In cases where manure is stored or processed, potential residues of chloramphenicol would be subject to degradation processes; however, published information about the kinetics of degradation of chloramphenicol under various conditions of storage and processing of animal excreta is extremely limited (see section 8.5). De Liguoro et al. (2003) demonstrated that processes that occur between the production of faeces and the application of manure to the soil are very effective in reducing the load of tylosin and oxytetracycline in the environment. Tylosin degraded rapidly in manure of treated Simmental calves, and was no longer detected 45 days after the last treatment. However, the half-life of oxytetracycline in manure was 30 days, and after 5 months maturation, oxytetracycline at a concentration of 820 µg/kg of manure was still detected.

In outside storage areas in particular, chloramphenicol would also be washed out and diluted by rainfall and possibly end up in basins retaining urine and manure fluids, where the conditions of stability of this compound are unknown and might be different from those prevailing in solid manure.

There are many ways of processing manure before its use as fertilizer. In some regions, farmyard manure is the traditional manure. It is a decomposed mixture of cattle dung and urine, together with straw and litter used as bedding material and feed not consumed by the animals. The waste material is collected regularly and placed in trenches finally plastered over with cow dung or earth slurry. It becomes ready for application as fertilizer after 3-4 months (Indiaagronet, 2003).

Frequently, the processing of livestock manure by anaerobic digestion in "bio-digesters" is a key component in integrated aquaculture systems. The main products from the biodigester are biogas and effluent, which is a potential fertilizer because the anaerobic digestion process results in conversion of organic nitrogen of the manure to ionized ammonia (NH₄⁺) (San Thy & Preston, 2003). Effluent may be superior to raw manure in supporting a higher yield of biomass in agricultural crops (Le Ha Chau, 1998a, 1998b). Fish may also grow faster when ponds are fertilized with biodigester effluent instead of unprocessed manure (Pich Sophin & Preston, 2001). Chloramphenicol is not likely to be stable under the described conditions of processing of manure.

The literature offers a wide range of numbers characterizing the quantity of manure produced by different species of animal under different conditions of husbandry, according to the way of processing the manure (e.g. capacity, initial loading, daily volumes added, water addition and retention times in biodigesters), and the amounts added to soil and/or ponds either in terms of dry matter or nitrogen per 10 000 m². The amount of manure or biodigester solids or effluents added also largely depends on the aquatic species and its density in the pond. It may be added directly or suspended in special manure bags. Fertilization is a basic part of pond preparation since plankton has to be available in adequate quantities before stocking with fish or shrimp.

The values given for production of excreta by terrestrial farm animals in the western hemisphere in Table 15 have been transformed into metric units from the non-metric units used in the original literature (Barker et al., 2003). Other sources give different values that are up to 25% higher or lower. Unfortunately, the term "manure" is rarely defined.

Table 15. Production of faeces and urine by farm animals

From Barker et al. (2003).

^a As voided.

Manure output by domestic animals is affected by many factors, which could differ widely from region to region in the world. In the tropics, a common method of comparing farm livestock is the tropical livestock unit (TLU) (LEAD Virtual Center, 2004). The TLU is based on the cow with a body weight of 250 kg and other animals are compared with it using (weight)^3/4 for scaling. The TLUs for selected tropical livestock are given in Table 16.

Table 16. TLU equivalents of some farm animals

TLU, tropical livestock unit.

The annual production of manure by one TLU (250 kg live weight) in an extensive system is roughly estimated to be about 1000 kg of dry matter. This estimate, which is not greatly different from the values given in Table 15, is based on the assumptions of a daily feed intake varying from 2 to 2.5% of body weight and a digestibility of feed varying from 40-60% (LEAD Virtual Center, 2004b).

In order to avoid over-sophisticated calculations performed using a purely hypothetical background, the following simplifying assumptions may be made. When pigs or cattle are treated with chloramphenicol they receive a dose of 50 mg/kg bw (per-oral, intramuscular or subcutaneous administration in pigs, and intramuscular or subcutaneous administration in cattle, except non-ruminating calves in which the per-oral route is useful) daily over 7 days. Chicken may be treated orally with drinking-water containing chloramphenicol (250 mg/l) over 7 days. Water intake (Guyer, 1996) by chickens is approximately 0.3 l/day per animal.

The resulting total doses would be approximately 87.5 g for a cow with a body weight of 250 kg, 26.25 g for a pig with a body weight of 75 kg, and 0.525 g per chicken. Assuming that a maximum of 50% of the dose is excreted as parent drug or hydrolysable conjugate of the parent drug in the combined mixed excreta within the treatment period plus an additional 3 days, these amounts would be contained in approximately 152 kg of beef cattle excreta, 62 kg of pig excreta and 0.7-1.5 kg of chicken excreta.

The expected concentrations of chloramphenicol equivalents, therefore, would range in the order of 0.29 g equivalents (chloramphenicol plus conjugates) per kg of bovine excreta, 0.21 g per kg of pig excreta and 0.18-0.38 g per kg of chicken excreta. Therefore one could expect to find chloramphenicol equivalents at a concentration of roughly 0.3g per kg of combined liquid and solid excreta from some major farm animals.

The degradation of chloramphenicol in manure has not been studied systematically. Since the molecule has a number of functional groups (e.g. the aromatic nitro group, the dichloro-acetyl side-chain) that could be attacked by known biotic processes, the behaviour of the compound cannot be predicted. As with other aromatic nitro compounds, the nitro group is expected to be particularly sensitive under anaerobic conditions (Hallas & Alexander, 1983). Chloramphenicol—like several other antibiotics (Johnson, 1994; Sanz et al., 1996; Lallai et al., 2002)— is, on the other hand, itself a powerful inhibitor of many anaerobic degradation processes. There are a few examples in the literature of situations in which the concentrations and persistence of other antibiotics and feed additives have been determined in various kinds of manure.

Runsey et al. (1977) conducted experiments in which chlortetracycline was used in a combination of feed additive for feedlot beef cattle. Fresh manure, stored manure, runoff water, manure weathered on pasture, and soil from pasture fertilized with manure were analysed for the additives. Seventeen percent of the chlorotetracycline fed to cattle appeared in fresh manure and 11% appeared in manure stored for 12 weeks. Aga et al. (2003) used an ELISA method to screen manure samples collected from hog lagoons and cattle feedlots for the presence of tetracycline residues. The concentrations measured varied from below the LOD (0.5 ng/kg) to 200 mg/kg. The degradation of tetracyclines in soil-applied manure was also followed using ELISA.; detectable concentrations were found for up to 28 days. Analysis of selected manure extracts by liquid chromatography with mass spectrometry (LC-MS) showed lower concentrations of total tetracyclines compared with the values obtained by ELISA, indicating the presence of other structurally related compounds or transformation products of tetracyclines. De Liguoro et al. (2003) followed the levels of oxytetracycline and tylosin over time in faeces, bedding and manure, and then in the soil of a manured field and surrounding drainage courses, after oral treatment of calves. Fifty Simmental calves were treated for 5 days with oxytetracycline at 60 mg/kg bw per day. After 15 days, the animals were treated for 5 days with tylosin at 20 mg/kg bw per day. Tylosin degraded rapidly and was no longer detected in manure 45 days after cessation of treatment, and no trace of the compound was detected in soil or surrounding water (LOD, 10 µg/l). The half-life of oxytetracycline in manure was 30 days, and the compound was still detectable in this matrix (concentration, 820 µg/kg) after 5 months maturation. Hamscher et al. (2002) investigated the distribution and persistence of tetracyclines and tylosin in a field fertilized twice with liquid manure. On the first fertilization, the manure contained tetracycline at 4.0 mg/kg and chlortetracycline at 0.1 mg/kg. Similar concentrations were applied again 1 year later. Soil sampling was performed 1 and 7 months after the first application and 1 month after the second application. At the third sampling time, the highest average concentrations of tetracycline of 86.2 (soil sublayer, 0-10 cm), 198.7 (10-20 cm), and 171.7 µg/kg (20-30 cm) and chlortetracycline at 4.6-7.3 µg/kg (all three sublayers) were found, indicating that tetracylines persisted and accumulated in the soil. Oxytetracycline and tylosin could not be detected in any sample analysed.

Campagnolo et al. (2002) analysed samples from swine waste storage lagoons and surface water and groundwater obtained from sites proximal to large-scale swine and poultry operations for multiple classes of antimicrobial compounds, and found multiple antimicrobial residues (commonly at concentrations of >100 µg/l) in storage lagoons. Schlusener et al. (2003) developed sensitive methods for the analysis of macrolides and ionophores and tiamulin in manure. The maximum concentrations found in manure samples were tiamulin at 43 µg/kg and salinomycin at 11 µg/kg. Ingerslev & Halling-Sorensen (2001) studied the biodegradability of olaquindox, metronidazole, and tylosin, in soil-manure slurries with 50 g of soil per litre. None of these substances persisted in the biodegradation experiments. Degradation half-lives for the primary degradation were: tylosin, 3.3-8.1 days; olaquindox, 5.8-8.8 days; and metronidazole, 13.1-26.9 days. Loke et al. (2000) studied the stability of tylosin A in manure under methanogenic conditions. Tylosin A is the major component (usually about 90% and not less than 80%) of tylosin. The half-life was less than two days. The authors could not determine whether the decrease in the concentration of tylosin A under aerobic and anaerobic conditions is caused by sorption, or by abiotic or biotic chemical degradation.

Haller et al. (2002) analysed six grab samples taken in Switzerland from manure pits on farms where medicinal feed had been applied and found total sulfonamide concentrations of up to 20 mg/kg of liquid manure.

The feed additive roxarsone is excreted unchanged by poultry. In experiments conducted by Garbarino et al. (2003) it was also found to be stable in fresh dried litter. However, when water was added to litter at about 50% w/w and the mixture was allowed to compost at 40°C, roxarsone disappeared and arsenate was formed within about 30 days. Increasing the amount of water and the incubation temperature increased the rate of degradation. The degradation process was most likely to be biotic.

According to the calculations performed in section 7, a concentration of 0.3g/kg of manure as estimated in section 8.4 would be high enough to cause significant residues in tissues of, for example, chickens scavenging on unprocessed manure within approximately ten half-life periods of the chloramphenicol residues. In practice, however, the excreta of treated animals would probably be diluted with excreta from untreated animals.

Assuming half-lives of <1 day to several days (depending on conditions such as temperature, water content, content of bedding material and particles), manure should no longer play a role as a potential source of contamination several weeks after discontinuation of the use of chloramphenicol in terrestrial animals. Since chloramphenicol is relatively unstable under anaerobic conditions, it would most likely not survive the process of farmyard manure production described above and the biodigester process. However, the compound could probably survive for several weeks or longer if administered to soil or grassland shortly after excretion by treated animals. Quantifiable amounts of residues could be taken up by free-ranging animals for a certain period of time. As mentioned in section 5.5, chloramphenicol could persist for many years in dry dusts formed during the mixing of chloramphenicol with dry feeds. The high concentrations of chloramphenicol present in small quantities of dust could occasionally cause residues in some animals, even a long time after the last use of chloramphenicol on a farm.

If unprocessed manure were to be directly added to aquatic systems, two different "worst-case" scenarios could be discussed:

Hypothetical "worst case" scenario 1: the daily excreta of 10 pigs with a body weight of 75 kg are washed directly into a pond with an area of 1000 m² and a depth of 1 m. After treatment of all animals as assumed in section 5.4, 0.5 × 26.25 × 10 = 0.13 kg of chloramphenicol equivalents (parent drug plus conjugates) would be added by this means to the pond during the days following treatment, resulting in a maximum concentration of <0.13 kg/1000 m³ or <130 µg/l. The half-life would be <10-20 days. Water exchange rates need also be considered in this scenario. There are no data enabling an estimate to be made of the quantities that could be taken up, e.g. through the gills, by aquatic species under these conditions. However, one cannot exclude the formation of traces of detectable residues in tissues. The problem would no longer exist several months after discontinuation of treatment of terrestrial animals.

Hypothetical "worstcase"scenario 2: unprocessed undiluted manure of treated animals is used in pond preparation. In practice the amounts would vary widely depending on, for example, the nitrogen requirements of the particular system. Assuming, for example, that in the preparation phase of a pond with an area of 10 000 m² a maximum primary dose of 3000 kg of unprocessed manure contaminated with chloramphenicol is applied when the water depth is 10 cm, followed by two maximum secondary doses of 400 kg each applied when the water depth is 30 cm and 100 cm, respectively (e.g. Pathak et al., 2000), the maximum total dose of chloramphenicol would be <0.3 × 3800, that is, <1140 g, depending on the degree of decomposition of active chloramphenicol residues. This amount would probably be quickly dissolved in the water and result in an initial concentration of <1.14 kg/10 000 m³ or <114 µg/l, which would decrease owing to degradation and water exchange. In this example, the true concentrations of chloramphenicol could be much less than 114 µg/l since the excreta would have been collected over a certain period of time, which would favour decomposition of chloramphenicol. Again, one could not exclude the possibility that the remaining chloramphenicol could lead to concentrations of residues in aquatic species at greater than the LOQ. However, such problems would rapidly disappear after discontinuation of the therapeutic use of chloramphenicol in terrestrial animals.

Another source of environmental contamination is the direct therapeutic use of chloramphenicol in aquatic species. In particular, its use in medicated feed had the potential to leave residues in the pond environment. Addition of medicated feed containing chloramphenicol at 2.5 g/kg of feed, five times per day for 3 days could mean, in practice, the introduction of up to 30 kg of feed/day per 10 000 m² (e.g. Cruz-Lacierda et al., 2000) depending on the density of the aquatic species. With this amount of feed, 22.5 g of chloramphenicol would be introduced into a pond with an area of 1000 m², for example; at least 15% of this dose would not be consumed (Primavera, 1994) by the aquatic species. Depending on the stability of the feed particles in water (Obaldo, 2001), and of the coating in particular, chloramphenicol could leach out at a rate of <0.5-5% per h. Assuming more stable particles and good quality coating, all the chloramphenicol contained in the feed could also fall down to the mud on the pond bottom and persist there with half-lives of up to several weeks, depending on conditions, such as oxygen content and salinity.

In summary, all the hypothetical considerations performed in this section suggest that after the implementation of a complete ban on the use of chloramphenicol in farm animals, the problems discussed in this section would vanish within several months.

The problems of potential significant carry-over in, for example, feed mills are not discussed in this document.

Assuming occasional contamination of fish or shellfish containing chloramphenicol at a concentration of 0.5 µg/kg, the additional chloramphenicol burden for average and preferential eaters of fish and shellfish could be estimated using data on consumption habits. Such data are not available at the international level. However, some data have been published for certain regions and for certain populations with very high consumption of fish and shellfish.

A survey of 212 people living in Singapore was conducted by Burger et al. (2002) to examine the relative importance of fish, shellfish, and other meat in the diet. From the authors' discussion of their findings in the context of international surveys on fish consumption, it appears that people in the Far East eat significantly more fish than do most people whose families have lived for generations in industrialized societies. In the study by Burger et al., it was found that people ate, on average, fish for about ten meals per week, chicken for eight meals per week, and shrimp and pork for about six meals each per week. While only 8% of people never ate fish, 18% ate fish at all 21 meals per week, and >20% ate shellfish for all 21 meals. Therefore, it seems to be appropriate to base an estimate of high seafood intake by preferential eaters on data obtained in surveys of people of Asian origin.

Sechena et al. (2003) described and quantified rates of seafood consumption, and acquisition and preparation habits of 202 first- and second-generation Asian-American and Pacific Islanders from 10 ethnic groups (Cambodian, Chinese, Phillippine, Hmong, Japanese, Korean, Lao, Mien, Samoan, and Vietnamese) in King County, Washington in 1997. Participants in the study were all consumers of seafood; only one person did not eat shellfish, which was otherwise the predominant seafood consumed by these people (45.9% of all seafood consumed). Rates of fish consumption were skewed considerably for all fish groups, indicating that a few respondents had a larger consumption rate than other respondents. The 90th percentile of all consumption rates for "all seafood" consumption was 3.928 g/kg bw per day.

Using this conservative estimate of seafood intake and multiplying it by the median concentration (0.5 µg/kg) of chloramphenicol found in the published alerts from the Food Standards Agency of Ireland, mentioned above, would result in an estimated daily intake of chloramphenicol of approximately 2 × 10­⁹ g/kg bw, or 0.12 µg for a person with a body weight of 60 kg. This estimate of intake could be slightly low since other products of animal origin could also occasionally contain traces of chloramphenicol.

Systemic drug absorption after ocular administration is well known and has been reported in hundreds of research papers for a large number of substances by numerous research groups. For a better understanding of these variable findings, it is important to consider the anatomy and physiology of the eye.

After topical ocular administration of drugs, the highly vascularized conjunctival and nasal mucosa are the major sites of systemic absorption. Drug absorption by this route bypasses the first-pass effects in the gastrointestinal tract and in the liver. However, local ophthalmic bioavailability can be very low, since the tear fluid is rapidly drained from the lower conjunctival sac through the puncta and the lachrymal duct into the lachrymal sac, from where it passes through the nasolachrymal duct into the inferior nasal meatus. From here, the fluid moves to the nasopharynx where it is swallowed into the gastrointestinal tract. It has been reported that drainage of the administered dose via the nasolacrimal system into the nasopharynx and the gastrointestinal tract takes place when the volume of fluid in the eye exceeds the normal lacrimal volume of 7-10 µl. Thus, the portion of the dose that is not eliminated by spillage is drained quickly and the time that the dose is in contact with the surfaces of the cornea and sclera is reduced to approximately 2 min (Saettone, 2002). According to Saettone (2003), the rate of loss of drug from the eye can be such that only 1-5% or less of the drug applied topically as a solution reaches the inner eye. For example, using timolol as a probe, Alvan et al. (1980) found that 12-88% of the dose administered was lost when 16 volunteers received two drops of 0.5% timolol ophthalmic solution in each eye, twice daily for 2 weeks. These anatomical and physiological properties of the eye explain the short pre-corneal residence time and the poor bioavailability of many eyedrop solutions that do not contain a viscosity agent in their formulation to prolong residence time (Sirbat et al., 2000).

In summary, there are three possible systemic absorption sites for drugs administered topically to the eye—conjunctival, inferior nasal, and gastrointestinal mucosa—and total bioavailability, therefore, should be assessed by estimating drug concentration in plasma or urine.

Part of the current knowledge of systemic bioavailability of drugs administered topically to the eye has accumulated from observations of systemic adverse effects after the use of topical ophthalmic preparations (Gerber et al., 1990; Flach, 1994; Jones et al., 1996; Shiuey & Eisenberg, 1996; Diamond, 1997). Urtti & Salminen (1993) emphasize the need to take into account the problem of systemic drug absorption in designing ocular drug and dosage forms to minimize systemic absorption and increase the oculospecificity of drugs, for example, reducing volume and increasing viscosity of eyedrops, controlling drug release from depot preparations, prodrug-derivatization, and addition of vasoconstrictive agents.

In many other studies in humans and animals, the systemic availability of ophthalmic drugs has been convincingly demonstrated by pharmacokinetic research. A few examples of hundreds of papers may be given here. Anderson (1980) determined that systemic absorption of epinephrine and dipivefrin hydrochloride was 55-65% of the ocularly applied dose. Chiang et al. (1983) gave delta 9-tetrahydrocannabinol to rabbits by ophthalmic administration, measured plasma concentrations, and compared with intravenous data to establish bioavailability. Kumar et al. (1985) studied the systemic absorption, plasma concentrations (maximum plasma concentrations are achieved within 10-20 min after topical instillation) and cardiovascular effects of ophthalmic solutions of phenylephrine hydrochloride. Salminen (1990) reviewed literature on human plasma concentrations after instillation of ocular timolol, levobunolol, atropine, cyclopentolate, scopolamine, phenylephrine, betamethasone and technetium-99. Kaila et al. (1999) determined the mean bioavailability of ocularly administered atropine to be 63.5% (n = 6; range, 19-95%). Sasaki et al. (2000) using tilisolol as a model substance and carboxymethylcellulose sodium salt as viscous polymer developed an in vivo pharmacokinetic model that accounts for corneal diffusion in albino rabbits and predicts the concentration of beta-blockers in the anterior segments, and characterizes the systemic absorption of instilled drug with ophthalmic viscous vehicle. Vainio-Jylha et al. (2001) studied the concentration of betaxolol in plasma after its topical ocular use. The drug showed a biphasic concentration-time curve in plasma, the first peak occurring already after several minutes, and concentrations were detectable even at 12 h after dosing. Korte et al. (2002) estimated the systemic bioavailability of timolol in eyedrops containing 0.5% timolol, and compared the cardiopulmonary effects of intravenous and ophthalmic timolol. The peak concentration of ophthalmic timolol in plasma was measured in most subjects within 15 min after drug administration. The systemic bioavailability was 78.0% (n = 8). Ophthalmic timolol resembled intravenous timolol in terms of systemic bioavailability, plasma kinetics, and cardiopulmonary effects. A recent article reviews the principles of systemic absorption of insulin applied topically to the eye. The physiological and pharmaceutical considerations for formulation development and the strategy of improving the systemic absorption and bioavailability of insulin are also discussed (Lee et al., 2002).

The above examples were given since with chloramphenicol alone, a substance that typically easily passes through all biological membranes and barriers, the systemic bioavailability after topical application is a controversial subject of discussion. However, this discussion has taken place in the absence of adequate studies using sensitive analytical methods.

In an early study by Trope et al. (1979), five children aged <9 years received eyedrops containing chloramphenicol, which were administered every 2 h to each eye for 5-7 days. Systemic absorption was not detected with the available assay methods. Walker et al. (1998) used HPLC with a limit of detection of 1 mg/l to investigate whether serum accumulation of chloramphenicol occurred after topical therapy in 40 patients. The mean dose of chloramphenicol received from eyedrops after 1 week of treatment was 8.0 mg, and after 2 weeks, 15.3 mg. As the authors had expected, chloramphenicol failed to accumulate to detectable levels. Contrary to these findings, chloramphenicol applied topically to the eye in ointment produced bacteriostatic concentrations of chloramphenicol in the aqueous humor, which lasted for several hours (George & Hanna, 1977). Hanna et al. (1978) reported that repeated drops of solutions containing 0.5% chloramphenicol for several hours were required to produce a concentration of chloramphenicol of 1 µg/ml in the aqueous humor. Yamada & Hiraki (1995) studied the ocular pharmacokinetics in rabbits of a combination formulation containing 0.25% chloramphenicol (CP) under various experimental conditions. When they sealed the cornea with cyanoacrylate glue to block transcorneal absorption, the absorbed fraction of chloramphenicol was <10% of that in the control, indicating that most of the chloramphenicol in the aqueous humor was derived from the transcorneal route. Ismail & Morton (1995) evaluated the bioavailability of three commercial chloramphenicol ophthalmic products supplemented with [¹⁴C]chloramphenicol. Samples of 50 µl were applied to the corneas of isolated bovine eyes. Their results indicate a greater bioavailability of chloramphenicol in ophthalmic ointments than in a liquid preparation, which gave extremely low levels of chloramphenicol in the aqueous humour and corneal.

Typical concentrations of chloramphenicol in eyedrops and ointments are 5 µg/µl (0.5%) and 10 µg/µl (1%), respectively. The recommended frequency of application varies. A dosage regimen that has been recommended on the basis of clinical trial suggests four daily treatments. Under these conditions, acute bacterial conjunctivitis was 88% healed within 9 days of treatment (Laerum et al., 1994).

The locally absorbed fraction of the drug dose enters the anterior chamber by penetration through the three layers and two membranes of the cornea. When a drop (50-75 µl) is applied to the eye it becomes diluted owing to reflex tearing, and the volume that is in excess of the normal lacrimal volume is drained from the eye. The average maximum capacity of the human conjunctival sac is 25-30 µl (Peterson, lecture notes). In consequence, it can be estimated that 66-75% of the administered dose is lost immediately, and that a maximum of 0.33 × 5 × 50 = 82.5 µg per application would be available for possible absorption through the cornea. Ointments have prolonged contact with the cornea, which improves absorption. Chloramphenicol apparently exhibits good penetration if the residence time is sufficiently long. Beasley et al. (1975) gave topical applications of an ophthalmic solution containing 0.5% chloramphenicol to patients at various times before cataract surgery. Aqueous humor obtained at the time of surgery contained intact chloramphenicol at a concentration of 3.5-6.7 µg/ml 1-2 h after topical administration. Aqueous humor is the watery fluid produced by the ciliary body that fills the space between the lens and cornea of the eye, which serves as a nutrient delivery system for the avascular cells in the cornea and eventually drains into a vein or lymphatic vessel. The normal human aqueous humor production during daytime is 2.75 ± 0.65 µl/min (mean ± standard deviation (SD)), while at night production is approximately half that amount (Larsson, 1998). The volume of the anterior chamber is dependent on age. In normal healthy subjects in two age groups, the volume of the anterior chamber was 247 ± 39 µl in the younger group (aged 20-30 years, n = 51) and 160 ± 39 µl in the older group (aged ≥60 years, n= 53) (Toris et al., 1999).

Using these values, the fraction of the bioavailable dose that is absorbed through the cornea per application of one drop (0.6-1.6 µg or 2.4-6.4 µg per day) during a treatment period can be calculated. This value probably represents a low estimate of systemic absorption, as it does not include absorption occurring at other possible sites described above. Nevertheless, this low estimate of systemic absorption of chloramphenicol from eyedrops is up to 50 times higher than the intake estimate calculated in section 7 and is of about the same order of magnitude as the intake corresponding to the highest contaminated seafood sample found in 2002.

No toxicological data on chloramphenicol were submitted to the Committee at its present meeting. The current evaluation was made on the basis of an extensive review of the scientific literature, particularly that published since the forty-second meeting, and with a focus on the toxicological data in humans.

Reports on uptake and metabolism in humans and animals showed that chloramphenicol is rapidly absorbed when administered orally and that it is extensively metabolized. A study with human bone marrow in vitro also showed evidence of metabolism in this tissue.

A number of studies with chloramphenicol and several metabolites of chloramphenicol have shown that they are cytotoxic to bone marrow in vitro.

In order to assess the genotoxicity of chloramphenicol, the Committee reassessed the results of tests reported at its thirty-second and forty-second meetings, and also considered new studies available in the published literature. Chloramphenicol was shown to cause DNA damage in a human fibroblast cell line and in primary cultures of rat hepatocytes, but not in human bone-marrow cells in vitro. The results of tests for reverse mutation in bacteria were mostly negative. In mammalian cells in vitro, chloramphenicol consistently gave positive results in tests for chromosomal aberrations, but results of tests for gene mutations and for sister chromatid exchange were inconsistent. Overall, these results indicated that chloramphenicol was genotoxic in vitro.

In tests for genotoxicity in vivo, chloramphenicol caused chromosomal aberrations in the bone marrow of mice, but gave negative results in tests for micronucleus formation in the bone marrow of mice and rats. It is not clear why contrasting results were obtained in these two assays, but the Committee considered that it was prudent to regard chloramphenicol as a mutagen in somatic cells in vivo.

According to data on heritable mutation reviewed by the Committee at previous meetings, chloramphenicol gave negative results in tests for dominant lethal mutation in mice and in Drosophila melanogaster.

At its present meeting, the Committee reviewed studies on genotoxicity with chloramphenicol and its metabolites in human bone marrow cells or peripheral blood lymphocytes in vitro. Only nitrosochloramphenicol and dehydrochloramphenicol induced DNA strand breaks, while chloramphenicol and other metabolites were without effects. This confirmed the results of previous studies that showed that some of the metabolites of chloramphenicol are genotoxic.

No adequate studies were available to evaluate the carcinogenicity of chloramphenicol in experimental animals. Chloramphenicol has been classified as "probably carcinogenic in humans" by IARC (IARC, 1990).

A number of toxicological studies in rodents were conducted in an effort to develop a model for chloramphenicol-induced aplastic anaemia in humans. While bone-marrow depression was confirmed in these studies, it did not progress to the characteristic aplastic anaemia of humans and was therefore not considered to be a suitable model for the disease in humans. The reversible bone-marrow depression that is seen in animals and humans receiving chloramphenicol can be attributed to its cytotoxicity.

A number of reports of epidemiological studies on the oral and injectable use of chloramphenicol in humans were reviewed; they confirmed that chloramphenicol was toxic to the bone marrow. In many cases, the toxic effects could be reversed by reducing or discontinuing treatment with chloramphenicol. There are, however, cases of aplastic anaemia that appear to be unrelated to dose and that are associated with a high mortality rate. In humans, the aplastic anaemia that is attributable to treatment with chloramphenicol is often fatal and is an idiosyncratic reaction that may have an immunological component. There is also evidence that some of the survivors of aplastic anaemia induced by chloramphenicol subsequently develop leukaemia.

The ophthalmic use of chloramphenicol represents the lowest therapeutic dose of the compound. Epidemiological data relating to the ophthalmic use of chloramphenicol in humans suggest that this form of administration is unlikely to be associated with aplastic anaemia. While any occurrence of aplastic anaemia associated with this form of administration is extremely rare, it is not possible to quantify the absolute risk of the ophthalmic use of chloramphenicol in humans because of the low background occurrence of idiopathic aplastic anaemia.

No adequate studies were available to fully assess potential reproductive toxicity with chloramphenicol. However, chloramphenicol has been shown to be embryotoxic and fetotoxic in a number of laboratory animal species.

In a case-control study in humans, the authors concluded that oral treatment with chloramphenicol in the second and third months of pregnancy presents little, if any, teratogenic risk. However, it is difficult to determine if any effects might occur during the first month of pregnancy.

Residue data

Most countries in the world do not permit the use of chloramphenicol in food-producing animals, in order to protect the health of consumers. Despite such restrictions, chloramphenicol has been detected in food samples collected in national monitoring programmes during the past 2 years and these residues have caused safety concerns. Shrimps, prawns, food products from aquatic animals, honey, royal jelly, meat and offal, sausage casings, rabbit and poultry meat and milk powder were among the commodities in which the drug was detected.

Chloramphenicol—an environmental contaminant?

While the results of monitoring clearly indicate intentional uses of chloramphenicol in some cases, it has also been argued that very low levels of chloramphenicol, such as those found in poultry and in products from aquaculture, could result from environmental contamination. The possibility that chloramphenicol could persist in the environment, or even be formed by soil microorganisms was discussed by the Fourteenth Session of CCRVDF, held in 2003 (Joint FAO/WHO Food Standards Programme, 2003).

Chloramphenicol was first described as an antibiotic produced by cultures of an actinomycete isolated from soil by Ehrlich et al. (1947). The soil samples were collected from a mulched field near Caracas, Venezuela, and from a compost soil on the horticultural farm of the Illinois Agricultural Experiment Station at Urbana. In studies conducted in 1952, the adsorption, stability, and rate of production of chloramphenicol in soil under different laboratory conditions were determined. When sterilized soil was inoculated with Streptomyces venezuelae and was incubated for long periods, the presence in soil of chloramphenicol formed by the microorganism after a lag phase of several weeks was demonstrated. The highest concentration measured was 1.12 mg/kg. However, when workers of the same group and in the same year analysed samples of normal soils collected from 91 cultivated and grassland sites from nine states of the USA and from 13 other countries, no chloramphenicol was identified in extracts from these soils. In this initial study (1952), the limit of detection of chloramphenicol was 0.3 mg/kg (turbidimetric assay using Shigella sonnel). In other experiments with an improved LOD of 0.05 mg/kg, and including the 91 samples of the previous study, these results were confirmed. Chloramphenicol was found and identified in soils only when organic material was added before sterilization and seeding with Streptomyces venezuela.

Whether antibiotics are produced in soil in detectable amounts by indigenous soil organisms has remained a subject of scientific dispute for several decades. It was only recently demonstrated that an antibiotic could be synthesized in detectable amounts in soil. Using biosensor methods with very low LODs, Streptomyces rimosus was found to produce oxytetracycline in untreated soil. However, similar studies have not been carried out for chloramphenicol.

With this background information and on the basis of an extensive search of the current literature, the Committee at its present meeting examined the following two hypothetical scenarios to explain potential environmental contamination of foods of animal origin with chloramphenicol:

Scenario 1

Assuming that:

• Chloramphenicol is naturally produced in the soil;
• Farm animals (e.g. pigs, chickens) ingest certain amounts of soil in their daily intake of dry matter;
• This may result in an uptake of chloramphenicol and subsequently in residues of chloramphenicol in tissues and products of those animals that are not associated with uses of chloramphenicol as a veterinary drug.

The Committee conducted a number of simple model calculations to estimate hypothetical intakes of chloramphenicol as a function of soil intake of animals. On the basis of data in the literature, it was assumed that 2% of the dry matter intake of pigs and chickens was soil. The concentration of chloramphenicol in soil was set at one of the following values:

• <0.05 mg/kg, corresponding to the LOD of the methods used by the authors of the historical studies, who were unable to detect chloramphenicol above this limit when they analysed a large number of soil samples from different countries; or
• 1 mg/kg, roughly corresponding to the highest concentration of chloramphenicol produced by S. venezuelae in inoculated sterilized soil samples supplemented with organic matter; or
• 25 mg/kg, roughly corresponding to the highest concentrations of chloramphenicol produced under experimental conditions with soils enhanced with tryptone and under the most favourable conditions.

The following steps were performed in the calculations:

• Calculation of average daily live-weight gain;
• Calculation of daily dry matter intake as function of body weight/live-weight gain;
• Calculation of soil intake as a fixed fraction of dry matter intake;
• Calculation of chloramphenicol intake using the above assumed chloramphenicol concentrations in soil;
• Estimation of the resulting tissue concentrations on the basis of the known pharmacokinetic behaviour of chloramphenicol.

The Committee concluded that concentrations of chloramphenicol in soil, as they were found under laboratory conditions in the presence of organic material, would suffice to explain occasional traces of chloramphenicol in tissues and products of free-ranging and/or scavenging livestock animals. With the LOD achieved in the 1950s, however, it was not possible to demonstrate the production of detectable amounts of chloramphenicol in soil. No further empirical data have been obtained since 1952. The possibility that chloramphenicol, produced naturally by soil microorganisms, could lead to the residues found in food-producing animals cannot be ruled out, but remains an unexplored hypothesis that is currently not supported by experimental data.

Scenario 2

Assuming that:

• Residues observed are caused by the exposure of some food-producing animals to chloramphenicol that persists in the environment. Such environmental sources result from historical uses as veterinary drug.
• Any persisting residues of chloramphenicol in farm environments are most likely to originate from excreta of treated animals, and a possible source of contamination could be farming systems in which manure is used as fertilizer, e.g. in integrated farming. Integrated farming of fish/shrimp and livestock combines aquaculture with production of pigs, ducks, chickens, and/or other livestock animals. Manure produced by livestock may be used as soil fertilizer. Additionally, it is possible that manure may be used as a nutrient in fish/shrimp ponds. The nutrients contained in manure are taken up by bacteria and micro-algae, which themselves feed filtrating organisms, mostly zooplankton. Some of these organisms are then consumed by fish or shrimp.

The Committee reviewed published literature in order to investigate the conditions of integrated farming as a potential cause of chloramphenicol residues in food of animal origin. Chloramphenicol was in use in farm animals as a veterinary drug before authorizations were withdrawn in many countries and regions. Before these restrictions, significant amounts of manure, probably containing intact chloramphenicol, had been used as fertilizers. Concentrations of chloramphenicol in fresh manure and certain patterns of its use in integrated farming of aquatic species could, in principle, also explain low concentrations of residue in certain farm animals, such as scavenging chickens, free-ranging pigs and in aquatic animals. However, when reviewing the available information on the half-life of chloramphenicol under different environmental conditions, no evidence was found to show that chloramphenicol could persist in the environment for periods longer than several months, except in dry dusts. Therefore, if there was any risk of food contamination resulting from historical use in the farm environment these problems should disappear within several months of cessation of the use of chloramphenicol. Similar considerations apply to the persistence of chloramphenicol in aquaculture after past uses of the drug as medicated feed that had been directly applied in ponds.

Analytical methods

In the past decade, several methods have been developed for the screening, quantification and confirmation of chloramphenicol in foods.

Screening for chloramphenicol could be performed with validated ELISA kits. The majority of these kits had a LOD of <1 µg/kg. However, confirmatory methods needed to be used in order to avoid false-positive results. For confirmatory purposes, highly sensitive methods based on GC-MS, either electron impact or negative chemical ionization mode, have been used. More recently, LC-MS/MS methods allow the determination and identification of chloramphenicol in food commodities such as honey, meat (chicken, turkey, pork and beef), fish and shellfish, at concentrations of <1 µg/kg. The LOD and LOQ are as low as 0.05 µg/kg and 0.1 µg/kg, respectively.

Conclusions

The Committee concluded that:

• There was no evidence supporting the hypothesis that chloramphenicol is synthesized naturally in detectable amounts in soil. Although this possibility is highly unlikely, data generated with modern analytical methods would be required for confirmation.
• There was evidence that the low concentrations of chloramphenicol detected by food monitoring programmes in the year 2002 could not originate from residues of chloramphenicol persisting in the environment after historical veterinary uses of the drug in food-producing animals. Owing to the high variability in the half-life of chloramphenicol under different environmental conditions, however, such a mechanism might occasionally cause low-level contamination in food.

Valid analytical methods are available to monitor low concentrations of chloramphenicol in foods; however, confirmatory methods require sophisticated and expensive equipment.

Since the advent of sensitive routine analytical methods for the quantitative determination of residues of chloramphenicol in foods of animal origin at concentrations far less than 1 µg/kg, illegal use of this drug is no longer attractive. Although only limited kinetic residue data exist that describe the terminal elimination from "deep compartments" (Arnold & Somogyi, 1986), these data suggest that the withholding times necessary to allow residues to deplete below 1 µg/kg with reasonable statistical certainty are already in the order of 15 to much longer than 30 days, depending on species, product, dose and route of administration. Therefore, the relatively frequent observation of residues far less than 1 µg/kg in monitoring programmes has stimulated a discussion on whether traces of chloramphenicol in foods could occasionally originate from environmental contamination.

The possible production of chloramphenicol in natural soils was investigated 50 years ago at the time of the discovery of chloramphenicol as a product of a soil microorganism. With the methodologies of that era it was not possible to convincingly demonstrate the production of appreciable amounts of chloramphenicol in soil. The question has never again been examined using adequate analytical methods. However, reliable laboratory experiments conducted by the group that discovered chloramphenicol have shown that in the presence of organic material as it may well occur on farmland chloramphenicol could be formed in concentrations up to 1 mg/kg. However no further empirical data have been generated since that time. These concentrations in soil which were found under laboratory conditions would suffice to explain occasional traces of chloramphenicol in tissues and products of free ranging and/or scavenging livestock animals.

Chloramphenicol was in common use as a veterinary drug before authorizations were withdrawn in many countries and regions. Before such restrictions, enormous amounts of manure likely to contain active chloramphenicol were used as fertilizers. Concentrations of chloramphenicol in fresh manure and certain patterns of its use could in principle explain low residue concentrations in certain farm animals, e.g. in scavenging chickens, free-ranging pigs and in aquatic animals. However, no convincing evidence has been found to show that chloramphenicol could persist in the environment for long times—except in dry dusts.

The occasional daily dietary intake of chloramphenicol by preferential eaters of fish and shellfish contaminated at the levels reported in the year 2002 (median value, 0.5 µg/kg) is more than one order of magnitude lower than the systemically bioavailable fraction of a daily dose of a typical ophthalmic formulation used in human medicine.

As there is evidence that chloramphenicol is a genotoxin in vivo, it is prudent to assume that chloramphenicol could cause some effects, such as cancer, through a genotoxic mechanism for which there is no identifiable threshold dose.

Epidemiological studies in humans show that treatment with chloramphenicol is associated with the induction of aplastic anaemia, which may be fatal. It was not possible to establish any dose-response relationship or threshold dose for the induction of aplastic anaemia.

The Committee noted that aplastic anaemia induced by chloramphenicol is a rare idiosyncratic response in humans, which may have an immunological component. In common with many other idiosyncratic immune system-mediated adverse reactions, no animal model could be developed. As a consequence of these considerations, and because the mechanism of chloramphenicol-induced aplastic anaemia remains unknown, the Committee could not identify any studies in animals or epidemiological studies that would assist the further toxicological evaluation of chloramphenicol.

The Committee concluded that it was not appropriate to establish an ADI for chloramphenicol.

Taking into account the present gaps in scientific knowledge, the Committee could not completely rule out the possibility that occasionally foods are contaminated from environmental sources. The easiest way to rule out this possibility would be a thorough investigation of chloramphenicol concentrations in soil using modern analytical methods.

The Committee was unable to propose options for regulating traces of chloramphenicol in foods. The human exposure which may have resulted from consumption of contaminated seafood in the years 2001-2003 was probably lower than the systemic exposure resulting from the use of ophthalmic formulations. However, the resulting risk could not be estimated.

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Summary of data from feeding experiments

Table 1. Feeding experiments with pigs in south-east Asia

RSM, rubber seeed meal; FWFS, fresh water fish silage; LW, live weight.

Table 2. Feeding experiments with chickens in south-east Asia

RSM, rubber seeed meal.
* Estimated from the data assuming 90% dry matter for broken rice and soya beans and 5% for duckweed.
^b Feed conversion rate (kg of feed/kg gain).
^c Carcass weight.
^d Dry matter intake.
^e The authors report the following relationship between dry matter intake (y) and live-weight gain (y): y= 0.415x - 9.75.

Model for the calculation of the concentrations in muscle of chloramphenicol ingested with soil.

A = A₀ × e^-kt

Alternatives:

The average plasma concentration at steady state after multiple doses can be calculated from (Rowland & Tozer, 1995):

where F is the fraction absorbed (dimensionless), D is the daily dose (µg/kg bw), and Cl is the body clearance

Values for Cl have to be taken from the literature.

Use of this model has been proposed by Sanders & Laurentie (2004).

Like the present monograph, this alternative model assumes:

Table 1. Daily oral dose necessary to maintain an average steady-state concentration of chloramphenicol of 1 µg/kg in muscle

^a It is assumed that either feed or water is the only source of contamination.

• The linearity of pharmacokinetics as function of the dose;
• A fraction of dose absorbed by oral route of 100% which is the worst-case scenario;
• A muscle/plasma ratio of chloramphenicol concentrations equal to 1.

The model gives the following results:

Using the selected model, it can be shown that the conditions described for pigs in Table 1, while producing an average steady-state concentration of chloramphenicol of 1 µg/kg, would at the same time cause a fluctuation at steady state of between approximately 0.06 µg/kg (minimum, shortly before the next dose) and 4.4 µg/kg (maximum, shortly after the last dose).

Clearance, volume of distribution and elimination half-life are linked through the equation:

Using, for example in the model for pigs, a clearance value of 4.16 (ml/min × kg) and a volume of distribution of 1400 ml would correspond to a half-life of 233 min or 3.89 h.

FOOTNOTES:

1. A detailed description of the model used for the calculations is given in Appendix II

    See Also:
       Toxicological Abbreviations
       Chloramphenicol (WHO Food Additives Series 23)
       Chloramphenicol (WHO Food Additives Series 33)
       CHLORAMPHENICOL (JECFA Evaluation)
       Chloramphenicol (IARC Summary & Evaluation, Volume 50, 1990)