WHO FOOD ADDITIVES SERIES: 53
First draft prepared by
J. Wongtavatchai
Faculty of Veterinary Medicine, Chulalongkorn University, Bangkok, Thailand
J.G. McLean
Camberwell, Victoria, Australia
F. Ramos
Laboratório de Bromatologia, Nutrição e Hidrologia, Faculdade de Farmácia, Universidade de Coimbra, Portugal
and
D. Arnold
Berlin, Germany
Chloramphenicol is a broad-spectrum antibiotic with historical veterinary uses in all major food-producing animals and with current uses in humans and companion animals. Chloramphenicol was evaluated previously by the Committee at its twelfth, thirty-second and forty-second meetings (Annex 1, references 17, 80 and 110). A number of other agencies have also reviewed chloramphenicol (e.g. International Agency for Research on Cancer (IARC), 1990; European Committee for Veterinary Medicinal Products, 1994; United States Food and Drug Administration, 1985). Concerns have been expressed about the genotoxicity of chloramphenicol and its metabolites, its embryo- and fetotoxicity, its carcinogenic potential in humans and the lack of a dose-response relationship for aplastic anaemia caused by treatment with chloramphenicol in humans. Deficiencies identified in data on the toxicity of chloramphenicol include information necessary for the assessment of carcinogenicity and effects on reproduction. An acceptable daily intake (ADI) has never been allocated and consequently a maximum residue limit (MRL) has not been assigned. This has resulted in the restriction of the use of chloramphenicol in veterinary medicine to non-food use.
Chloramphenicol was originally isolated from the soil organism Streptomyces venezuelae in 1947, but is now produced synthetically. Three common forms are used for systemic therapy, depending on the route of administration; a free base form of chloramphenicol, chloramphenicol palmitate and chloramphenicol succinate. Other formulations are also available for topical use. Chloramphenicol usually acts as a bacteriostatic, but at higher concentrations or against some very susceptible organisms it can be bactericidal. It is used in the treatment of human infection with Salmonella typhi (typhoid) and other forms of salmonellosis, and other life-threatening infections of the central nervous system and respiratory tract (Parfitt, 1999). In veterinary medicine, chloramphenicol is used for the treatment of a variety of infections in animals, particularly those caused by anaerobic bacteria or those that are resistant to other antimicrobial agents. Chloramphenicol in animals is well absorbed by both oral and parenteral routes (Plumb, 2002).
There is good evidence for a haemotoxic effect of chloramphenicol in humans, with two forms of toxicity being described. The first is a commonly occurring, dose-related reversible bone-marrow depression, which develops during treatment and is reversible following the withdrawal of the drug. The second is a severe aplastic anaemia, which is non-dose-related and often irreversible.
This monograph summarizes the recently published literature and submitted unpublished information on the toxicity of chloramphenicol.
The usual therapeutic range for chloramphenicol in serum in most animal species is 5-15 µg/ml. After dosing, chloramphenicol is widely distributed throughout the body. The volume of distribution of chloramphenicol reported in companion animals is 1.8 l/kg in the dog, 2.4 l/kg in the cat and 1.41 l/kg in the horse. Hepatic metabolism by a glucuronidative mechanism is the principle pathway for which chloramphenicol undergoes biotransformation to an inactive metabolite, chloramphenicol glucuronide. Only about 5-15% of the drug is excreted unchanged in the urine. Dogs excrete only about 6% of unchanged drug into the urine. Cats have a limited ability to form glucuronide conjugates with drugs and therefore excrete chloramphenicol more slowly than other animals, with 25% or more of the administered dose excreted unchanged in the urine. The elimination half-life of chloramphenicol is 1.1-5.0 h in dog, <1 h in foals and ponies, and 4-8 h in cats (Adams, 1995; Plumb, 2002).
The pharmacokinetic properties of orally administered chloramphenicol in broiler chickens indicate that chloramphenicol is rapidly absorbed. At a dose of 30 or 50 mg/kg bw, the drug reached the maximum plasma concentration at 0.72 h or 0.60 h, it was eliminated with a mean half-life (t1/2 beta) of 6.87 or 7.41 h and had a bioavailability of 29% or 38% respectively. A concentration of chloramphenicol of >5 µg/ml was achieved in plasma at 15 min, and persisted up to 2 or 4 h after administration of chloramphenicol at a dose of 30 or 50 mg/kg bw. When chickens received an oral dose of chloramphenicol at 50 mg/kg bw once daily for 4 days, three metabolites, dehydrochloramphenicol; nitrophenylaminopropanedione-chloramphenicol (NPAP-chloramphenicol); and nitrosochloramphenicol were found in kidney, liver and muscle. The study found a slow clearance of residues, particularly of the NPAP and nitrosochloramphenicol residues, which were detected in tissues at 12 days after dosing (Anadon et al., 1994).
Plasma concentrations of chloramphenicol were determined in four calves given four oral doses of chloramphenicol palmitate, each corresponding to a dose of chloramphenicol of 25 mg/kgbw, at 12 h intervals. After the fourth dose, the plasma concentration of chloramphenicol reached a steady state of 5-6 µg/ml. The half-life of elimination was 4.5 h. Dehydrochloramphenicol at a concentration of 3-7 µg/ml was also detected in the plasma. The authors suggested that dehydrochloramphenicol, which is a metabolite produced by intestinal bacteria and suggested to be associated with fatal aplastic anaemia in human, may occur in edible tissues of animals treated with chloramphenicol (Gassner & Wuethrich, 1994).
Several metabolites of chloramphenicol were identified in urine samples obtained from male Wistar rats and from a human volunteer given tritiated chloramphenicol at a dose of 10 mg/kg bw by mouth. In rats, the two most abundant metabolites detected in the first 24 h by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) were chloramphenicol-base and chloramphenicol-acetylarylamine. The remaining metabolites were unchanged chloramphenicol, chloramphenicol-oxamic acid, chloramphenicol-alcohol, chloramphenicol-glucuronide and chloramphenicol-oxamylethanolamine. Similar end-products were also found in the human volunteer. The amount of chloramphenicol-oxamylethanolamine, which is an end-product of chloramphenicol biotransformation that was previously reported in birds, represented 0.74% and 1.37% of the ingested radioactivity found in the rat and human urine samples. The formation of chloramphenicol-oxamylethanolamine as an end-product of the metabolism of chloramphenicol by the liver was proven by the release of chloramphenicol-oxamylethanolamine after incubation of tritiated chloramphenicol with hepatocyte microsomes from rats treated with phenobarbital (Cravedi et al., 1995).
Chloramphenicol-aldehyde as a metabolic product of chloramphenicol was identified in a study in four children with major infections treated with chloramphenicol at a dose of 50 mg/kg bw per day). The residues in samples of urine collected during the treatment were analysed using HPLC and GC-MS. Results indicated the existence of compounds with characteristics corresponding to the synthesized chloramphenicol-aldehyde derivatives. The author concluded that chloramphenicol-aldehyde, a metabolite that was toxic to bone marrow and previously observed only in rat hepatic tissue, was a new metabolite in humans (Holt, 1995).
A study performed in vitro in human bone-marrow cells from 72 donors showed that chloramphenicol succinate was metabolized to chloramphenicol and other metabolites. In all 72 samples, the HPLC analysis of cell-free supernatant obtained from samples of bone marrow incubated with chloramphenicol succinate for 3 h at 37°C revealed a substance with a retention time corresponding to that of chloramphenicol. Other metabolites, nitrosochloramphenicol and unidentified metabolites, were also presented in some bone marrow samples. The study referred to the ultimate toxic derivatives of chloramphenicol produced in the bone marrow in situ as resulting from the metabolic biotransformation of the prodrug, thus indicating the marrow as both the site of metabolic conversion and the target of injury (Ambekar et al., 2000).
Catalan et al. (1993) reported that chloramphenicol induced sister chromatid exchange in bovine lymphocyte cultures, an effect that is indicative of DNA damage and repair, and also observed a delay in the cell cycle.
The cytotoxicity and genotoxicity of chloramphenicol and six metabolites were investigated in human bone-marrow cells (RiBM cells) in vitro. The six metabolites tested in the study were: nitrosochloramphenicol, chloramphenicol-glucuronide, chloramphenicol base (NAPD), and an alcohol derivative (hydroxy-amphenicol, HAP), dehydrochloramphenicol and nitrophenyl-aminopropanedione-chloramphenicol (NPAP-chloramphenicol). The cytotoxic effect was demonstrated by inhibition of incorporation of tritiated thymidine into DNA. The genotoxic effect was evaluated by the induction of DNA single-strand breaks. Cytotoxic effects were found with three metabolites, nitrosochloramphenicol, dehydrochloramphenicol and NPAP-chloramphenicol, at concentrations ranging from 2 × 105 to 2 × 104 mol/l. Nitrosochloramphenicol appeared to be the most potent cytotoxic compound tested, while chloramphenicol-glucuronide and HAP were not cytotoxic in RiBM cells. A similar cytotoxic response was reported earlier in human peripheral blood lymphocytes, but dehydrochloramphenicol was the most inhibitory compound. Genotoxic potential was observed with nitrosochloramphenicol and dehydrochloramphenicol at a concentration of 1-2 × 104 mol/l, with a dose-response pattern; chloramphenicol and other metabolites were devoid of genotoxic effect at concentrations up to 4 × 103 mol/l. On the basis of the response found in RiBM cells compared with the previous investigation using peripheral blood lymphocytes, the authors concluded that RiBM cells were much less susceptible to the genotoxic effect of chloramphenicol metabolites than were human lymphocytes (Lafarge-Frayssinet et al., 1994; Robbana-Barnat et al., 1997).
An increase in apoptosis in marrow progenitor cells was reported in patients with aplastic anaemia (Philpott et al., 1995). The first evaluation of apoptosis in toxicity caused by chloramphenicol was assessed in vitro in a study using a monkey kidney-derived cell line and human haematopoietic progenitor cells from human neonatal cord blood. At a concentration of 2-5 mmol/l, chloramphenicol caused apoptosis in dividing cells of both systems. In a subsequent study of myelotoxicity in vivo, morphological evidence of apoptosis was seen in erythroid and myeloid precursors in femoral marrow of B6C3F1 mice given chloramphenicol at a dose of 200 mg/kg bw. The authors suggested that effect of chloramphenicol is at the differentiation stage of the committed marrow progenitor cells, rather than the replication stage of the stem cells, and therefore this response appears to be paralleling the reversible bone-marrow depression seen in the treated patient (Holt et al., 1997, 1998).
The observation that chloramphenicol induces apoptosis in haemopoietic stem cells was confirmed by additional studies using models in vitro and in vivo. Phenotypic analyses using flow cytometry (with a fluorescence-activated cell sorter, FACS) have demonstrated the induction of apoptosis in purified human bone-marrow CD34+ cells treated with chloramphenicol (Kong et al., 1999). A link between this cytotoxicity and chloramphenicol-induced apoptosis was confirmed in vivo in BALB/c mice treated orally with a single high dose of chloramphenicol at 4000 mg/kg bw or with thiamphenicol. Apoptosis in femoral mononuclear cells sampled at 36 h after dosing, as indicated by morphological evidence for increased numbers of apoptotic nucleated marrow cells, was only induced by chloramphenicol, while thiamphenicol gave a negative result. The authors suggested that the induction of apoptosis in marrow progenitor cells might account for chloramphenicol-induced toxicity associated with aplastic anaemia in humans (Turton et al., 2002b).
Inhibition of protein synthesis in the mitochondria of bone-marrow cells has been considered as a mechanism by which bone-marrow depression is induced by chloramphenicol. The underlying cytotoxicity may be caused by the similarity between mitochondrial ribosomes and bacterial ribosomes, both of which are 70S. Thus chloramphenicol can also inhibit mitochondrial protein synthesis in mammalian cells, particularly in erythropoietic cells, which appear to be sensitive to the drug (Sande & Mandell, 1993; Kucers et al., 1997). It was reasoned that the inhibition of mitochondrial protein synthesis suppressed the division of mitochondria and resulted in the formation of megamitochondria. Investigation of the toxicity caused by chloramphenicol in mouse hepatic cells in vivo, however, showed that antioxidants prevented the formation of megamitochondria (Matsuhashi et al., 1996). The role of antioxidants in reducing the cytotoxic effects of chloramphenicol was also reported to occur in vitro in a study using a monkey kidney-derived cell line and haematopoietic progenitor cells from human neonatal cord blood. Also, in cells in culture, the cytotoxic effects of chloramphenicol on apoptosis and suppression of progenitor cell growth were not pronounced when cells were co-cultured with antioxidants such as mercaptoethylamine or vitamin C (Holt et al., 1997). Both studies suggested that toxicity caused by chloramphenicol relates intimately to oxidative stress, with a possible link between a metabolic event—the production of free radicals—and bone marrow suppression.
The cytotoxic potential of chloramphenicol with regard to cell membrane function was examined in a study investigating inhibition of protozoan motility. The effect of chloramphenicol on the locomotion of the protozoan Tetrahymena pyriformis, a model widely used for the evaluation of toxicity in excitable tissue, was tested. Chloramphenicol appeared to depress the motility of the test organism more effectively than did chloramphenicol succinate, the hydrophilic form of chloramphenicol. Results suggested that chloramphenicol, with its hydrophobic free form, has the ability to partition into the lipid bilayer of the cell membrane and thus the potential to cause membrane-mediated toxic effects. The authors postulated that such effects might explain the acute toxicity of chloramphenicol in excitable tissues, such as myocardium, and are a possible mechanism for chloramphenicol-induced cardiovascular collapse in neonates, or "grey baby syndrome" (Wu et al., 1996).
In contrast to the membrane-mediated toxic effects observed in Tetrahymena spp., chloramphenicol had no adverse effects on the morphologic characteristics and migration of canine corneal epithelial cells in vitro. A monolayer of cultured cells from the canine corneal epithelium was treated with a number of different antibiotics after a defect had been made on the monolayer. The toxicity of the antibiotics was determined by the morphologic characteristics and the migration of treated cells. Pure antibiotics were used at a concentration similar to that in tears, obtained with topical use of the commercially available antibiotic products in humans. Comparison between control cells and cells treated with antibiotics indicated that chloramphenicol had no cytopathologic effects on the monolayer and cellular morphologic characteristics, and migration of the treated cells was similar to that of control cells (Hendrix et al., 2001).
Two types of chloramphenicol-induced toxicity in humans have been widely discussed. The first is a frequently occurring, dose-related, bone-marrow depression that develops during treatment with chloramphenicol. The condition is seen as a mild anaemia, with decreased haemoglobin concentrations and reticulocytopenia, with the bone marrow showing reduced erythroid precursors, increased myeloid : erythroid cell ratio and vacuolation of erythroid cells. The patient returns to normal after drug withdrawal. Inhibition of protein synthesis in bone-marrow cells has been proposed as the mechanism of these effects (Kucers et al., 1997). The second is a severe, non-dose-related aplastic anaemia, which is irreversible. Aplastic anaemia is evident as severe pancytopenia in peripheral blood, with an acellular or hypocellular bone marrow. This might also result in leukaemia in humans (Dollery, 1999; Turton et al., 2002a).
Severe bone-marrow failure induced by chloramphenicol in man is relatively infrequent. Susceptibility to chloramphenicol-induced aplastic anaemia and leukaemia in man is considered to involve a genetic element. It has been suggested that chloramphenicol-induced aplastic anaemia and leukaemia are related to the DNA damage caused by nitrosochloramphenicol, which is a product of the reduction of the para-nitro group of chloramphenicol. The ability to reduce the para-nitro group to the nitroso derivative is genetically determined, and thus gives rise to an individual metabolic predisposition to such drug-induced conditions. The assumption is supported by investigations in vitro and in vivo demonstrating that the haematological response to chloramphenicol in mice is partly strain-dependent (Festing et al., 2001). However, the exact biochemical mechanism responsible for aplastic anaemia in man has not yet been elucidated.
A battery of toxicological studies was performed in an attempt to develop a rodent model for chloramphenicol-induced aplastic anaemia in humans. However, recent studies have confirmed several previous reports that no suitable or reliable laboratory animal model of aplastic anaemia exists, although administration of chloramphenicol succinate in rodent models induces haematological changes comparable to the chloramphenicol-induced reversible, dose-dependent bone-marrow depression seen in humans (Young & Maciejewski, 1997; Holt et al., 1998; Yallop et al., 1998; Turton et al., 1999).
Haematoxicity induced by chloramphenicol was investigated in a study in CD-1 weanling mice. Animals were given chloramphenicol at a dose of 1400 mg/kg bw by gavage daily for 10 days, and blood samples were taken at 1, 4 and 15 days after the last dose. Haematological data at day 1 after dosing showed a significant reduction in erythrocytes, erythrocyte volume fraction and haemoglobin values, which returned to normal by day 4 or 15. The investigator suggested that the reversible, dose-dependent anaemia seen in man could develop in CD-1 mice given chloramphenicol succinate (Turton et al., 1999).
In a study of the potential of chloramphenicol succinate and thiamphenicol to induce aplastic anaemia, female BALB/c mice were given chloramphenicol succinate at 2000 mg/kg bw per day or thiamphenicol at 850 mg/kg bw per day by gavage for 17 days. On days 1, 13, 22, 41, 98 and 179 after the final dose, blood and marrow samples were collected for haematological examination and assays for haematopoietic stem cells. Chloramphenicol succinate and thiamphenicol were found to have similar effects. Significant reductions in values for peripheral blood parameters (erythrocyte count, erythrocyte volume fraction and haemoglobin concentration) and bone marrow parameters (erythroid colony forming units and granulocyte-macrophage colony forming units) were found in samples at day 1 after dosing. At the later sampling times, values for all the observed parameters gradually returned to normal, and there was no evidence of marrow suppression by the end of the experiment. On the basis of this observation, the authors determined that chloramphenicol succinate and thiamphenicol induced reversible anaemia in BALB/c mice; however, aplastic anaemia does not appear in the BALB/c mouse (Turton et al., 2000).
The induction of haematoxicity by administration of chloramphenicol was attempted in another rodent species, after the induction in mice was unsuccessful. Guinea-pigs were examined for susceptibility to bone-marrow depression induced by chloramphenicol succinate. In a dose range-finding study, chloramphenicol succinate administered at a dose of 825 mg/kg bw for 16 days induced changes comparable to the reversible bone-marrow depression seen in humans, but there was no evidence of late-stage marrow depression, as would be seen in marrow aplasia. The authors concluded that rodents are not susceptible to myelotoxicity induced by chloramphenicol. The guinea-pig, like the mouse and rat, serves as a model for early events, but is not a good model for aplastic anaemia induced by chloramphenicol in man (Turton et al., 2002a).
Despite the well-recognized potential toxicity of chloramphenicol in humans, the drug is considered by most experts to be of low toxicity in adult companion animals when they are appropriately dosed. The development of aplastic anaemia as seen in humans does not appear to be a significant problem in animals. However, a dose-related reversible bone-marrow suppression is seen in all species, primarily after long-term therapy. Early signs of bone-marrow toxicity can include vacuolation of many of the early cells of the myeloid and erythroid series, lymphocytopenia, and neutropenia. Other adverse effects that may be noted in animals treated with chloramphenicol include anorexia, vomiting, diarrhoea and depression. Cats tend to be more sensitive to developing adverse reactions to chloramphenicol than are dogs; cats given chloramphenicol at a dose of 50 mg/kg bw every 12 h for 2-3 weeks do develop in high incidence of adverse effects (Plumb, 2002).
A bone marrow disorder was reported in a study of toxicity in a dog that was dosed orally with chloramphenicol at 300 mg/kg bw per day for 14 days. The results showed a decrease in the number of total erythroid cells, with proportional increase in myeloid cells, yielding a significantly increased myeloid to erythroid cell ratio (Baig et al., 1994).
Breeder turkey hens given drinking-water containing chloramphenicol at a concentration of 500 mg/l for 4 days showed a decrease in egg production. The toxic effects of chloramphenicol were mortality and cessation of egg production, which were more severe when a combination of chloramphenicol and monensin was given (Friedman et al., 1998).
Overall analysis of toxicity with chloramphenicol has suggested that the most serious toxic effect, aplastic anaemia reported in humans, is not seen in animals. However, reversible, dose-related bone-marrow suppression can be observed in all species given chloramphenicol in excessive doses or for prolonged periods. Other signs of toxicity caused by chloramphenicol are evident in animals in susceptible states, e.g. in neonatal animals or pregnant animals in which the hepatic biotransformation of chloramphenicol is impaired, or where the drug causes a decrease in protein synthesis in the fetus. However, owing to the potential toxicity of chloramphenicol in humans, and because of the possibility that metabolites of chloramphenicol might be found in the edible tissues of animals treated with chloramphenicol, the use of chloramphenicol in veterinary practice is not permitted in food-producing animals in many countries.
Toxicity caused by chloramphenicol in humans has been widely discussed because it induces bone-marrow depression. The more common dose-related bone-marrow depression is evident when the daily dose of chloramphenicol is >4 g in humans. Toxicity is reversible if the treatment is discontinued or the dosage is reduced. A more serious and unpredictable reaction is aplastic anaemia, which is not considered to be dose-related. Although the incidence of aplastic anaemia has been shown to correlate with several risk factors, it is estimated to occur with a frequency of 1 in 24 000-40 000 courses of treatment with chloramphenicol. Mortality from aplastic anaemia occurs in >50% of cases (Greenwood, 2000; Maluf et al., 2002).
Rappeport & Bunn (1994) suggested that aplastic anaemia in humans is an idiosyncratic reaction to chloramphenicol, which has an immunological basis and which is related to the nitrobenzene structure. This hypothesis is supported by clinical evidence showing that 40-50% patients with aplastic anaemia have a partial or complete response to a variety of immunosuppressive agents.
Young (2002) reviewed the pathophysiology of aplastic anaemia and reported that most cases can be characterized by a T-cell mediated destruction of bone-marrow haematopoietic cells.
This aberrant immune response may be a reaction to chemicals, drugs or viral infections, but endogenous antigens may also be involved. Many drugs can cause idiosyncratic haematopoietic failure; however, it is rare that patients, some of whom may have only ingested small quantities of the drug, show bone-marrow failure as a complication. Owing to the idiosyncratic nature of the response, it is difficult to study aplastic anaemia, and animal models do not exist (Young & Maciejewski, 1997). The therapeutic use of chloramphenicol has been followed by the development of aplastic anaemia in humans. This was particularly notable in the period following the introduction of chloramphenicol as a therapeutic agent in 1948, and before its association with aplastic anaemia had been recognized.
Other indications of toxicity associated with treatment with chloramphenicol in humans have been recognized. Circulatory collapse ("grey baby syndrome") has occurred in human neonates treated with chloramphenicol. This adverse reaction may be explained by the poor hepatic biotransformation of the drug in neonates as a result of slow glucuronidation of chloramphenicol. Toxic concentrations of chloramphenicol in blood and tissues develop secondary to an inability to conjugate the drug or to excrete the conjugate efficiently. However, the precise reason for the occurrence of cardiovascular collapse in grey baby syndrome is poorly understood. It has been stated that nitro-reduction derivatives of chloramphenicol might play a role in causing hypotension, and the hypothesis was assessed by perfusion of chloramphenicol through the isolated lobules of human placenta. A decrease in blood pressure was found at the time coinciding with a peak in concentration of nitric oxide, which is a product of the nitroreduction of chloramphenicol (Holt & Bajoria, 1999).
The potential for an adverse reaction induced by treatment with chloramphenicol is of critical importance in seriously ill or compromised patients. In patients with pre-existing haematologic abnormalities or hepatic failure, or in neonates, chloramphenicol is only used when no other effective antibiotics are available. Chloramphenicol has not been determined to be safe for use during pregnancy. The drug may decrease protein synthesis in the fetus, particularly in the bone marrow. Chloramphenicol is found in human milk at 50% of serum concentrations in humans and therefore the drug should be given with extreme caution to nursing mothers (Greenwood, 2000; Plumb, 2002).
The most serious adverse effect of treatment with chloramphenicol in humans is its association with acquired aplastic anaemia. Many population-based studies have been carried out to identify etiological factors associated with aplastic anaemia and to determine a link between the use of chloramphenicol and the development of marrow aplasia. Young & Alter (1994) reported that the published estimates of incidence of aplastic anaemia are significantly influenced by the methods used to acquire the data, and the diagnostic exclusion criteria. The incidence estimates reported in some former studies were too high owing to the inclusion of cases improperly classified as aplastic anaemia; the reported incidence of aplastic anaemia declines when rigorous diagnostic criteria have been applied. On the basis of an extensive review, the authors concluded that the incidence rate for aplastic anaemia is 2-6 cases per million population, with most cases of aplastic anaemia being classified as idiopathic (Young & Alter, 1994).
There are only a few recently documented cases of aplastic anaemia in patients that were sensitive to chloramphenicol. Possible etiologic factors associated with aplastic anaemia were identified in 151 Turkish patients who met the diagnostic criteria. The findings suggested that these cases of aplastic anaemia were most often idiopathic (99 out of 151 cases). The most common identifiable etiologic factor was the use of drugs (23 out of 151 cases), which were mainly non-steroidal anti-inflammatory agents, while chloramphenicol appeared to be specified in 1 out of 23 cases of drug use associated with aplastic anaemia. Exposure to benzene was the second most common causal agent in the studied cases (19 out of 151 cases) (Alnigenis et al., 2001).
In an investigation of potential risk factors associated with aplastic anaemia in the state of Parana, Brazil, the statistical evaluation of 125 cases of aplastic anaemia showed no positive association between use of chloramphenicol and development of aplastic anaemia. Instead, the causes of aplastic anaemia in Brazil were apparently identified as common factors related to the disease, such as exposure to certain chemicals. The incidence found in this study was similar to that reported in Thailand and Europe (Maluf et al., 2002).
Additional reports evaluating the correlations between the incidence of aplastic anaemia and use of chloramphenicol were documented in cases of aplastic anaemia in Nigeria and in Nepal. In a 5-year prospective study in Nigeria, it was estimated that aplastic anaemia developed in 0.002% of non-obstetric patients treated with chloramphenicol. Of 18 cases of aplastic anaemia diagnosed in Nepal, 16 were identified as being idiopathic and one was found to be associated with toxicity caused by treatment with chloramphenicol. Both studies concluded that chloramphenicol-induced aplastic anaemia is rare (Durosinmi & Ajayi, 1993; Sah et al., 1999).
It has been claimed that the topical ophthalmic use of chloramphenicol causes bone-marrow aplasia, but this issue has not been completely resolved. Recent observations have shown that the use of chloramphenicol as a topical eye medication is unlikely to introduce aplastic anaemia. Two extensive population-based studies in industrialized and developing countries presented no support for the claim that eyedrops containing chloramphenicol increase the risk of aplastic anaemia. The investigators found that there was no history of use of eyedrops containing chloramphenicol in more than 400 cases of aplastic anaemia examined. On the basis of this observation, the authors disagreed with the general recommendation stating that use of eyedrops containing chloramphenicol should be avoided because of an increased risk of aplastic anaemia (Wiholm etal., 1998).
Serum concentrations of chloramphenicol were monitored in a study in 40 patients treated with eyedrops containing chloramphenicol. HPLC with a minimum limit of detection (LOD) of 1 mg/l was used to measure the serum accumulation of chloramphenicol after topical therapy. After a course of treatment in which the mean dose of chloramphenicol received in 1 week of treatment was 8.0 mg, and in 2 weeks was 15.3mg, serum concentrations of chloramphenicol were below the limit of detection. The authors considered that the topical use of chloramphenicol was not a risk factor for induction of dose-related toxicity in bone marrow, and the suspension of use of topical chloramphenicol in ophthalmic practice was questioned (Walker et al., 1998).
Despite the failure of epidemiological studies to find an association between the topical use chloramphenicol and development of aplastic anaemia, the hypothesis of a metabolic predisposition in individuals predisposed to blood dyscrasias cannot be disregarded. Small doses of chloramphenicol similar to those used in topical therapy may cause this idiosyncratic reaction in certain individuals. In 1993, 23 cases of blood dyscrasias in patients treated topically with chloramphenicol for ophthalmic purposes were reported to the national register of drug-induced ocular side-effects in Oregon, USA (Fraunfelder et al., 1993).
In a critical review of the potential risk of developing aplastic anaemia attributable to topical use of chloramphenicol, the authors postulated that the risk posed by topical use of chloramphenicol may be similar to that of orally administered chloramphenicol. This is because topical administration achieves systemic effects by absorption through the conjunctival membrane or through drainage down the lacrimal duct followed by absorption from the gastrointestinal tract. In their view, it is not possible to justify subjecting patients to such potential risk, and therefore ocular chloramphenicol should be used only when there is no alternative (Doona & Walsh, 1995).
Contact sensitivity to chloramphenicol is rare in humans. Two cases of skin ulcer, secondary to contact dermatitis, were reported in a woman aged 48 years and a man aged 46 years. Both patients had applied chloramphenicol in the form of chloromycetin cream to their wounds for about 1 month and developed skin ulcer at the application sites. Hypersensitivity to chloramphenicol was confirmed by patch tests in both patients. The ulcer healed after use of the drug was discontinued (Matsumoto et al., 1998).
The teratogenic risk of chloramphenicol was studied in a population-based dataset of the Hungarian case-control surveillance of congenital abnormalities, 1980-1996. Retrospective investigation of the effects of oral treatment with chloramphenicol during pregnancy was implemented in 38 151 pregnant women who had healthy babies (control group) and 22 865 pregnant women who had congenitally abnormal newborns or fetuses. The case-control pair analysis of pregnant women who were treated in the second month or third month of pregnancy did not reveal any teratogenic potential of chloramphenicol in humans. The authors concluded that treatment with chloramphenicol during early pregnancy presents little, if any, teratogenic risk to the fetus in humans (Czeizel et al., 2000). However, the human embryo implants at day 6-7 of gestation and organogenesis begins at day 21; heightened susceptibility to malformations occurs during this period (Rogers & Kavlock, 2001). Therefore, this study may have overlooked the occurrence of early abnormalities.
Although the use of chloramphenicol in veterinary medicine has been restricted to non-food animals, residues have been found in samples taken from domestically produced animals in national monitoring programmes and foods, and in samples moving in international trade. For example, results of analyses carried out in Germany in the years 2000-2002 in accordance with directive 96/23/EEC showed that a small fraction (<0.2%) of all samples (n> 17 500) taken at farms and slaughter houses contained residues of chloramphenicol. The concentrations that were found in a total of 11 positive samples from fattening cattle, swine and poultry ranged from 0.3 to 3.3 µg/kg with eight values of <1 µg/kg (BVL, 2003). Unfortunately, comparable information was not available from many countries and, the limits of detection or quantification (LOD or LOQ) of the analytical methods used in some countries were—until recently—too high to allow quantification of traces of chloramphenicol.
The Health and Consumer Protection Directorate-General of the European Commission communicated ranges of concentrations of chloramphenicol in some food items. These had been reported by Member States during the years 2000-2003 (European Commission, 2004). The information included concentrations for the following commodities: skimmed milk powder (range, 0.021-1.23 µg/kg), milk products (range, 0.3-1.27 µg/kg), honey (range, 0.3-4.0 µg/kg; one sample at 38.7 µg/kg), washed pollen (0.58 µg/kg), shrimps (range, 0.1-7.7 µg/kg; two samples at 31.89 and 297 µg/kg), crabmeat (range, 0.3-1 µg/kg), crayfish (range, 0.14-6.3 µg/kg), casings (range, 0.5-2.9 µg/kg), rabbit meat (0.3 µg/kg), turkey breasts (0.82 µg/kg), and chicken breasts (range, 0.4-1.2 µg/kg).
The presence of residues of chloramphenicol has caused major food scares in the past 2 to 3 years. Shrimps, prawns, food products from other aquatic animals, honey, royal jelly, meat and offal, casings, rabbit, poultry meat and milk powder are among the commodities in which the drug was found. Many of the shipments of commodities in which residues of chloramphenicol were found originated in south-east Asia.
The Food Standards Agency of the United Kingdom, for example, has published a table with test results (Food Standards Agency, 2002/2003) obtained with samples of honey that were on sale in the United Kingdom. Using a method with a "reporting limit" (RL) of 0.3 µg/kg, the results ranged from "none detected above the RL set" to a maximum of 7.2 µg/kg. Several samples also contained streptomycin, for which the RL was 50 µg/kg. A group of 20 samples of honey that were analysed in the Netherlands (Voedsel en Waren Autoriteit, 2004) gave an average mass concentration of 1.9 µg/kg (range, 0.06-5.9 µg/kg). Positive findings of residues of chloramphenicol in honey from different geographic regions have also been reported in the scientific literature (Verzegnassi et al., 2003). Most of the contaminated honey originated from China. Reybroeck (2003) analysed samples of honey available on the Belgian market; samples were screened using an enzyme-linked immunosorbent assay (ELISA) (LOD, 0.1-0.3 µg/kg, depending on clean-up). Positive samples were subjected to high-performance liquid chromatography-mass spectrometry (HPLC-MS) confirmatory analysis (LOD, 0.1 µg/kg). Of the samples with known origin, only samples of Chinese origin contained residues of chloramphenicol (31 out of 40 samples). It has been hypothesized that contamination of honey with chloramphenicol could be related to treatments against foulbrood disease (Dharmananda, 2003).
While some of the positive findings are most likely to be the result of intentional uses of chloramphenicol rather than of environmental contamination, it has also been argued that very low concentrations of chloramphenicol detected in certain foods of animal origin, e.g. in poultry and in products from aquaculture, could perhaps be derived from environmental sources—either from chloramphenicol produced naturally by microorganisms or from residues resulting from past uses, which still persist in the environment.
Examples of low concentrations from information published by the Food Standards Agency of Ireland (Food Standards Agency of Ireland, 2002/2003) for the year 2002 are summarized in Table 1. The Food Standards Agency of Ireland has placed on the Internet information on alert/non-alert notifications of the Member States of the European Union concerning residues of chloramphenicol in shrimps, prawns, fish, fishery products and several other food commodities. A review of the data shows that at least nine of the Member States had communicated such notifications. In the majority of approximately 110 short summaries on notifications, no quantitative data on residue concentrations were communicated. However, an evaluation of a total of 47 quantitative results (33 for shrimp samples, 4 for prawn samples, 1 for fish and 9 for crabmeat, crayfish and surimi samples) showed the following distribution characteristics of the concentrations found: range, 0.1-34 µg/kg; median: 0.5 µg/kg.
Table 1. Range of concentrations of chloramphenicol found in samples of aquaculture products
|
Lower class limit |
Upper class limit |
Number of results |
|
0.1 |
0.5 |
25 |
|
>0.5 |
1.0 |
7 |
|
>1.0 |
1.5 |
2 |
|
>1.5 |
2.0 |
0 |
|
>2.0 |
2.5 |
2 |
|
>2.5 |
3.0 |
0 |
|
>3.0 |
3.5 |
3 |
|
>3.5 |
4.0 |
1 |
|
>4.0 |
4.5 |
1 |
|
>4.5 |
5.0 |
1 |
|
>5.0 |
34.0 |
5 |
From Food Standards Agency of Ireland (2002/2003).
Although these data are not representative and the samples were not all independent, it cannot be ruled out that there could exist two distributions of concentrations of residues:
The countries involved were not equally represented and the above numbers of samples are too low for any valid comparison between countries of origin. Another set of results was made available for evaluation by the Committee: the average value of a population of 50 samples of shrimp in which the presence of chloramphenicol was confirmed indicated an average value of 0.25 µg/kg, (range, 0.06-0.69 µg/kg), with two outlying values of 3.0 and 3.7 µg/kg (Voedsel en Waren Autoriteit, 2004).
The Centre of Analytical Services and Experimentation of Ho Chi Minh City conducted a validation study of GC and HPLC-MS methods for detection of chloramphenicol; a number of shrimp samples were analysed. During the first three quarters of 2002, a total of 44 samples were found to contain chloramphenicol (range of concentrations, 0.4-1.4 µg/kg) (Ngoc Son, 2002).
The Fourteenth Session of the FAO/WHO Codex Alimentarius Committee for Residues of Veterinary Drugs in Foods (CCRVDF) discussed the possibility that such traces of chloramphenicol found in food-producing animals could originate from environmental contaminations, rather than from intentional use. The report of the session reflects the discussion as follows (Joint FAO/WHO Food Standards Programme, 2003):
114. The request from Indonesia to consider the elaboration of an MRL for chloramphenicol in shrimp was addressed by the Joint Secretariat who discussed the possibility that this compound could find its way into animal tissues via other routes than its use as a veterinary drug. Limited data showed that chloramphenicol may persist in the environment or even be formed by soil microorganisms. Hypothetically, very low levels found in animal products could therefore not be related to the use of chloramphenicol as a veterinary drug. Several delegations stressed that it would be premature to draw any conclusions or to discuss a possible classification as a contaminant and that illegal use of the drug was a primary concern. It was noted that international trade had been disrupted severely during the past year by the rejection of products which had been contaminated at very low levels with chloramphenicol and some other veterinary drugs. The Committee noted the offer of the FAO Secretariat to JECFA to examine the potential persistence of chloramphenicol in the environment or its formation by soil microorganisms on the basis of data to be provided by Indonesia.
An attempt to investigate these possibilities has been made in the present monograph. After discussion of the development and current status of analytical methods, essentially two hypotheses for a possible environmental origin for very low concentrations of residues in foods were tested. In the first hypothetical scenario it is assumed that:
The second hypothetical scenario assumes that food-producing animals may currently still occasionally be exposed to persisting environmental residues of chloramphenicol resulting from historical veterinary uses.
This monograph also assesses the hypothetical dietary intakes resulting from low-level contamination of seafood with residues of chloramphenicol and compares these intakes with the lowest known human therapeutic exposures.
The ideal method for the analysis of residues of chloramphenicol should be sensitive, accurate and precise, and provide unambiguous information on the identity of the analyte. Furthermore, it should be as cost-effective and robust as possible. In practice, it is difficult to develop methods that combine all these characteristics. Therefore, the analytical strategies used for the control of residues of chloramphenicol in animal tissues frequently include the initial application of a screening method, followed by a confirmatory analysis of those samples that gave positive results with the screening method.
Typically, the three basic steps used in the majority of methods of analysis for chloramphenicol are:
This step usually includes homogenization and extraction of the tissue with suitable organic solvents, the separation of liquids and solids and the removal of lipids from the crude extract. Temperature control during storage and extraction of the sample is important in order to avoid metabolism in vitro, particularly in samples of liver and kidney, respectively, owing to the action of metabolizing enzymes (Parker & Shaw, 1988; Sanders et al., 1991). Analysis of liver and kidney requires enzymatic hydrolysis of conjugates of chloramphenicol; this step can apparently be omitted when working with trout tissues (Baradat et al., 1993; Mottier et al., 2003).
Use of the following organic solvents has been described for the extraction of chloramphenicol:
The lipids were removed through solvent partition, using:
—nHexane (Nagata & Saeki, 1992; Kijak, 1994; Epstein, 1994; Chevalier et al., 1995; Gude et al., 1995; Li et al., 2001; Perez et al., 2002; Pfenning et al., 2002a; Turnipseed et al., 2002; Storey et al., 2003; Gantverg et al., 2003; Pfenning et al., 2003);
—A mixture of nhexane and chloroform (1:1) (Sanders et al., 1991);
—nHeptane (Rupp et al., 2003; Stuart et al., 2003).
There are numerous procedures used to purify the primary extract in order to remove substances interfering with the detection and quantification step. Solid-phase extraction is the most widely used technique for purification in the analysis of residues of chloramphenicol in food matrices (Chevalier et al., 1995; Neuhaus et al., 2002; Turnipseed et al., 2002; Storey et al., 2003; Gantverg et al., 2003; Pfenning et al., 2003; Posyniak et al., 2003; Mottier et al., 2003; Impens et al., 2003).
There are, however, other purification techniques, for example, immunoaffinity chromatography (van de Water et al., 1989; Gude et al., 1995), which takes advantage of highly selective hapten-antibody interactions.
In past decades, several analytical methods have been developed and reviewed for the detection and quantification of chloramphenicol in foods and biological fluids.
Gas chromatography: the presence of polar functional groups in the chloramphenicol molecule requires a derivatization step, usually through a sylilation reaction, before gas chromatography analysis. The attachment of particular functional groups onto the chloramphenicol molecule may also lower the LOD of the electron capture detector (ECD) and MS. The silylation reaction is usually catalysed by acids or bases. Frequently used silylation reagents include:
ECD has been widely used for the analysis of chloramphenicol. The GC-ECD method for analysis of chloramphenicol in muscle of prawns, described by Munns et al. (1994) for example, reached a LOD of 1 µg/kg.
When GC is coupled with MS, the most frequently applied ionization techniques are chemical ionization (CI) and electron impact (EI).
Negative chemical ionization (NCI) presents a limited number of fragments; however, the molecular ion is always part of the spectrum. Owing to the presence of two chlorine atoms in the chloramphenicol molecule, GC-MS in NCI mode is one of the most reliable techniques, and it is the most commonly used method for the confirmation of residues of chloramphenicol. The LOD can be <0.1 mg/kg in muscular tissue (van Ginkel et al., 1990; Epstein, 1994; Borner et al., 1995). GC-MS in EI mode is less sensitive; however, it produces spectra of fragments, which are reproducible with different instruments, and can therefore be stored in electronic databases for reference purposes.
HPLC: The development of atmospheric pressure ionization (API) mass detectors, coupled HPLC, has become one of the most reliable and widespread techniques in the analysis of residues of chloramphenicol. This combination of liquid chromatography and mass spectrometry (LC-MS) enables the detection and quantification, without derivatization, of polar non-volatile analytes, such as chloramphenicol.
Neuhaus et al. (2002) have described an LC-MS/MS method with a LOD of 0.08 µg/kg and an LOQ of 0.3 µg/kg in prawns. Mottier et al. (2003) have developed a method for meat (chicken, turkey, pork and beef) and aquatic products (crab, prawn and fish), using liquid chromatography (electrospray ionization) tandem mass spectrometry (LC(ESI)-MS/MS), with isotopic dilution, reaching LOD and LOQ values of 0.003 µg/kg and 0.01 µg/kg, respectively.
Impens et al. (2003) have described an analytical strategy for the screening and confirmation of residues of chloramphenicol in prawn tissue, using ELISA for screening and GC-MS/MS and LC-MS/MS for confirmation; both selective techniques reached LODs of 0.1 µg/kg.
Gantverg et al. (2003) have developed a very sensitive method for the detection and confirmation of chloramphenicol in pork and beef muscle and in urine. After extraction, chloramphenicol was determined through LC-MS/MS in CI mode at negative atmospheric pressure, and by GC-MS in EI mode.
In summary, GC-MS and LC-MS/MS are, nowadays, the most reliable techniques for the determination of residues of chloramphenicol in edible animal tissues.
Selected examples of the development of analytical methods between 1990 and 2003 are listed in Table 2.
The European Union has recently defined minimum required performance limits (MPRLs) for analytical methods used for the determination of substances for which no permitted limit has been established, and in particular for those substances, like chloramphenicol, whose use is not authorized or specifically prohibited by Community legislation. For chloramphenicol, a MRPL of 0.3 µg/kg was established (European Commission, 2003).
Table 2. Overview of the development of analytical methods for residues of chloramphenicol in foods of animal origin
|
Analytical method |
Food matrix |
LOD |
LOQ |
Levels reported (µg/kg) |
Reference |
|
GC(NICI)-MS |
Muscle/egg |
0.1 |
>0.1 |
— |
van Ginkel et al. (1990) |
|
LC/UV |
Calf muscle |
1 |
— |
— |
Sanders et al. (1991) |
|
LC-MS |
Some aquatic species |
0.1 |
0.3 |
— |
Van de Riet et al. (1992) |
|
GC(NICI)-MS |
Bovine muscle |
0.6 |
— |
— |
Epstein (1994) |
|
GC(NICI)-MS |
Cows' milk |
0.5a |
— |
— |
Kijak (1994) |
|
GC-ECD |
Shrimp |
1 |
— |
— |
Munns (1994) |
|
GC(NICI)-HRMS |
Egg |
0.3 |
0.5 |
— |
Borner et al. (1995) |
|
GC-ECD |
Egg |
0.3 |
0.5 |
— |
|
|
HPLC (RP) |
Foie gras |
2.5 |
— |
— |
Chevalier et al. |
|
LC/UV |
Pasteurized milk |
50a |
— |
— |
Perez et al. (2002) |
|
LC-MS/MS ESI(-) |
Shrimp |
<0.5 |
— |
— |
Pfenning et al. (2002b) |
|
LC-MS/MS |
Shrimp |
0.08 |
0.3 |
— |
Neuhaus et al. (2002) |
|
GC-ECD |
Shrimp |
0.05 |
— |
0.65-0.72 |
Ngoc Son (2002) |
|
GC(NCI)-MS |
Shrimp |
0.3 |
— |
— |
|
|
LC-MS/MS APCI(-) |
Shrimp |
0.1 |
— |
— |
|
|
LC-MS/MS APCI |
Equine, porcine and bovine muscle |
0.02 |
— |
— |
Gantverg et al. (2003) |
|
GC-MS/MS |
Shrimp |
0.1 |
— |
— |
Impens et al. (2003) |
|
LC-MS/MS |
Shrimp |
0.1 |
— |
— |
|
|
LC(ESI)-MS/MS |
Chicken meat |
0.003 |
0.01 |
— |
Mottier et al. (2003) |
|
LC-MS/MS (ESI) |
Shrimp, crab |
0.1 |
— |
— |
Storey et al. (2003) |
|
GC-ECD |
Bovine muscle |
— |
0.25 |
— |
United States Department of Agriculture (2003) |
|
GC(NICI)-MS |
Bovine muscle |
— |
0.25 |
— |
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
|
Time (days) |
Concentration of chloramphenicol |
Number of cells |
PH |
Concentration of chloramphenicol |
Number of cells (105/g) |
PH |
Concentration of chloramphenicol |
Number of cells |
PH |
|
Experiment 1 |
Experiment 2 |
Experiment 3 |
|||||||
|
0 |
— |
2.00 |
5.90 |
— |
0.39 |
5.90 |
0.0 |
38.7 |
5.45 |
|
7 |
0.00 |
23.30 |
5.57 |
0.00 |
35.50 |
5.05 |
0.0 |
12.0 |
5.05 |
|
20 |
0.00 |
28.50 |
5.10 |
1.12 |
48.20 |
5.60 |
0.0 |
37.0 |
5.63 |
|
36 |
0.58 |
18.20 |
5.45 |
— |
— |
— |
— |
— |
— |
|
65 |
— |
— |
— |
0.56 |
62.50 |
5.63 |
0.5 |
30.0 |
5.45 |
|
93 |
0.50 |
168.00 |
5.75 |
— |
— |
— |
— |
— |
— |
|
100 |
— |
— |
— |
0.82 |
945.00 |
5.85 |
0.79 |
1230.0 |
5.78 |
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
|
Time (days) |
Recovery of chloramphenicol from soil (mg/kg) |
||
|
1/24 (1 h) |
1.900 |
1.700 |
2.200 |
|
1 |
1.300 |
1.300 |
1.200 |
|
2 |
0.850 |
1.000 |
0.890 |
|
3 |
0.770 |
0.740 |
0.820 |
|
4 |
0.550 |
0.240 |
0.470 |
|
5 |
0.600 |
0.690 |
0.600 |
|
7 |
0.250 |
0.290 |
0.440 |
|
11 |
0.063 |
0.069 |
0.270 |
|
17 |
0.130 |
0.130 |
0.150 |
|
29 |
0.041 |
0.093 |
0.041 |
|
45 |
<0.027 |
0.036 |
0.033 |
|
92 |
<0.027 |
<0.027 |
<0.027 |
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 Kow for chloramphenicol is 1.14. With regard to sorption coefficients to soil solids (Kd,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 Kd,solid (0.2-2 l/kg) (Tolls, 2001; Rabolle & Spliid, 2000).
Lai et al. (1995) estimated Kd,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
|
Condition of incubation |
Sediment |
Concentration of chloramphenicol (mg/l) |
||
|
100 |
200 |
400 |
||
|
Aerobic |
Freshwater eel pond |
6.4 |
9.2 |
9.7 |
|
Marine shrimp pond |
1.6 |
1.8 |
1.6 |
|
|
Anaerobic |
Freshwater eel pond |
20.7 |
21.1 |
20.4 |
|
Marine shrimp pond |
20.3 |
21.3 |
15.6 |
|
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)
|
Salinity |
% Chlorphenicol adsorbed within 1 h |
-ka |
t1/2 |
-ka |
t1/2 |
|
|
Aerobic |
Anaerobic |
Aerobic |
Anaerobic |
|||
|
24 |
22 |
25 |
0.067 |
10.0 |
0.573 |
1.2 |
|
30 |
18 |
24 |
0.031 |
23.0 |
0.642 |
1.1 |
|
36 |
17 |
18 |
0.012 |
57.8 |
0.578 |
1.2 |
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)
|
Salinity |
% Chloramphenicol adsorbed within 1 h |
-ka |
t1/2 |
-ka |
t1/2 |
|
|
Aerobic |
Anaerobic |
Aerobic |
Anaerobic |
|||
|
0 |
28 |
23 |
0.297 |
2.4 |
1.915 |
0.4 |
|
12 |
25 |
21 |
0.155 |
4.5 |
0.957 |
0.7 |
|
24 |
27 |
25 |
0.080 |
8.9 |
0.495 |
1.4 |
|
36 |
28 |
23 |
0.039 |
18.4 |
0.286 |
2.4 |
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
|
Production class |
Live weight (kg) |
Average daily gain (kg) |
Dry matter intake |
|
|
(kg/animal per day) |
(kg of dry matter/kg average daily gain) |
|||
|
Starter |
10-20 |
0.450 |
0.80-0.84 |
1.78-1.87 |
|
Grower |
20-50 |
0.700 |
1.61-1.91 |
2.04-2.73 |
|
Finisher |
50-100 |
0.820 |
2.63-3.29 |
2.94-4.01 |
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
|
Description of the group of animals |
Range of mean soil ingestion (% of dry matter intake) |
|||
|
Lower bound |
Upper bound |
|||
|
Mean |
Standard error |
Mean |
Standard error |
|
|
Lactating cows |
||||
|
Confined to concrete |
0.14 |
0.02 |
0.53 |
0.05 |
|
Housed in freestall barns with soil bedding |
0.35 |
0.06 |
0.64 |
0.18 |
|
Access to unpaved lots with no vegetation |
0.60 |
0.07 |
0.96 |
0.22 |
|
Yearling heifers and dry cows |
||||
|
Confined to concrete |
0.52 |
0.11 |
0.81 |
0.19 |
|
Access to unpaved lots with no vegetation |
0.25 |
0.04 |
2.41 |
0.26 |
|
Access to unpaved lots with sparse vegetation |
1.56 |
0.21 |
3.77 |
1.50 |
|
On pasture but receiving supplemental feed |
1.38 |
0.33 |
2.43 |
0.50 |
From Fries et al. (1982).
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:
The following steps were performed in the calculations1:
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
|
Average daily gain |
Dry matter intake |
Soil intake |
Day |
Body weight |
Concentration of chloramphenicol in soil (µg/g)( |
Cumulative intake of chloramphenicol (µg) |
|
0.164 + 0.00693 × |
35 |
2% of dry matter intake |
1 |
20 |
<0.05 |
<0.7 |
|
119 |
75 |
<0.05 |
<183 |
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:
The results are summarized in Table 11.
Table 11. Estimated amounts of chloramphenicol in the body resulting from soil ingestion, in pigsa
|
Concentration of chloramphenicol in soil (µg/g) |
<0.05 |
1 |
25 |
|
|
Cumulative intake of chloramphenicol (µg/animal) |
<183 |
3651 |
91 267 |
|
|
Daily dose (µg/kg bw) |
0.035 |
0.7 |
17.5 |
|
|
Half-life I (h) |
Half-life II (h) |
Estimated amount of chloramphenicol in the bodies of the pigs after 119 days (µg) |
||
|
2 |
— |
<0.0006-<2.62 |
0.013-52.5 |
0.32-1312 |
|
4 |
— |
<0.042-<2.66 |
0.84-53.3 |
21-1333 |
|
6 |
— |
<0.17-<2.80 |
3.5-55.9 |
87-1399 |
|
8 |
— |
<0.37-<2.99 |
7.4-59.9 |
186-1498 |
|
10 |
— |
<0.61-<3.23 |
12.1-64.6 |
303-1615 |
|
10 |
100 |
<1.26-<3.88 |
25.2-77.7 |
630-1942 |
|
100 |
— |
<12.8-<16.3 |
274-326 |
6838-8150 |
|
a |
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). |
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 pigsa
|
Concentration of chloramphenicol in soil (µg/g) |
<0.05 |
1 |
25 |
|
|
Cumulative intake of chloramphenicol (µg/animal) |
<183 |
3651 |
91 267 |
|
|
Half-life I (h) |
Half-life II (h) |
Estimated concentration of chloramphenicol in muscle (µg/kg) |
||
|
2 |
— |
<0.000-<0.025 |
0.000-0.5 |
0.003-12.5 |
|
4 |
— |
<0.000-<0.025 |
0.008-0.51 |
0.2-12.7 |
|
6 |
— |
<0.002-<0.027 |
0.033-0.53 |
0.83-13.3 |
|
8 |
— |
<0.004-<0.029 |
0.070-0.57 |
1.77-14.3 |
|
10 |
— |
<0.006-<0.031 |
0.115-0.62 |
2.89-15.4 |
|
10 |
100 |
<0.012-<0.037 |
0.24-0.74 |
6-18.5 |
|
100 |
— |
<0.12-<0.16 |
2.6-3.1 |
60.8-77.6 |
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 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
|
Average daily gain (g) |
Dry matter intake |
Soil intake |
Concentration of chloramphenicol in soil (µg/g) |
Body weight (kg) |
Cumulative intake of chloramphenicol (µg) |
Amount in body (µg) |
Concentration in muscle (µg/kg) |
||||
|
Day 1 |
Day 75 |
Day 1 |
Day 75 |
Day 1 |
Day 75 |
Day 1 |
Day 75 |
||||
|
3.599 + 0.01623 × bw |
3 × average daily gain |
2% of dry matter intake |
<0.05 |
150 |
1002 |
<0.018 |
<2.6 |
— |
<0.02 |
— |
<0.02 |
|
0.02 |
|||||||||||
|
1 |
150 |
1002 |
0.362 |
52 |
— |
0.45 |
— |
0.30 |
|||
|
25 |
150 |
1002 |
9.050 |
1308 |
— |
11.30 |
— |
8.10 |
|||
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 (t1/2 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:
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 compl