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    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 (g/g  MCC (g)
      of ADI   =    ----------------------                     
    (g/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
    g/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 g/ml. The Committee at its forty-seventh
    meeting used the mean MIC50 of 1.68 g/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 g/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 g/ml:

                               0.5 g/ga  220 gb
       Upper limit of ADI =    ------------------------
                               0.4c  1d  60 kge

                          =    4.6 g/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 g/kg bw on the basis of
    the microbiological end-point. This ADI is one-tenth the toxicological
    ADI of 0-50 g/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