THIAMPHENICOL First draft prepared by Dr R. Fuchs, Department of Experimental Toxicology and Ecotoxicology, Institute for Medical Research and Occupational Health, Zagreb, Croatia 1. Explanation 2. Biological data 2.1 Biochemical aspects 2.1.1 Absorption, distribution and excretion 2.2 Toxicological studies 2.2.1 Acute toxicity studies 2.2.2 Short-term toxicity studies 2.2.3 Long-term toxicity/carcinogenicity study 2.2.4 Reproductive toxicity studies 2.2.5 Special studies on embryotoxicity and teratogenicity 2.2.6 Special studies on genotoxicity 2.2.7 Special studies on immune responses 2.2.8 Special studies on microbiological effects 2.3 Observations in humans 3. Comments 4. Evaluation 5. References 1. EXPLANATION Thiamphenicol is a broad-spectrum antimicrobial agent, structurally similar to chloramphenicol, used orally to control infections in humans, pigs, poultry and non-ruminating cattle. It is bacteriostatic for both Gram-positive and Gram-negative aerobes and for some anaerobes. It has not been previously evaluated by the Committee. The molecular structure of thiamphenicol is shown below.2. BIOLOGICAL DATA 2.1 Biochemical aspects 2.1.1 Absorption, distribution and excretion 2.1.1.1 Rats After i.v. administration in rats the half-life of thiamphenicol was 46.3 minutes, compared to 21.5 minutes for chloramphenicol (Ferrari & Della Bella, 1974). Pretreatment of rats with phenobarbital increased thiamphenicol half-life slightly from 46.3 to 55.2 minutes, whereas the chloramphenicol half-life was reduced from 21.5 minutes to 9.3 minutes (Della Bella et al., 1968a). Following administration of 30 mg/kg to rats, thiamphenicol was eliminated in the urine almost entirely in the unchanged form: 62% following oral administration and 47% after i.m. administration (both at 48 hours). No significant glucuro-conjugation products were found. In the bile, 3.4% of the administered dose appeared unchanged and 10-12% appeared as conjugated products after 4 hours. Of the administered dose, 36% was eliminated in faeces after 75 hours, almost entirely as unchanged thiamphenicol. Distribution studies showed that levels in the kidney and liver were higher than in plasma, while brain concentrations were negligible (Gazzaniga, 1974). 2.1.1.2 Rabbits The amount of metabolites recovered in urine and bile within 7 hours after i.v. administration was about 73% for thiamphenicol and 60% for chloramphenicol, and the percentage of glucuronide to total amount recovered was 8% for thiamphenicol and 66% for chloramphenicol. The recovery of thiamphenicol into the bile was only 1%, mostly in unchanged form. About 3% of administered chloramphenicol was excreted into the bile and two-thirds of this consisted of metabolites (Uesugi et al., 1974). 2.1.1.3 Dogs Following intraduodenal administration of 70 mg/kg thiamphenicol or chloramphenicol, 30% of the thiamphenicol dose was found unchanged in urine within 8 hours, whereas only 8.3% of chloramphenicol was eliminated in the active form. After i.m. injection, the urinary elimination of active antibiotics within 8 hours was 24.2% for thiamphenicol but only 1.76% for chloramphenicol (Laplassote, 1962). 2.1.1.4 Pigs To determine plasma and tissue concentrations of thiamphenicol in pigs following dietary treatment, 16 male pigs, approximately 7 weeks old, were fed thiamphenicol in the diet at a concentration of 900 mg/kg (equivalent to 30 mg/kg bw), twice daily for a period of 5 days. Three pigs were maintained as controls and fed basal diet only. Venous blood samples were taken prior to treatment and at various time-points during the study. The animals were killed at 4, 6, 8 and 10 days after treatment and samples of kidney, liver, muscle, fat and lung taken. Concentrations of thiamphenicol in plasma were measured following solvent extraction, using HPLC. On the second day of the dosing period, scouring and swelling or redness of the anus/perineal area were noted in all treated animals. The signs resolved within 4-10 days. The maximum mean plasma level of thiamphenicol (1.28 mg/litre) was demonstrated 8 hours after the first dose. Mean levels were in the range of 0.22-0.80 mg/litre during the dosing period and declined over the withdrawal period to give concentrations below or close to the limit of detection (0.01-0.08 mg/litre) from 4 hours to 5 days after the end of treatment. The results of the analysis of tissue samples were not reported (Redgrave et al., 1991). 2.1.1.5 Humans After a 500 mg oral dose of each of thiamphenicol and chloramphenicol, the plasma levels appear to be similar. A high level of active thiamphenicol and a low level of chloramphenicol were found in the urine (53.1% and 9.2%, respectively, after 24 hours). The half-life of thiamphenicol is significantly increased in renal insufficiency, but is almost unaffected by liver cirrhosis (Azzolini et al., 1972). 2.2 Toxicological studies 2.2.1 Acute toxicity studies The acute toxicity of thiamphenicol and thiamphenicol-glycinate is given in Table 1. The predominant clinical signs in the animal species tested were sedation and piloerection after oral dosing, and dyspnoea, cyanosis, motility disorders and respiratory arrest after parenteral administration. 2.2.2 Short-term toxicity studies 2.2.2.1 Rats The oral toxicity of thiamphenicol, when administered as an aqueous suspension in 0.5% methocel for 13 week, has been investigated in four groups of rats (Sprague-Dawley 30/sex/group). Thiamphenicol was administered by oral gavage at dose levels of 30, 45, 65 or 100 mg/kg bw per day, while a fifth control group received 0.5% methocel only. The first 15 animals of each group were killed on completion of the treatment period, and the remaining 15 after a recovery period of 8 weeks. Mortality during the treatment period was elevated among animals of both sexes receiving 100 mg/kg bw per day, and during the recovery period mortality was similar in all groups. At levels of 65 and 100 mg/kg bw per day, pallor, hair loss, prostration, hunched posture and flaccid musculature were seen. After the recovery phase the incidence and the severity of symptoms were similar in all groups including controls. Body weight stasis or loss was seen in animals receiving 65 and 100 mg/kg bw per day, and after 8 weeks recovery body weight was similar in all groups. Food intake was reduced at dose levels of 45, 65 and 100 mg/kg bw per day, with immediate improvement after cessation of treatment. Table 1. Acute toxicity of thiamphenicol (TAP) and thiamphenicol-glycinate (TAP-G) Species Sex Route LD50 (mg/kg bw) Reference TAP TAP-G Mouse M & F oral > 5000 Bonanomi, 1978 M & F i.p. > 3000 1550 Bonanomi, 1978 M & F i.v. 450 Bonanomi, 1978 Rat M & F oral > 5000 Bonanomi, 1978 M & F i.p. > 3000 1750 Bonanomi, 1978 M & F i.v. 470 Bonanomi, 1978 In all treated animals there were changes in erythrocyte parameters, differential and total leukocyte counts and clotting parameters, all of which were dose-related. After the recovery period erythrocyte and leukocyte counts were still low in males treated with 65 and 100 mg/kg bw per day. Plasma levels of urea, triglycerides and total protein were altered from week 7 in animals treated with 45 mg/kg bw per day and after the end of treatment also in males receiving 30 mg/kg bw per day. Parameters associated with liver and kidney function were affected at the two higher doses. After 8 weeks full recovery was considered to have occurred. The weights of all the main organs were reduced at dose levels of 65 and 100 mg/kg bw per day, but after the recovery period only the testis weight was still reduced. Postmortem examination revealed effects on the gastro- intestinal tract and spleen in both sexes and in the liver, thymus and testis of males at the highest dose level only. After 8 weeks only the testis weights were still reduced. The erythroid/myeloid cell ratio was increased in both sexes at doses of 65 or 100 mg/kg bw per day, and after 8 weeks was still slightly higher than usual. Treatment-related changes were seen in tissues with high cell turnover rates; most of them recovered after a period of respite from treatment. Animals in the groups treated with 30 and 45 mg/kg per day showed no histopathological findings except hepatocytic reduced basophilia (male, 45 mg/kg bw per day) and increased splenic extramed- ullary haematopoiesis (female 45 mg/kg bw per day). Testicular germinal epithelial deficit was present at doses above 45 mg/kg, and caecal oedema and adnexal atrophy were present after the recovery period at the highest dose level. The NOEL was 30 mg/kg bw per day (Marubini et al., 1991). In a 6-month toxicity study in the rat, thiamphenicol was administered as an aqueous suspension in 2% gum arabic by stomach tube 6 days/week to 180 Wistar rats (30/sex/group) at dose levels of 0, 40 or 120 mg/kg bw per day. Body weight and food intake were recorded twice weekly in the first 4 weeks of treatment, and thereafter only once a week. At the end of weeks 4, 8, 16 and 24, ten animals from each group were killed following collection of 2-hour urine sample. Haematology, clinical chemistry assays and urinanalysis were performed on all tested animals. Histological examinations were carried out on the lungs, spermatozoa, blood and bone marrow smears and samples of main organs. There was a decrease in food intake at a dose level of 120 mg/kg bw per day and a dose- and time-related decrease in body weight gain in females. No effect was observed on erythropoiesis or on hepatic or renal function. Urinanalysis showed the presence of albumin and haemoglobin in the high-dose rats. No gross pathological variations in organ weights were observed, but irritative changes in the gastrointestinal mucosa and high incidence of monolateral spontaneous hydronephrosis were seen both in treated and control animals. Histopathological examination performed on control and high-dose rats revealed a slight effect on the morphology of spermatozoa in the high-dose group at the 8th, 16th and 24th week of treatment (Della Bella et al., 1968b). 2.2.2.2 Rabbits Thiamphenicol-glycinate was administered subcutaneously to groups of rabbits ("Fauve de Bourgogne" seven/sex/group) at dose levels of 0, 25, 50 or 100 mg/kg bw per day, 6 days/week for 12 weeks. Animals were weighed weekly and submitted to haematological (erythrocyte and leukocyte counts with differentials) and biochemical (urea, reducing sugars, chlorides) examinations before treatment, after 6 weeks and at the end of the treatment period. After macroscopic examination of the viscera, the various organs were examined histologically. During the treatment two control animals and three in the high-dose group died (no autopsies were carried out). A reduction in polymorphonuclear neutrophils in male rabbits in all treated groups and a slight fall in erythrocyte levels in high-dose females were observed. Anatomical and pathological examination provided no evidence of damage attributable to thiamphenicol-glycinate administration (Brunaud, 1965). 2.2.2.3 Dogs In a 7-week study in beagle dogs (four/sex/group), thiamphenicol was administered orally in gelatin capsules at dose levels of 0, 40 or 80 mg/kg bw per day. Two males and two female dogs were killed at the end of the 7-week treatment, and the remaining animals were kept for further 12 weeks without treatment before being killed. Behaviour and body weights were recorded, haematology and clinical chemistry examinations and urinanalysis were conducted on all animals pretest and at various intervals during the study. Complete gross postmortem examination, organ weighing and histopathological evaluation were conducted on all animals. The animals in the two treated groups developed diarrhoea soon after the beginning of treatment, which spontaneously regressed in the low-dose group but persisted in the high-dose group. At 80 mg/kg bw per day reduced food consumption with loss of weight and muscular asthenia occurred, and vomiting was observed in some cases. Four of these dogs were killed at the end of the 4th week. Slight loss of weight was observed in two dogs in the low-dose group. Decreases in haematocrit, haemoglobin concentration and erythrocyte count were seen in both treated groups, but did not appear to be dose-related, and returned to normal on withdrawal of treatment. Slight increase in proteinuria was observed in the last few weeks of treatment. At 40 mg/kg bw per day superficial erosion of the gall bladder mucosa and at 80 mg/kg bw per day haemorrhagic ulcers in the gall bladder, diffuse muco-membranous enteritis and thymic involution were seen in dogs killed at the end of treatment period. In dogs killed after the 12-week recovery period no differences were observed between treated and control animals. Histological examination after 7 weeks revealed in the high-dose group severe cholecystitis, chronic sclerosing pancreatitis, enteritis, severe depletion of haematopoietic marrow and lymphoid thymus depletion. Two dogs in the low-dose group showed depletion of germinal epithelium in the testes and multinuc- leated cells in seminiferous tubules, which were not seen in the high- dose group. None of the changes observed at 7 weeks were detectable in the animals kept for 12 weeks after cessation of treatment (Bonanomi et al., 1978). Thiamphenicol was administered orally in gelatin capsules to 24 beagle dogs (four/sex/group) at dose levels of 30, 60 or 120 mg/kg bw per day for a period of 4 weeks. Physical observations, ophthalmo- scopic examinations, and body weight and food consumption measurements were performed before treatment and over selected intervals during the treatment. Haematology, clinical chemistry and urinanalysis were conducted on all animals pretest and at study termination. Complete gross postmortem examinations, organ weight and histopathological evaluation were conducted on all animals. The body weights of the high-dose animals were slightly lower than those of controls at week 3 in both sexes, and at week 4 in males only. At 60 and 120 mg/kg bw per day absolute and relative liver weights in male dogs were greater than in controls, and relative liver weights were also increased in females. Microscopically, hepatocellular hypertrophy was present in the liver of mid- and high-dose animals, which correlated with the increase in liver weights in these groups. No other parameter evaluated showed evidence of adverse treatment-related effects. The NOEL was 30 mg/kg bw per day (Kelly & Daly, 1990). Thiamphenicol was administered orally in gelatin capsules to 56 beagle dogs (seven/sex/group) at dose levels of 15, 30 or 60 mg/kg bw per day. After 6 months of treatment four animals/sex/group were sacrificed, and the remaining three animals/sex/group were kept for a 2-month recovery period. Physical observations, ophthalmoscopic examinations, body weight, food consumption, haematology and clinical chemistry examinations and urinanalysis were conducted on all animals pretest and on all surviving animals at selected intervals during the treatment and recovery period. Complete gross postmortem examinations, organ weight and histopathological evaluation were conducted on all animals. One control male was found dead during the study. One male and one female dog in the highest dose group were moribund and had to be killed. Clinical signs prior to death included lethargy, poor food consumption, emaciation, tremors and dehydration. Physical findings related to thiamphenicol administration were tremors, lethargy, irregular gait and excessive licking or chewing in the high-dose group. Tremors were also present in the mid-dose animals. These signs were seen during the last two months of the study and were not present at the end of the recovery period. Body weights of the high-dose males during the study were 4 to 18% lower than those of controls. Decreased erythrocyte counts and mean haematocrit values were seen in high-dose males at weeks 6 and 13 and at termination of the study, and in high- and mid-dose females at week 13 and at termination of the study. After the recovery period no differences were observed in the haematological parameters between control and treated animals. No treatment-related effects were seen in the bone marrow smear examinations. Mean serum cholesterol and phospholipid levels of the mid-and high-dose males at week 6 and 13 and at termination of the study were greater than control values. The same parameters were elevated in high-dose females at the end of the study. Mean serum glucose levels of males at 60 mg/kg bw per day and females at 30 and 60 mg/kg bw per day were significantly increased. Mean fibrinogen values of high-dose females were elevated at the end of the study. Relative liver weights were increased at mid- and high-dose levels. Pathological lesions related to treatment were seen in sections of the thymus (exacerbation of involution), bone marrow (decreased cellularity), liver (centrilobular necrosis and pigment deposition), testes (focal and diffuse tubular atrophy) and oesophagus (ulceration) from high-dose animals of both sexes, mostly occurring in animals killed in a moribund condition. No alterations were noted in any of the tissues examined microscopically from animals that were allowed to recover after treatment. The NOEL was 15 mg/kg bw per day (Kelly & Daly, 1991). 2.2.2.4 Pigs A study designed to determine tolerance in pigs to treatment with thiamphenicol at three times the recommended dose for 5 days and at the normal recommended dose for 15 days was conducted with 16 weaned large white hybrid pigs (two pigs/sex/group). Thiamphenicol was administered in the diet at dose levels of 30 or 90 mg/kg bw per day for 5 days or 30 mg/kg bw per day for 15 days. The control group of animals was fed basal diet only. Clinical signs, body weight and food consumption were recorded. Blood, urine and faecal samples were obtained before dosing and at various time-points during the study. No significant treatment-related clinical abnormalities were noted. Body weight changes and food consumption were within normal limits. No consistent treatment-related differences in haematological and biochemical parameters or in urinanalysis values were observed. The authors concluded that treatment with thiamphenicol had no significant adverse effects on general health, body weight, food consumption or standard clinical pathology parameters (Roberts et al., 1989). In a 4-week toxicity study, groups of pigs (Large White hybrid, four/sex/group) were fed 25, 50 or 100 mg thiamphenicol/kg bw per day. The control group of animals was fed basal diet only. Clinical signs, body weight and food consumption were recorded. Blood, urine and faecal samples were obtained before dosing and during week 4 for clinical pathological investigations. At the end of the 4-week dosing period, pigs were killed, and selected tissues were processed for histological examination. In all groups treated with thiamphenicol, swelling and erythema of the anus, vulva/testes and perineal area, tail and hocks were observed on the second day of the dosing period. These effects were consistent with scouring and irritancy, e.g., as a result of disruption of normal gastrointestinal flora activity, and disappeared within 1 to 13 days. All pigs remained in good health thereafter. Slight reductions in body weight gain and food consumption were noted at 50 and 100 mg/kg bw per day. At week 4 there was a slight reduction in mean PCV, haemoglobin concentration and erythrocyte counts in animals receiving 100 mg/kg bw per day and a treatment-related reduction of urinary pH in all groups dosed with thiamphenicol. At the end of the study, increases in liver and kidney weights in pigs fed 50 and 100 mg/kg bw per day were observed. On histological examination, treatment-related changes were found in the highest dose group only: an increase in vacuolation and fat in renal tubular epithelium and, in some animals, minimal diffuse hepatocyte vacuolation and hepatocyte fat (Cameron et al., 1990). 2.2.3 Long-term toxicity/carcinogenicity study 2.2.3.1 Rats As a range-finding study for the dose selection in a 2-year carcinogenicity study, four groups of F-344 rats (10/sex/group) were given drinking-water containing 0, 125, 250 or 500 mg/litre thiamphenicol (equal to 9, 17 or 36 mg/kg bw per day for males and 12, 29 or 39 mg/kg bw per day for females) for 13 weeks. The examinations at the end of the study covered clinical observations, water consumption, body weight changes, haematological parameters, serum biochemistry, organ weights and gross and microscopic appearance. In haematological examinations anaemic changes were observed in males treated with 250 mg/litre or more and high-dose females. Similar changes, such as increased MCV and increased related counts, were observed in males of the 125 mg/litre group and females of the 250 and 125 mg/litre groups. At autopsy, enlargement of the caecum was seen in treated groups of both sexes. Histologically, the highest dose animals showed decreased haematopoiesis of the bone marrow, decreased spermato- genesis of the testis and sperm granulomas of the epididymis. Sperm granulomas in the epididymis were also seen in some of the animals in the 250 mg/litre group. Based on the results of this pilot study, a 2-year carcinogenicity study of thiamphenicol was performed in rats. Three groups of F-344 rats (50/sex/group) were given drinking-water containing 0, 125 or 250 mg thiamphenicol/litre (equal to 8 or 16 mg/kg bw per day for males and 9.7 or 19 mg/kg bw per day for females) for 104 weeks. All surviving animals subjected to 4-week withdrawal of the test chemical after the end of the treatment were killed for full histopathological examinations. The high-dose animals showed decreased body weight gain, but the incidence of rumours in treated groups was not significantly higher than that of controls (Maekawa, 1996; summary report only was available). 2.2.4 Reproductive toxicity studies 2.2.4.1 Rats In a fertility study on male Wistar rats, oral treatment with thiamphenicol for 2 or 3 months (30 animals per dose level, 10 per treatment time), at dose levels of 120, 180 or 240 mg/kg bw per day, resulted in reduction in the number of tubular germinal cells, which was more marked at the highest dose level. Ten animals of each group were treated for 4 weeks, ten for 8 weeks and the last ten for 12 weeks. At the end of each test period 5 animals of each dose group were killed and necropsied, while the remaining 5 rats were mated with normal females. At 240 mg/kg bw per day extensive testicular hypotrophy, together with severe depletion of the germinal epithelium 21 days after withdrawal of treatment, was seen. Histological changes coincided with a reduction of the fertility index, which gradually recovered within 50 days. Litters from matings between treated males and normal females were normal in number and weight, and no morpho- logical abnormalities were observed. The concentration ratio of thiamphenicol between testes and plasma after administration of 240 mg/kg bw per day thiamphenicol was 1, indicating the absence of accumulation in testes (Della Bella et al., 1967). Groups of 21 Sprague-Dawley rats were given thiamphenicol orally (30, 60 or 120 mg/kg bw per day daily) from day 15 of gestation to day 21 postpartum. In groups receiving 60 and 120 mg/kg bw per day a higher post-implantation loss, slight weight reduction at birth and increased rate of perinatal mortality were observed. No malformations were observed. Development of pups was inhibited during the lactation period with a consistent dose-dependent relationship. From day 30 postpartum a good recovery was observed in all groups. Sexual behaviour and fertility of F1 animals were normal, and the F2 generation showed no signs of abnormal development (Bonanomi et al., 1980). 2.2.5 Special studies on embryotoxicity and teratogenicity 2.2.5.1 Rats Teratogenicity studies were carried out on 195 mature female Wistar rats (15/group) given thiamphenicol orally at dose levels of 40, 80 or 160 mg/kg bw per day from days 1 to 21 of pregnancy and 80 or 960 mg/kg bw per day from days 1 to 7, 7 to 14 or 14 to 21 of gestation. Thiamphenicol did not induce any teratogenic effects in any of the four studies carried out. When the treatment period was 1-21 days, a dose-related increase in resorptions was noted, and newborns had a high mortality rate in the second and third week of life, particularly in the 40 mg/kg bw per day group. In rats treated on days 1-7 of gestation, a non-dose-related increase in resorptions and an increased mortality in newborns in the third week after birth were observed. When the treatment period was from 7 to 14 days, complete resorption of fetuses occurred at 160 mg/kg bw per day. The mean number of newborns per litter was reduced at 80 mg/kg bw per day and there was a high mortality of newborns in the first week. An increased mortality among newborns of the group treated with 80 mg/kg bw per day during days 14-21 of gestation was observed (Bonanomi & De Paoli, 1969). When the inhibition of mitochondrial functions induced by thiamphenicol was compared with the inhibition of overall embryonic development, it appeared that mitochondrial respiration was the rate-limiting step for the embryotoxic effects of thiamphenicol. Because of the lack of specificity of these effects, prenatal mortality rather than teratogenic effects was seen (Bass et al., 1978). 2.2.5.2 Rabbits A teratogenicity study was performed with 50 New Zealand white rabbits administered thiamphenicol orally at dose levels of 5, 30, 60 or 80 mg/kg bw per day from the 8th to the 16th day of pregnancy. The highest dose resulted in a complete resorption of implantation due to the toxic effects on the mothers. Data obtained in all treated groups showed moderate fetal toxicity with a dose-related increase in abortion rate and resorption. No skeletal malformations were found in fetuses (Bonanomi et al., 1974). Thiamphenicol in 0.5% Methocel K15M was administered daily by oral gavage to female rabbits (16/group) from day 6 to day 18 of gestation at doses of 1.25, 2.5 or 5.0 mg/kg bw per day. Control animals received the vehicle alone. The females were killed on gestation day 29 and subjected to postmortem examination. All fetuses were examined for external and internal abnormalities and skeletal changes. No clinical signs attributable to treatment were observed. Mild signs of maternal toxicity were observed in mid- and high-dose animals, indicated by reduction in body weight changes during the treatment period. Necropsy findings in females on gestation day 29 were incidental, with no relation to treatment. Litter parameters and sex ratios did not show any significant difference between groups. Mean fetal weight was decreased in the high-dose group. Two fetuses in the mid-dose group and one in the control group were malformed, with cleft palate, abnormally shaped head, extra digits and incomplete flexure of the hind limbs. The number of small fetuses in the high-dose group was higher than in the control group. The few anomalies seen during the internal examination were not considered to be treatment-related. Skeletal examination revealed no differences between high-dose fetuses and controls. The authors concluded that thiamphenicol administered by oral gavage at concentrations of 1.25, 2.5 and 5 mg/kg per day had no effects on pregnancy or embryo-fetal development. However, the Committee concluded that the NOEL for embryotoxicity under the conditions of this experiment was 1.25 mg/kg per day (Sisti, 1994). 2.2.6 Special studies on genotoxicity The results of genotoxicity assays on thiamphenicol are given in Table 2. Table 2. Genotoxicity assays on thiamphenicol Test system Test object Concentration Results Reference Ames test1 S. typhimurium 0.5-50 Negative Pinasi TA98, TA100, µg/plate2 et al., 1990a TA1535, TA1537, TA1538 Gene conversion Saccharomyces 2.8-140.3 mM3 Negative Marca & and mitotic cerevisiae Bonanomi, crossing over1 1979 Gene mutation Chinese hamster 50-5000 Negative Pinasi assay1 V79 cells µg/ml4 et al., 1990b Chromosomal Cultured human 700-3250 Negative Mosesso & aberrations1 lymphocytes µg/ml5 Driedger, 1989 DNA repair Primary rat 500 and 1000 Negative Bichet, 1985 test hepatocytes µg/kg6 In vivo Mouse bone 2500 and 5000 Negative Pinasi micronucleus marrow mg/kg7 et al., 1990c assay 1 Both with and without rat liver S9 fraction 2 Methyl methanesulfonate and cyclophosphamide were used as positive controls 3 2-Nitrofluorene, 9-aminoacridine, sodium azide and 2-aminoanthracene were used as positive controls 4 Ethyl ethanesulfonate and N-dimethylnitrosoamine were used as positive controls 5 Mitomycin-C and cyclophosphamide were used as positive controls 6 2-Aminofluorene was used as positive control 7 Cyclophosphamide was used as positive control 2.2.7 Special studies on immune responses The effects on spontaneous nephritis in NZB × OUW hybrid mice (32-39 males/group) were investigated in a study involving lifetime administration of thiamphenicol in feed at dose levels of 25, 50 and 250 mg/kg bw per day. Body weight, urinary protein, limited haematology, organ weights and histopathology investigations were reported. The prolonged treatment with thiamphenicol at dose rates of > 50 mg/kg reduced the severity of the spontaneous renal disease and significantly extended lifespan, compared to untreated controls. The immunosuppressive effect of thiamphenicol was demonstrated histologically by a reduction in immune-complex deposition in the glomeruli. No evidence of malignancy or premalignant signs was seen (Simpson et al., 1979). 2.2.8 Special studies on microbiological effects 2.2.8.1 In vivo In a study of thiamphenicol-induced changes in mouse intestinal microflora, 100 female albino mice were divided into 10 subgroups, and five of these groups were treated with thiamphenicol at concentrations of 40 µg/kg diet for 35 days. Samples of intestinal content were taken from the caecum for bacteriological analysis before the treatment and after 7, 14, 28 and 35 days. Microorganisms were isolated by preparing serial dilutions of intestinal content, and the most representative bacteria of the mouse intestinal microflora were cultured on specific media. Their sensitivity to thiamphenicol was assessed by calculating the minimal inhibitory concentration (MIC) of the drug. The results show that the mean counts of various microorganisms did not differ significantly between control mice and those fed with thiamphenicol. The numbers of the bacterial populations did vary at the different sampling times, and in some cases the difference from pre-treatment results was significant, but confidence limits were the same in treated and control mice killed at the same time. The investigation shows that there were no appreciable differences in the type or amount of bacterial flora related to thiamphenicol administration. Differences in the distribution of genera such as Diplococcus sp. and Escherichia sp. were similar to those in controls. The thiamphenicol MIC values for the numerous strains tested indicate that addition to feed at concentrations corresponding to the proposed MRL of 40 µg/kg feed does not select for drug-resistant strains and has no effect on the qualitative or quantitative composition of the intestinal microflora. The MIC remained unchanged throughout the 35 days of the study (Poli, 1994). 2.2.8.2 In vitro In vitro antibacterial activity of thiamphenicol against 489 bacterial isolates from infected animals was determined by the agar dilution method. In the case of mycoplasmas, however, MICs were determined by the broth dilution method. Depending on bacterial strains tested estimations were made under aerobic or anaerobic conditions. The results are presented in Table 3 (Albini, 1989). Data on the sensitivity of normal components of human intestinal microflora are presented in Table 4 (Sutter & Finegold, 1976; Schioppacassi, 1992). Table 3. Antibacterial activity of thiamphenicol against 489 animal pathogens Organisms Number of MIC (µg/ml) Range isolates MIC50 MIC90 Bacteroides spp. 11 2 16 1 - 128 Bordetella spp. 9 32 32 16 - 32 Campylobacter spp. 17 8 16 4 - 16 Clostridium spp. 37 2 4 0.25 - 16 Corynebacterium spp. 10 2 16 2 - 16 Escherichia coli 61 128 >128 16 - > 128 Haemophilus pleuropneumoniae 7 0.5 1 0.5 - 1 Micrococcus spp. 6 0.5 0.5 0.5 - 8 Mycoplasma spp. 9 1 2 0.125 - 4 Pasteurella spp. 71 1 2 0.25 - 128 Salmonella spp. 34 32 32 8 - > 128 Staphylococcus spp. 94 8 32 4 - > 128 Streptococcus spp. 123 2 4 0.5 - > 128 2.3 Observations in humans Reversible dose-related bone marrow suppression is seen after thiamphenicol treatment and is attributed to its inhibitory effect on mitochondrial protein synthesis (Nijhof & Kroon, 1974). Reversible inhibition of myeloid and erythroid colony growth by thiamphenicol, resulting from an inhibition of mitochondrial protein synthesis, is consistent with the reversible bone marrow suppression induced by this drug (Yunis & Gross, 1975). A study of clinical reports on the use of thiamphenicol in 16 631 cases from 1968 to 1977 in Japan revealed that blood disorders occurred in 41 (0.46%) out of 8848 patients receiving thiamphenicol glycinate therapy and 28 (0.36%) out of 7783 patients receiving thiamphenicol. The disorders were dose-dependent, occurring mainly in erythrocytes, and disappeared spontaneously on discontinuation of the drug (Tomoeda & Yamamoto, 1981). The para-nitro group of chloramphenicol has been shown to have a central role in the pathogenesis of aplastic anaemia, probably as a result of its reduction to the highly toxic nitroso metabolite, which is a potent inhibitor of DNA synthesis. Thiamphenicol does not show this activity and the absence of the para-nitro group is therefore advanced as evidence that thiamphenicol cannot induce aplastic anaemia (Murray et al., 1983). Thiamphenicol has been used extensively in human medicine for over 25 years. Total human exposure to thiamphenicol up to 1987 has been estimated at 130-650 million exposures, assuming an average course of therapy of 7.5-15 g (Personal communication from Dr S. Biressi, Zambon Group SpA, Italy to Dr R.D. Agostino, Farmaquest Co.; submitted to WHO by Zambon Group SpA, Italy). Epidemiological studies have not established any causal association between thiamphenicol treatment and irreversible aplastic anaemia (TAP Pharmaceuticals Inc., 1987). Statistical analysis of data obtained in these studies support the argument that the risk of aplastic anaemia from exposure to thiamphenicol is similar to the background risk of idiopathic aplastic anaemia (from 1 in 200 000 to 1 in 800 000) (Kelly & Kaufman, 1989). Table 4. Antibacterial activity of thiamphenicol against 261 strains of anaerobic bacteria isolated from humans (From: Sutter & Finegold, 1976) Bacteria No. of Cumulative % susceptible to indicated concentration (µg/ml) strains tested <0.1 0.5 1.0 2.0 4.0 8.0 16.0 32.0 64.0 128.0 Bacteroides fragilis 42 5 21 71 100 Bacteroides 59 9 24 51 90 100 melaninogenicus Other Bacteroides 21 19 24 57 76 86 91 95 100 and Selenomonas Fusobacterium 8 100 nucleatum Other Fusobacterium 12 42 92 100 Peptococcus and 17 35 71 100 Gaffkya Peptostreptococcus 15 33 47 93 100 Table 4. (cont'd). Bacteria No. of Cumulative % susceptible to indicated concentration (µg/ml) strains tested <0.1 0.5 1.0 2.0 4.0 8.0 16.0 32.0 64.0 128.0 Anaerobic and 6 50 83 100 microaerophilic streptococci Gram-negative cocci 7 43 86 100 Eubacterium 7 43 57 100 Arachnia propionica 2 50 100 Propionibacterium 4 50 75 100 Actinomyces 16 25 56 94 100 Lactobacillus 10 10 30 50 90 100 Clostridium 8 100 perfringens Other Clostridium 27 4 22 63 78 96 100 3. COMMENTS Studies on thiamphenicol available for evaluation included data on pharmacokinetics, acute toxicity, short-term toxicity, reproductive toxicity, developmental toxicology, genotoxicity, and limited information on long-term toxicity. Studies on microbiological effects of thiamphenicol and epidemiological data on humans were also considered by the Committee. The Committee noted that many of the studies were conducted utilizing protocols that would not meet contemporary standards, and therefore the substance was evaluated under the procedures developed for drugs with a long history of use (Annex 1, reference 104). The pharmacokinetic data showed that the drug is rapidly absorbed when administered by oral or parenteral routes. After intravenous administration in rats, the half-life was estimated to be 46 minutes. The main route of excretion in humans and animals is in the urine; approximately 60% of an oral dose of 30 mg/kg bw was excreted unchanged in the urine over a 24-hour period. Single oral doses of thiamphenicol were of low toxicity to mice and rats (LD50 > 3000 mg/kg bw). Short-term oral toxicity studies with thiamphenicol were performed in rats, dogs and pigs, the results are described in the following paragraphs. In a 13-week study in rats at dose levels of 30, 45, 65 or 100 mg/kg bw per day, increased mortality was observed among animals given 100 mg/kg bw per day. In a 6-month study in rats, where the highest dose used was 120 mg/kg bw per day, increased mortality was not reported. In both studies, decrease in body weight gain during treatment occurred at doses of > 65 mg/kg bw per day. Dose-related decreases in red blood cell parameters, differential and total white blood cell counts, and clotting parameters were observed in the 13-week study, but the same effects were not reported in the 6-month study. Testicular germinal epithelial cell depletion was seen at doses above 45 mg/kg bw per day in the 13-week study, and a dose of 30 mg/kg bw per day was considered to be the NOEL. Dogs were given 40 or 80 mg thiamphenicol/kg bw per day for 7 weeks. At both dose levels decreases in body weight were observed, as well as reversible decreases in haematocrit, haemoglobin concentration and erythrocyte count. At 40 mg/kg bw per day, superficial erosion of the gall bladder mucosa was observed. The higher dose level resulted in haemorrhagic ulcers in the gall bladder, diffuse mucomembranous enteritis and early thymic involution. Two dogs in the low-dose group had testicular germinal epithelial cell depletion and multinucleated cells in the seminiferous tubules. When thiamphenicol was given to dogs at doses of 30, 60 or 120 mg/kg bw per day for 4 weeks, the body weights of the high-dose animals were slightly lower than those of controls. In the mid- and high-dose groups, increases in absolute and relative liver weights were observed. Hepatocellular hypertrophy was present in the liver of dogs given 60 or 120 mg/kg bw per day. In a 6-month study dogs were given thiamphenicol at doses of 15, 30 or 60 mg/kg bw per day. The body weights of high-dose males during the study were up to 18% lower than those of controls. The main haematological findings were decreases in red blood cell parameters at the highest dose level. Increases were noted in mean serum cholesterol level and phospholipid concentrations in males (30 and 60 mg/kg bw per day groups) and females (60 mg/kg bw per day group), and in the mean serum glucose concentration of males (60 mg/kg bw per day group) and females (30 and 60 mg/kg bw per day groups). The relative liver weights at the mid- and high-dose levels were increased. Histopatho- logical lesions related to treatment were seen in the thymus (early involution), bone marrow (decreased cellularity), testes (focal and diffuse tubular atrophy) and oesophagus (ulceration) of high-dose animals. The NOEL was 15 mg/kg bw per day. In a 4-week study, pigs were treated with 25, 50 or 100 mg thiamphenicol/kg bw per day. In the highest-dose group, slight reduction in body weight gain, as well as reductions in mean packed cell volume, haemoglobin concentration and erythrocyte counts, were observed, and histological examination showed vacuolation in renal tubular epithelial cells and mild diffuse hepatocyte vacuolation. In all treated groups treatment-related reduction in urine pH was observed. A summary report of a two-year carcinogenicity study in rats, including a range-finding study, was available to the Committee. Rats were given 125 or 250 mg/kg thiamphenicol in drinking-water (equal to 8 or 16 mg/kg bw per day for males and 10 or 19 mg/kg bw per day for females) for 104 weeks. The highest-dose animals showed a decrease in body weight gain, but there was no significant increase in the incidence of tumours in treated groups compared to control animals. In a long-term study in mice (32-39 males/group), which was designed primarily to investigate the effects of thiamphenicol on immune responses, thiamphenicol was administered orally at doses of 25, 50 or 250 mg/kg bw per day. No evidence of neoplastic or preneoplastic changes was observed. In a study to determine the effect of thiamphenicol on fertility in rats, the drug was administered orally at doses of 120, 180 or 240 mg/kg bw per day for 2 or 3 months. Thirty male rats were used per dose level (10 per treatment period). From each treatment-period group, half of the animals were killed for histopathological examination at the given time intervals, while the remaining males were mated with untreated females. Reductions in the number of germinal epithelial cells in testes of all treated animals were observed. These changes were present up to 21 days after termination of treatment, and full recovery was observed by 50 days. Histological changes correlated with the fertility index. Litters from matings between treated males and non-treated females were normal in number and no physical abnormalities were reported. Thiamphenicol was given orally to rats from day 15 of gestation to day 21 post-partum at doses of 30, 60 or 120 mg/kg bw per day. In the mid-and high-dose groups, there were higher post-implantation losses and increased rates of perinatal mortality. Physical develop- ment of pups was inhibited during the lactation period in a dose- dependent manner. Sexual behaviour and fertility of F1 animals were normal, and animals in the F2 generation showed no abnormalities. In four teratogenicity studies in rats, thiamphenicol was administered orally at dosages of 40, 80 or 160 mg/kg bw per day from days 1 to 21 of pregnancy or of 80 or 960 mg/kg bw per day over critical days of gestation (either 1-7, 1-21, 7-14 or 14-21). No teratogenic effects were observed. In all animals treated from days 1 to 21, a dose-related increase in resorption was noted and newborn pups had an elevated mortality rate. A teratogenicity study was performed in rabbits using oral doses of 5, 30, 60 or 80 mg/kg bw per day from days 8 to 16 of gestation. Complete resorption of embryos occurred at 80 mg/kg bw per day. In other treated groups, moderate fetal toxicity and dose-related increases in abortion rate and resorption were reported. No malformations were found in fetuses. In another teratogenicity study, rabbits received oral doses of thiamphenicol at doses of 1.25, 2.5 or 5 mg/kg bw per day from days 6 to 18 of gestation. Mild maternal toxicity was observed in mid- and high-dose animals in the form of depressed body weights during the treatment. No effects were observed on embryo-fetal development. The NOEL was 1.25 mg/kg bw per day. Thiamphenicol gave negative results in five in vitro genotoxicity tests and in an in vivo micronucleus assay using mouse bone marrow. The Committee considered data from human epidemiological studies and concluded that there was no evidence that thiamphenicol can induce aplastic anaemia, in contrast to the structurally related compound, chloramphenicol. The Committee considered data from several in vitro studies on the minimum inhibitory concentration (MIC) of thiamphenicol for a wide range of animal and human pathogens as well as genera representative of the human gut flora. The modal MIC50 value (minimum inhibitory concentration of thiamphenicol giving complete inhibition of growth of 50% of cultures) was 1.68 µg/ml for 261 bacterial strains isolated from humans. The following species were found to be the most sensitive: Bacteroides, Fusobacteria, Propionibacteria and Actinomyces. The Committee also noted that 40 µg thiamphenicol/kg food given to mice over 35 days did not alter the intestinal microflora in this species. The Committee calculated a microbiological ADI for thiamphenicol using the following formula: Upper limit of MIC50 (µg/g) × mass of colonic content (g) microbiological = ADI fraction of oral × safety × human body dose available factor weight (kg) 1.68 × 220 = 0.4 × 1 × 60 = 15 µg/kg bw In calculating a microbiological ADI the Committee took the following factors into consideration: * Concentration: 1.68 µg/ml was the modal MIC50 for micro- biological effects on human intestinal microflora (the density was assumed to be 1 g/ml). * Availability: the Committee calculated the available portion of thiamphenicol as follows: 100% ingested - 60% excreted via = 40% bioavailable urine within in the intestinal 24 hours tract 1 - 0.6 = 0.4 * Safety factor: the Committee concluded that the data deriving from the microbiological studies (substantial amount of MIC data covering a variety of microorganisms and in vivo data from animal studies) provided sufficient information on microbio- logical effects of thiamphenicol. It therefore adopted a safety factor of 1 in the calculation. 4. EVALUATION Taking into account the available toxicological and antimicrobial data and the ADI based on antimicrobial activity, the Committee concluded that the toxicological data provided the most appropriate end-point for the evaluation of thiamphenicol. The Committee established a temporary ADI of 0-6 µg/kg bw for thiamphenicol, based on the NOEL of 1.25 mg/kg bw per day for maternal toxicity in the teratogenicity study in rabbits and a safety factor of 200. The ADI was designated "temporary" because only a summary report of the carcinogenicity study in rats was available. Detailed reports of the carcinogenicity study and the range-finding study used to establish dose levels in that study are required for evaluation in 1999 (see Annex 4). 5. REFERENCES Albini, E. (1989). In vitro antibacterial activity of thiamphenicol against bacterial strains recently isolated from animal infectious diseases. 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Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Bonanomi, L. & De Paoli, A.M. (1969). Teratogenicity study of oral thiamphenicol in the rat. Unpublished report from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Bonanomi, L, Pacei, E., & De Paoli, A.M. (1974). Teratogenicity study of oral thiamphenicol in the rabbit. Unpublished report from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Bonanomi, L, De Paoli, A.M., & Gazzaniga, A. (1978). Toxicity study after repeated oral administration of thiamphenicol in the dog. Unpublished report from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Bonanomi, L., Aliverti, V., Ornaghi, F., Losa, M., & Motta, F. (1980). Thiamphenicol perinatal and postnatal toxicity study in the rat. Unpublished report from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Brunaud, M. (1965). Thiamphenicol glycinate hydrochloride. Long-term toxicity in the rabbit by subcutaneous route. Unpublished report from Clin-Byla Laboratories, Paris, France. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Cameron, D.M., Crook, D., Brown G., Gopinath, C., Farmer, H., & Offer, J.M. (1990). Z 2041: 4-Week toxicity study in pigs. Unpublished report No. ZBN 12/891518 from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Della Bella, D., Bonanomi, L., De Paoli, A.M., & Gazzaniga, A. (1967). Fertility study in male rat treated with thiamphenicol by the oral route. Unpublished report from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Della Bella, D., Ferrari, V., Marca, G., & Bonanomi, L. (1968a). Chloramphenicol metabolism in the phenobarbital induced rat. Comparison with thiamphenicol. Biochem. Pharmacol., 17, 2381-2390. Della Bella, D., Bonanomi, L., De Paoli, A.M., & Gazzaniga, A. (1968b). Toxicity study of thiamphenicol after repeated oral administration in the rat. Unpublished report from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Ferrari, V. & Della Bella, D. (1974). Comparison of chloramphenicol and thiamphenicol metabolism. Postgrad. Med. J., 50, 17-22. Gazzaniga, A. (1974). Thiamphenicol - pharmacokinetics and metabolism in animals. Unpublished report from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Kelly, C.M. & Daly I.W. (1990). A four week oral toxicity study in the dog via capsule administration with Z 2041. Unpublished report No. 88-3391 from Bio/dynamics Inc., East Millstone, NJ, USA. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Kelly, C.M. & Daly, I.W. (1991). A six month oral toxicity study in the dog via capsule administration with Z 2041 with a two month recovery period. Unpublished report No. 88-3392 from Bio/dynamics Inc., East Millstone, NJ, USA. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Kelly, J.P. & Kaufman, D.W. (1989). Ant-infective drug use in relation to the risk of agranulocytosis and aplastic anemia: A report from the international agranulocytosis and aplastic anemia study. Arch. Intern. Med., 149, 1036-1040. Laplassote, J. (1962). Recherches expérimentales sur le thiopénicol: activité antibactérienne, concentrations humorales, élimination. Comparaison avec le chloramphénicol. Therapie, 16, 104-108. Marca, G. & Bonanomi, L. (1979). Gene conversion and mitotic crossing-over in Saccharomyces cerevisiae 6117 with thiamphenicol with and without metabolic activation. Unpublished report from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Maekawa, A. (1996). Summary report on two-year rat carcinogenicity study of thiamphenicol. Submitted by Sasaki Institute, Tokyo, Japan to the Japanese Ministry of Health and Welfare. Marubini, M., Motta, F., Cerioli, A., & Finn, J.P. (1991). Z 2041. Thirteen week oral toxicity study in rats followed by a recovery period of 8 weeks. Unpublished report No. 1219 from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Mosesso, P. & Driedger, A. (1989). Chromosome abberations in human lymphocytes cultured in vivo. Unpublished report No. LSC-RTC 116028-M-02989 from Life Science Research Roma Toxicology Centre SpA, Rome, Italy. 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Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Pinasi, C., Ferrini, S., & Ceriani, D. (1990c). Genotoxicity evaluation of Z 2041 in the "in vivo" mouse micronucleus assay. Unpublished report No. 1233 from Toxicology Department, Zambon Research, Bresso, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Poli, G. (1994). Qualitative and quantitative changes in the mouse intestinal microflora induced by chronic thiamphenicol at maximum residual level (MRL) dose. Unpublished report from Facolta di Medicina Vetarinaria, Universita di Milano, Milan, Italy. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Redgrave, V.A., Cameron, D.M., Anderson, A., & Maxwell, J.G. (1991). Blood concentrations of thiamphenicol in pigs following dietary administration of Z 2041. Unpublished report No. ZBN 11/901351 from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. 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Susceptibility of anaerobic bacteria to 23 antimicrobial agents. Antimicrob. Agents Chemoter., 10, 736-752. TAP Pharmaceuticals Inc. (1987). Issues relating to the safety of thiamphenicol with special attention to the matter of aplastic anemia. Unpublished report from TAP Pharmaceuticals Inc., Corte Mader, Canada. Submitted to WHO by Zambon Group SpA, Bresso, Milan, Italy. Tomoeda, M. & Yamamoto, K. (1981). The hematologic adverse reaction experience with thiamphenicol in Japan. In: Safety Problems Related to Chloramphenicol and Thiamphenicol Therapy, Raven Press, New York, pp. 103-110. Uesugi, T., Ikeda, M., Hori, R., Katayama, K., & Arita, T. (1974). Metabolism of thiamphenicol and comparative studies of its urinary and biliary excretion with chloramphenicol in various species. Chem. Pharm. Bull., 22, 2714-2722. Yunis, A.A. & Gross, M.A. (1975). Drug-induced inhibition of myeloid colony growth: protective effect of colony stimulating factor. J. Lab. Clin. Med., 86, 499-504.
See Also: Toxicological Abbreviations THIAMPHENICOL (JECFA Evaluation)