INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION TOXICOLOGICAL EVALUATION OF CERTAIN VETERINARY DRUG RESIDUES IN FOOD WHO FOOD ADDITIVES SERIES: 43 Prepared by the Fifty-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) World Health Organization, Geneva, 2000 IPCS - International Programme on Chemical Safety ANTIMICROBIAL AGENT The Committee examined the results of studies of antimicrobial activity in vitro in which relevant gut microflora were used to calculate ADIs for the antimicrobial agent that was reviewed. The equation used for deriving the ADIs is based on that described in the report of the forty-seventh meeting of the Committee (Annex 1, reference 125). The upper limit of the ADI was derived as follows: Upper limit MIC50 (µg/g × MCC (g) of ADI = ---------------------- (µg/kg bw) FA × SF × BW (kg) where: MIC50 = The minimum concentration of an antimicrobial drug that completely inhibits the growth of 50% of cultures of a given microorganism, as judged by the naked eye, after a given period of incubation. For the purposes of the evaluation, the MIC50 value(s) for the strain(s) of the relevant species tested. Alternatively, the lowest MIC50 value for the most sensitive species can be used. MCC = Mass of colonic content; a value of 220 g is used. FA = Fraction of an oral dose available to act upon microorganisms in the colon SF = The safety factor used to account for uncertainty about the amount and relevance of data for MIC values available for review; may range from 1 to 10, a value of 1 being used when extensive, relevant microbiological data are available. BW = Body weight; a value of 60 kg is used. THIAMPHENICOL (addendum) First draft prepared by F.R. Ungemach Institute of Pharmacology, Pharmacy and Toxicology Veterinary Faculty, University of Leipzig, Leipzig, Germany Explanation Biological data Short-term study of toxicity Study of carcinogenicity Study of teratogenicity in rabbits Special studies on microbiological effects Comments Evaluation References 1. EXPLANATION Thiamphenicol was evaluated by the Committee at its forty-seventh meeting (Annex 1, reference 125), at which time a temporary ADI of 0-6 µg/kg bw was established on the basis of a NOEL of 1.25 mg/kg bw per day for maternal toxicity in a study of teratogenicity in rabbits and by applying a safety factor of 200. The ADI was designated 'temporary' since only a summary report of the study on carcinogenicity in rats was available. In order to evaluate the carcinogenic potential of thiamphenicol further, the Committee requested detailed reports giving the individual data from the carcinogenicity study and from the range-finding study from which the doses were derived. Since the previous evaluation, the results of a 13-week study of toxicity and of a subsequent two-year study of the carcinogenicity of thiamphenicol in rats have been published, and the data for individual animals in both studies have been submitted. The additional information is summarized and discussed in this addendum. Furthermore, the study of developmental toxicity in rabbits and the studies on the microbiological effects of thiamphenicol which were used by the Committee at its forty-seventh meeting to establish the toxicological and microbiological ADIs were re-evaluated to determine the most appropriate toxicological and microbiological end-points. 2. BIOLOGICAL DATA 2.1 Short-term study of toxicity In a study of toxicity with repeated doses, which was designed to find the range of doses to be used in a subsequent study of carcinogenicity, Fischer 344 rats were treated orally with thiamphenicol for 13 weeks. The appropriate doses for this study were determined in two pilot studies of single and repeated oral doses of three to four weeks' duration. Data on individual animals were not submitted. After a single oral dose of 4, 6, 8, or 10 g/kg bw of thiamphenicol given by gavage to groups of five rats of each sex, no relevant clinical signs, no suppression of body-weight gain, and no pathological findings were observed during the 14-day observation period, indicating that this substance has low acute toxicity. When groups of two to five male and female rats were given 0, 500, 1000, 2000, or 2500 mg/L of thiamphenicol in drinking-water (equal to 0, 67, 130, 270, and 330 mg/kg bw per day) for three to four weeks, body-weight gain was severely suppressed at doses of 1000 mg/L and higher. At the lowest dose, there was slight suppression of body-weight gain and slight pathological alterations, such as thymic atrophy, dilatation of the caecum, suppression of haematopoiesis, and testicular degeneration. A dose of 500 mg of thiamphenicol per litre of drinking-water was selected as the highest level for the 13-week study (Ando et al., 1997). The 13-week study was not performed in accordance with good laboratory practice, but the data for individual animals were provided for evaluation, allowing an independent assessment. In this study, groups of 12 Fischer 344 rats of each sex received drinking-water ad libitum containing thiamphenicol at a dose of 0, 125, 250, or 500 mg/L for 13 weeks, equal to 9, 17, and 36 mg/kg bw per day for males and 12, 21, and 39 mg/kg bw per day for females. The animals were observed daily for clinical signs of adverse reactions. Body weight and food and water consumption were recorded weekly. At the end of treatment, all survivors were checked for haematological and serum parameters, organ weights and gross and histopathological appearance. No deaths were recorded in any of the groups. A dose-related reduction in body-weight gain was observed in all treated groups, reaching significance at the intermediate and high doses. Animals at the high dose also showed increased relative liver and kidney weights, decreased relative thymus weight, slight suppression of bone-marrow erythropoiesis and testicular lesions with vacuolation of germinal epithelium, spermatogranulomas, and reduced sperm count in the epididymides; similar effects were observed occasionally at the intermediate dose. Slight changes in haematological parameters (increased mean corpuscular volume, decreased erythrocyte and platelet counts) and serum biochemical parameters (decreased total protein, calcium, and cholesterol in females, increased albumin:globulin ratio, urea nitrogen, and alkaline phosphatase in females) were seen in a dose-dependent manner at the two higher doses. Thiamphenicol at all three doses caused enlargement of the caecum, which is a common side-effect of long-term oral administration of antibacterial agents in rabbits. The lowest dose caused no other relevant substance-related adverse in organs or tissues, and the minor haematological and serum bioc, were almost within the range of normal variations and were inconsistent with regard to dose and sex. A dose of 250 mg/L of thiamphenicol in drinking-water (equal to 17 mg/kg bw per day) was slightly toxic, whereas 125 mg/L (equal to 9 mg/kg bw per day) caused no histopathological lesions in the target organs of thiamphenicol, such as bone marrow and testicular tissue. The minor changes in haematological and serum biochemical parameters at the lowest dose were biologically insignificant. The NOEL was 125 mg/L, 9 mg/kg bw per day (Ando et al., 1997; Maekawa, 1998a). 2.2 Study of carcinogenicity The doses used in a two-year carcinogenicity study in Fischer 344 rats were based on the findings of the dose range-finding study described above. The previous Committee had access only to a summary report of the results of this study, but detailed individual data have since been submitted which allowed independent assessment of the results and of the conclusions summarized in a recent publication. The design of the study did not conform to good laboratory practice, but the study was of adequate quality and duration. Randomly allocated groups of 50 rats of each sex received thiamphenicol in their drinking-water at a concentration of 0, 150, or 250 mg/L for 104 weeks; the drinking-water was supplied ad libitum. On the basis of water consumption, the doses of thiamphenicol were calculated to be equal to 5 and 11 mg/kg bw per day for males and 7 and 14 mg/kg bw per day for females. The complete dissolution of thiamphenicol in the water at all doses and the stability of the solutions at room temperature were checked. The plasma concentrations of thiamphenicol were not determined. The animals were observed daily for clinical signs and deaths, and food and water consumption and body weights were checked once a week for the first 13 weeks and then every four weeks. Four weeks after cessation of the treatment, all survivors were killed and autopsied. The organs were weighed and gross and microscopic anatomical pathological examinations were performed. Reduced body-weight gain was observed at the high dose, which resulted in siginificantly lower body weights of females at the end of the experiment. Food consumption and water intake were not significantly different in the treated and control groups and remained constant throughout the experiment. The mean length of survival of thiamphenicol-treated animals was not different from that of controls; in fact, by the end of the experiment, slightly more treated animals (72-78%) than controls (68% of males and 72% of females) were still alive. The only difference in organ weights was increased pituitary weights in females at the high dose. The incidence and severity of non-neoplastic lesions such as nephropathy, bile-duct proliferation, microgranuloma in liver, bone marrow, and lymph nodes, extramedullary haematopoiesis in spleen, and atrophy of accessory genital glands were similar in all groups, including the controls. These pathological findings were considered to be spontaneous and typical for aged Fischer 344 rats. The histopathological survey showed no evidence of effects on target organs such as bone marrow and testicular tissue. Furthermore, no changes were reported that reflected the slight, biologically insignificant alterations in some haematological and biochemical parameters observed at the lowest dose in the 13-week study in rats. The total tumour incidence was 100% in males in all groups, whereas in females the percentage of tumour-bearing animals showed a nonsignificant dose-related increase, from 84% in controls to 86% at the low dose and 90% at the high dose. Tumours were found in many organs and tissues in all groups, and the organ distribution and histological characteristics of the tumours were similar to those reported to occur spontaneously in rats of this strain, with the exception of a higher incidence of histiocytic sarcomas of the bone marrow in some control and treated animals. The reason for the more frequent occurrence of this rare tumour remains unclear, but it does not appear to be related to treatment in view of the higher incidence in controls and the lack of dependence on dose. The incidence of pituitary adenomas was increased in females, from 12/50 in controls to 16/49 at the low dose and, significantly, to 19/50 in high-dosed animals. The more frequent occurrence of this tumour was, however, not associated with any increase of the incidence of preneoplastic lesions or differences in induction time as compared with controls. In males, the incidence of pituitary adenomas was decreased in a dose-dependent manner. At the high dose, a slight statistically nonsignificant increase in the incidence of thyroid follicular adenomas and subcutis fibromas was observed in males and increases in the incidences of thyroid C-cell adenomas, clitoral gland adenomas and uterine endometrial stomal polyps in females; however, the incidences of these tumours were within the range of the spontaneous incidence in Fischer 344 rats. The Committee concluded that thiampenicol is not carcinogenic in Fischer 344 rats when given continuously in the drinking-water at slightly toxic doses up to 14 mg/kg bw per day for two years. The NOEL was 5 mg/kg bw per day (Kitamura et al., 1997; Maekawa, 1998b). 2.3 Study of developmental toxicity in rabbits The study of developmental toxicity in rabbits treated by daily oral administration of thiamphenicol at a dose of 0, 1.25, 2.5, or 5 mg/kg bw on days 6-18 of gestation, which was evaluated by the Committee at its forty-seventh meeting (Annex 1, reference 125), was re-evaluated. In this study, thiamphenicol caused mild toxicity in the dams, as indicated by a dose-dependent suppression bw throughout the administration period. This effect reached a maximum within the first week and was partly reversed after cessation of treatment. This slight maternal toxicity was not associated with overt toxic effects in embryos or fetuses. Decreased mean fetal weight was seen at the highest dose as compared with controls, but this effect was presumed to be the consequence of increased litter weight and a larger number of fetuses (Sisti et al., 1994). At its forty-seventh meeting, the Committee used the NOEL for maternal toxicity of 1.25 mg/kg bw per day to establish a temporary ADI, although the body-weight gain of the pregnant animals was significantly decreased at this dose at days 7-9 of treatment and did not reach the values of the control group until the end of the experiment. Rabbits are, however, known to be highly sensitive to orally administered antimicrobial agents that cause gastroenteritis and poor digestion, with negative consequences on body-weight gain. 2.4 Special studies on microbiological effects The Committee at its forty-seventh meeting evaluated the antibacterial activity of thiamphenicol on the basis of published literature (Annex 1, reference 125). No new data have been provided. In the available studies, the minimum inhibitory concentrations (MICs) of a wide range of pathogens and of bacterial species representative of human gut flora indicate that anaerobic bacterial strains are especially sensitive to thiamphenicol. Actinomyces, Fusobacterium and Propionibacterium were found to be the most sensitive species, with MIC50 values of 0.5-1.0 µg/ml. The Committee at its forty-seventh meeting used the mean MIC50 of 1.68 µg/ml for all 261 strains of 16 bacterial species of human origin included in the study as the microbiological end-point to calculate the ADI. According to the most recent approach of the Committee for the assessment of microbiological risks of residues of antibacterial substances (see section 2.3 of the report of the present meeting: Annex 1, reference 140), the microbiological ADI should be established on the basis of the MIC50 of the most sensitive, relevant bacterial species of the human gut flora. Fusobacterium, with a MIC50 of 0.5 µg/ml, was considered to be one of the most relevant microorganisms of the human gut flora, of which 20 strains had been tested. 3. COMMENTS In a pilot study to determine the range of doses to be used in a carcinogenicity study, thiamphenicol was administered in the drinking-water to Fischer 344 rats of each sex at a dose of 0, 125, 250, 0r 500 mg/L (equal to 9, 17, and 36 mg/kg bw per day in males and to 12, 21, and 39 mg/kg bw per day in females), for 13 weeks. Thiamphenicol caused no deaths. At 125 and 250 mg/L in drinking-water, numerous adverse effects were reported, including dose-dependent suppression of body-weight gain, a slight reduction in erythropoiesis in bone marrow, reduced erythrocyte and platelet counts, decreased thymus weight and increased liver and kidney weights relative to body weight, testicular lesions with degeneration of the seminiferous tubules, reduced sperm count, and spermatogranulomas in the epididymides. In addition, minor changes were observed in serum biochemical parameters, with reduced total serum protein, an increased albumin:globulin ratio, and decreased cholesterol concentration. In all treated animals, thiamphenicol caused dose-related enlargement of the caecum, which is a common side-effect of long-term oral treatment of rodents with antibiotics. At the lowest dose, no other relevant treatment-related adverse effect was seen on tissues or organs or on haematological or serum biochemical parameters. The results indicated that 250 mg/L in drinking-water (equal to 17 mg/kg bw per day) was slightly toxic, whereas 125 mg/L (equal to 9 mg/kg bw per day) was the NOEL. On the basis of the findings in the pilot study, doses of thiamphenicol of 0, 125, and 250 mg/L in drinking-water (equal to 5 and 11 mg/kg bw per day in males and 7 and 14 mg/kg bw per day in females) were selected for the two-year study of carcinogenicity in Fischer 344 rats. The highest dose caused a reduction in body-weight gain, whereas food and water consumption remained unchanged. The mean survival time of thiamphenicol-treated animals was not different from that of controls. The incidence and severity of non-neoplastic lesions were similar in all groups, and the lesions were considered to be spontaneous and typical of aged Fischer 344 rats. The weight of the pituitary gland was increased in females. The incidence of malignant tumours was not significantly increased in treated animals over that in controls, and the distribution of tumours by organ and the histological characteristics and frequency of the tumours observed in all groups were similar to those reported to occur spontaneously in this strain of rats. The incidences of tumours at some sites were slightly increased in animals at the highest dose but did not exceed the spontaneous incidence reported in historical controls. The increased incidence of pituitary adenomas reached significance only in females and was not associated with any increase in the incidence of preneoplastic lesions or differences in induction time. On the basis of these findings, the Committee concluded that there was no evidence for the carcinogenicity of thiamphenicol in the two-year study in rats. This conclusion was further supported by the absence of genotoxic effects of thiamphenicol in five tests for genotoxicity in vitro and a test for micronucleus formation in mice treated in vivo that had been evaluated by the Committee at its forty-seventh meeting. At its present meeting, the Committee noted that a histopathological survey had shown no evidence that thiamphenicol was toxic in target tissues such as bone marrow and testis. Furthermore, no changes were reported that reflected the slight, biologically insignificant alterations in some of the haematological and biochemical parameters observed at the lowest dose in the 13-week study of toxicity in rats. The NOEL in the two-year carcinogenicity study was therefore 5 mg/kg bw per day. The Committee reconsidered the study of developmental toxicity in rabbits that was used at the forty-seventh meeting to establish the ADI, on the basis of the NOEL of 1.25 mg/kg bw per day for maternal toxicity. The Committee considered that this effect was not an appropriate toxicological end-point because of the known sensitivity of rabbits to orally administered antimicrobial agents. The Committee considered that the NOEL of 5 mg/kg bw per day in the two-year study of carcinogenicity in rats was the most relevant toxicological end-point. The Committee further reconsidered the microbiological data on thiamphenicol that were reviewed at its forty-seventh meeting. At that time, the mean MIC50 of all 261 strains of 16 bacterial species of human origin that had been studied was used to calculate the ADI. The most recent approach of the Committee for assessing the microbiological risks of residues of antimicrobial substances is described in section 2.3 of the report of the present meeting (Annex 1, reference 140). At its present meeting, the Committee calculated the upper limit of the ADI on the basis of antimicrobial activity from the formula given on p. using the MIC50 of the most sensitive, relevant bacterial species of the human gut flora, Fusobacterium, which was 0.5 µg/ml: 0.5 µg/ga × 220 gb Upper limit of ADI = ------------------------ 0.4c × 1d × 60 kge = 4.6 µg/kg bw where a is the MIC50 value for Fusobacterium spp. as the most sensitive, relevant bacterial species, b is the mass of the colonic content, c is the bioavailable fraction of thamphenicol in the gastrointestinal tract, d is the safety factor, and e is the human body weight. 4. EVALUATION The Committee established an ADI of 0-5 µg/kg bw on the basis of the microbiological end-point. This ADI is one-tenth the toxicological ADI of 0-50 µg/kg bw that would be derived on the basis of the NOEL of 5 mg/kg bw per day in the two-year study of carcinogenicity in rats and a safety factor of 100. 5. REFERENCES Ando, J., Ishihara, R., Imai, S., Takano, S., Kitamura, T., Takahashi, M., Yoshida, M. & Maekawa, A, (1997) Thirteen-week subchronic toxicity study of thiamphenicol in F344 rats. Toxicol. Lett., 91, 137-146. Kitamura, T., Ando, J., Ishihara, R., Iijima, T., Nishimura, S., Yoshida, M., Takahashi, M. & Maekawa, A. (1997) Lack of carcinogenicity of thiamphenicol in F344 rats. Food Chem. Toxicol., 37, 1075-1080. Maekawa, A. (1998a) Thirteen-week toxicity study of thiamphenicol in rats. Individual data. Unpublished report. Submitted to WHO by Sasaki Institute, Tokyo, Japan. Maekawa, A. (1998b) Carcinogenicity study of thiamphenicol in rats. Individual data. Unpublished report. Submitted to WHO by Sasaki Institute, Tokyo, Japan.
See Also: Toxicological Abbreviations