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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 41





        Prepared by:
          The 50th meeting of the Joint FAO/WHO Expert
          Committee on Food Additives (JECFA)



        World Health Organization, Geneva 1998




    GENTAMICIN (addendum)

    First draft prepared by
    Dr P. Olsen & Dr L. Dragsted
    Institute of Food Safety and Toxicology
    Danish Veterinary and Food Administration, Copenhagen, Denmark

    1.   Explanation
    2.   Biological data
         2.1  Toxicological studies
              2.2.1     Special studies on human intestinal flora
              2.2.2     Special studies on genotoxicity
         2.2  Structural similarities with known carcinogens
    3.   Comments
    4.   Evaluation
    5.   References

    1.  EXPLANATION

         Gentamicin is an aminoglycoside antibiotic. This compound was
    previously evaluated by the Committee at its forty-third meeting
    (Annex 1, reference 113), when a temporary ADI of 0-4 µg/kg bw was
    allocated. The Committee recommended temporary maximum residue limits
    (MRLs) of 100 µg/kg for muscle and fat, 200 µg/kg for liver, and 1000
    µg/kg for kidney, in both cattle and pigs, and 100 µg/L for cow's
    milk, all of the values being expressed as parent compound.

         The Committee required the following information for evaluation
    in 1997: (i) results of studies on the effects of gentamicin on
    specific genera of microorganisms obtained from the human intestine;
    (ii) additional data to assist in the assessment of carcinogenic
    potential, which should include the results of assays for gene
    mutations in mammalian cells and chromosomal aberrations  in vitro 
    and  in vivo, and details of an investigation on possible structural
    similarities between gentamicin and known carcinogens; and (iii) a
    validated chemical analytical method with a limit of quantification
    below the MRL recommended for milk. These results were not available
    to the Committee at its forty-eight meeting (Annex 1, reference 128),
    and the temporary acceptable daily intake established at the
    forty-third meeting of the Committee (Annex 1, reference 113) and the
    temporary MRLs recommended at that meeting were extended until 1998.
    This information has now become available and is summarized and
    discussed in this monograph addendum.

    2.  BIOLOGICAL DATA

    2.1  Toxicological studies

    2.1.1  Special studies on human intestinal flora

         Studies on the effects of gentamicin on bacterial isolates from
    human intestinal microflora  in vitro were available; studies have
    not been performed  in vivo. The minimal inhibitory concentration
    (MIC) of gentamicin was determined for anaerobic and aerobic bacteria
    isolated from fresh stools of 25 healthy adult volunteers. All tests
    were performed at the department of Medical Microbiology of St Radboud
    University Hospital in Nijmegen, The Netherlands. No statement was
    made about accordance with GLP procedures. Ten isolates each of
     Enterococcus spp., coliforms,  Proteus spp.,  Bacteroides spp.,
     Lactobacillus spp,  Bifidobacterium spp,  Prevotella spp,
     Eubacterium spp.,  Clostridium spp.,  Fusobacterium spp., and
    anaerobic gram-positive cocci were tested, including five control
    strains. The range of species tested was considered to be
    representative of the human gut bacterial flora, mainly governed by
    anaerobes.

         The anaerobic bacteria were tested by the agar dilution method
    recommended by the National Council for Clinical Laboratory Standards.
    Brucella agar base supplemented with haemin and vitamin K was used,
    and gentamicin was tested in serial double dilutions. A density of
    1 × 106 bacterial cells per spot was used, and incubation was for
    48 h at 37°C under anaerobic conditions. The control strains used were
     Bacteroides fragilis (ATCC 10584) and  Peptostreptococcus 
     anaerobius (ATCC 27337). Facultative anaerobic bacteria was tested
    by a broth dilution technique on microtitre plates, as recommended by
    the National Council for Clinical Laboratory Standards. Isosensitest
    Broth (Oxoid CM 491) was used as the medium, and gentamicin was tested
    in serial double dilutions. Bacterial cells were tested at a density
    of 1 × 104 per well, and bacterial growth was determined after 24
    and 48 h of incubation. The control strains used were  Escherichia 
     coli (ATCC 25922),  Enterococcus faecalis (ATCC 29212), and
     Proteus mirabilis (ATCC 14273). The MIC value was defined as the
    lowest concentration of anaerobic or facultative anaerobic bacteria
    that prevented visible growth in the test medium. The MIC values
    determined are shown in Tables 1 and 2. The sensitivity of human
    intestinal bacteria to gentamicin was presented as the geometric mean
    MIC value for each genus and for the overall population of strains.
    The geometric mean MIC values of gentamicin ranged from 0.04 µg/ml for
     Proteus spp. to > 128 µg/ml for  Prevotella and  Bacteroides spp.
    Among the anaerobes, the most sensitive to gentamicin were
     Eubacterium spp., with MIC values ranging from 2 to 32 µg/ml and a
    geometric mean MIC value of 6.06 µg/ml. The overall geometric mean MIC
    value for gentamicin for all genera tested was 8.86 µg/ml (Lohuis &
    Aerts, 1996).

    2.1.2  Special studies on genotoxicity

         Gentamicin was tested  in vitro for its ability to induce
    forward gene mutation in Chinese hamster ovary cells at concentrations
    of 128ś5000 µg/ml and chromosomal aberrations in these cells at
    concentrations of 800-5000 µg/ml, both with and without metabolic
    activation. It was also tested  in vivo for its ability to induce
    nuclear anomalies in CD-1 mouse bone-marrow cells at intravenous doses
    of 20ś80 mg/kg bw, the highest dose being the maximum tolerated dose
    (Table 3). All of the studies were performed according to appropriate
    standards for study protocol and conduct. There was no indication of
    mutagenic activity. Two additional studies on chromosomal aberrations
     in vitro and  in vivo (Won, 1996a,b) were received for evaluation,
    but they were not taken into consideration because of shortcomings in
    the study protocol and reporting.

    2.2  Structural similarities with known carcinogens

         The structural relationship of gentamicin with known carcinogens
    was analysed by investigating the chemical structure of gentamicin for
    features commonly found to be associated with mutagenic and/or
    carcinogenic potential ((structural alerts). Structurally, gentamicin
    comprises three aliphatic six-membered rings, two tetrahydropyrane
    rings (purpurosamine and garosamine), and one cyclohexane ring
    (2-deoxystreptamine). The latter is bound to the tetrahydropyrane
    rings through ether linkages in the form of glucosidic bonds. The ring
    system is substituted with amino groups, hydroxyl groups, aliphatic
    side chains, and aliphatic side chains containing primary or secondary
    amino groups. The three analogues of gentamicin vary with respect to
    an aliphatic side chain containing a primary or a secondary amino
    group (Figure 1). Two compilations of structural alerts exist in the
    literature. The first contains a list of 10 structural features that
    are considered to indicate an increased probability of carcinogenic
    activity in animals (US Food and Drug Administration, 1994). The
    second is based on several comparisons of the chemical structure,
    mutagenicity to  Salmonella typhimurium, and carcinogenicity of 522
    rodent carcinogens and 55 human carcinogens. The electrophilicity,
    mutagenicity, and carcinogenicity of the tested chemicals was compared
    on the basis of this data bank, and a list of 19 molecular structural
    alerts (mainly for genotoxic carcinogens) was drawn up (Tennant &
    Ashby, 1991; Ashby & Paton, 1993), many of which were similar to those
    in the list of the US Food and Drug Administration.

         Amino groups are considered as alerts in both lists, but only
    when they are attached to an aromatic ring system; none of the amino
    groups in gentamicin is attached to aromatic rings. None of the other
    structural features listed in the two compilations was found in the
    chemical structure of gentamicin. The author concluded that gentamicin
    contains none of the structural alerts associated with potential
    carcinogenic or mutagenic activity (Inveresk Research, 1997).

    Table 1. Antimicrobial activity of gentamicin in vitro on 
    110 strains of facultative anaerobic and anaerobic bacteria 
    (10 isolates per strain) isolated from human stools 

                                                                       
    Species                               Geometric mean 
                                                                       
                                          MIC (µg/ml)    Range
                                                                       
    Facultative anaerobes
    Enterococcus faecium and              0.87           0.06-2
      Enterococcus faecalis.
    Coliforms: Escherichia coli,          0.05           0.03-0.13
      Citrobacter freundii, 
      Klebsiella pneumoniae and 
      Klebsiella oxytoca  
    Proteus mirabilis and                 0.04           0.03-0.13
      Proteus vulgaris 
    Anaerobesa
    Bacteroides spp.                      > 128          > 128
    Lactobacillus spp.                    29.86          16-64
    Bifidobacterium spp.                  19.70          4-126
    Prevotella spp.                       > 128          > 128
    Eubacterium spp.                      6.06           2-32
    Clostridium spp.                      111.4          64-> 128
    Fusobacterium spp.                    78.79          4-> 128
    Anaerobic gram-positive cocci         27.86          8-126
                                                                       
    From Lohuis & Aerts (1996)
    a Species not specified


    Table 2. Antimicrobial activity of gentamicin in vitro on some 
    strains used for quality control 

                                                                          
    Species                                  MIC (µg/ml)
                                                                          
                              Aerobic incubation      Anaerobic incubation
                                                                          
    Enterococcus faecalis     4                       -
      (ATCC 29212)
    Escherichia coli          0.125                   -
      (ATCC 25922)
    Proteus mirabilis         < 0.0625                -
      (ATCC 14273)
    Bacteroides fragilis      -                       > 128
      (ATCC 10584)
    Peptostreptococcus 
      anaerobius              -                       -
      (ATCC 27337)
                                                                          
    From Lohuis & Aerts (1996)



        Table 3. Genotoxity of gentamicin sulfate

                                                                                                        

    Test system       Test object                  Concentration          Result          Reference
                                                                                                        

    In vitro
    Forward           Chinese hamster              128, 320, 800,         Negativea,b     Poul (1997a)
     mutation         ovary cells (hprt locus)     2000, 5000 µg/ml

    Chromosomal       Chinese hamster              800, 2000, 5000        negativea,c     Poul (1997b)
     aberration       ovary cells (CHO-K1)         µg/ml

    In vivo
    Micronucleus      Bone marrow of               20, 40, 80 mg/,        Negatived       Holmstrom &
     formation        CD-1 mice                    kg bw intravenously                    Innes (1997)
                                                                                                        

    a   With and without metabolic activation (S)
    b   Benzo[a]pyrene (+S9 mix) and ethyl methanesulfonate (-S9 mix) used as positive controls; in the 
        absence of metabolic activation the mutation frequency was twice the control value (unusually low
        mutation frequency) at concentrations of 5000, 2000, and 125 µg/ml in one assay but not in the 
        second assay.
    c   Methyl methanesulfonate (+S9 mix) and cyclophosphamide (-S9 mix) used as positive controls
    d   Cyclophosphamide used as positive control
    


    FIGURE 1

         It was noted that existing long-term studies on the
    aminoglucosides neomycin, dihydrostreptomycin, and aminosidine in
    experimental animals were not included in the databases on known
    carcinogenes, which formed the basis for tabulating the structural
    alerts. Consequently, the lists may have limited value for predicting
    the possible carcinogenic potential of the gentamicin structure. Since
    no carcinogenic effect has been observed with the aminoglycosides that
    have been tested in long-term studies (Annex 1, reference 111;
    Committee for Veterinary Medicinal Products, 1996), however, it is
    reasonable to assume that inclusion of these studies in the databases
    would not have altered the conclusion about structureśactivity
    relationships.

    3.  COMMENTS

         The Committee considered the results of studies of mutagenicity
     in vitro and  in vivo, analyses of the structural similarities of
    gentamicin to known carcinogens, and the effect of gentamicin on
    specific bacterial species obtained from the human gut. The studies
    were carried out according to appropriate standards for study protocol
    and conduct.

         Gentamicin was tested  in vitro for its ability to cause gene
    mutations and chromosomal aberrations in Chinese hamster ovary cells
    and  in vivo for its ability to induce nuclear anomalies in mouse
    bone-marrow cells at doses of 20ś80 mg/kg bw by intravenous
    administration. The results of these tests were negative, and the
    Committee concluded that gentamicin is unlikely to be genotoxic.

         Possible structural similarities between gentamicin and known
    carcinogens were analysed. None of the structural features listed in
    the available databases for carcinogenicity were found within the
    chemical structure of gentamicin. In view of this finding and since

    the aminoglycosides that have been tested (neomycin,
    dihydrostreptomycin, and aminosidine) do not elicit a tumorigenic
    effect in rats, the Committee considered that gentamicin is unlikely
    to have carcinogenic activity. This conclusion is supported by the
    results of the tests for genotoxicity in mammalian cells  in vitro 
    and  in vivo.

         The effect of gentamicin on the growth of 110 bacterial strains
    obtained from the human gastrointestinal tract was evaluated after
    incubation  in vitro. MIC values were determined for 80 isolates of
    the eight predominant groups of human intestinal anaerobic microflora,
     Bacteroides spp.,  Lactobacillus spp.,  Bifidobacterium spp.,
     Prevotella spp.,  Eubacterium spp.,  Clostridium spp.,
     Fusobacterium spp., and anaerobic gram-positive cocci. In addition,
    data were provided for 30 isolates of the facultative anaerobes
     Enterococci spp.,  Proteus spp., and coliforms. The MIC values for
    these isolates ranged from 0.06 to > 128 µg/ml. Although the
    facultative anaerobic bacteria were the most sensitive organisms, the
    Committee agreed that they should not be used in the calculation of
    the ADI because they are not predominant species in the human
    intestine. Instead, the Committee used the MIC for the most sensitive
    relevant genera isolated from the human gastrointestinal tract, in
    this case  Eubacterium spp. The geometric mean for this strain was
    6 µg/ml at an inoculum density of 106 colony-forming units.
     Eubacterium spp. were also used in the evaluation at the forty-third
    meeting, when an MIC value of 0.8 µg/ml was used for establishing the
    temporary ADI. Although the MIC values identified at the forty-third
    and the present meeting were different, the Committee concluded that
    the value obtained using isolates from the human gut flora was more
    relevant for establishing an ADI.

         In calculating an ADI based on the antimicrobial activity of
    gentamicin, the Committee used the formula described on p. 28, as
    follows:

                   Upper limit     6 µg/ga × 220 g
                                =
                      of ADI        1b × 1c × 60 kg

                                =  22 µg/kg bw

    a  For the purpose of this evaluation, the MIC50 value is the
         geometric mean MIC for gentamicin against the 10 strains of the
         most sensitive relevant genus isolated from the human intestinal
         tract, in this case  Eubacterium spp.

    b  Absorption of gentamicin after oral administration is poor;
         therefore, a factor of 1 was used to represent 100% availability
         in the gastrointestinal tract.

    c  A safety factor of 1 was used because sufficient, relevant
         microbiological data were available.

    4.  EVALUATION

         The Committee established an ADI of 0-20 µg/kg bw on the basis of
    a microbiological end-point. The Committee noted that the lowest NOEL
    identified at the forty-third meeting in toxicological studies was 10
    mg per kg bw, which is 500 times the microbiological ADI.

    5.  REFERENCES

    Ashby, J. & Paton, D. (1993) The influence of chemical structure on
    the extent and sites of carcinogenesis for 522 rodent carcinogens and
    55 different human carcinogen exposures.  Mutat. Res., 286, 3-74.

    Committee for Veterinary Medicinal Products (1996) Aminosidine. Status
    report (EMEA/MRL/050/95-Final), http://www.eudora.org/emea.html. The
    European Agency for the Evaluation of Medicinal Products, London,
    United Kingdom.

    Holmstrom, L.M. & Innes, D.C. (1997) Gentamicin, micronucleus test in
    bone marrow of CD-1 mice. Unpublished report No. 14888 from Inveresk
    Research, Tranent, Scotland, United Kingdom. Submitted to WHO by
    Inveresk Research, Tranent, Scotland, United Kingdom.

    Inveresk Research (1997) Gentamicin: Structureśactivity relationships.
    Unpublished report submitted to WHO by Inveresk Research, Tranent,
    Scotland, United Kingdom.

    Lohuis, J. & Aerts, R. (1996) Susceptibility of anaerobic and aerobic
    isolates from human faeces towards gentamicin. Unpublished report No.
    96R/0478 from Intervet, Boxmeer R & D Laboratories. Submitted to WHO
    by Inveresk Research, Tranent, Scotland, United Kingdom.

    Poul, J.M. (1997a) Evaluation of gentamicin sulfate in the Chinese
    hamster ovary cell/hypoxanthine-guanine-phosphoribosyl-transferase
    (CHO/HGPRT) forward mutation assay. Unpublished report No. RTOX9703
    from Centre National d'Etudes Vétérinaires et Alimentaires,
    Laboratoire des médicaments vétérinaires, Unité de toxicologie,
    F-35133 Fougčres, France. Submitted to WHO by Inveresk Research,
    Tranent, Scotland, United Kingdom.

    Poul, J.M. (1997b) Evaluation of gentamicin sulfate in the in vitro
    chromosome aberration assay using CHO-K1 cells. Unpublished report No.
    RTOX9702 from Centre National d'Etudes Vétérinaires et Alimentaires,
    Laboratoire des médicaments vétérinaires, Unité de toxicologie,
    F-35133 Fougčres, France. Submitted to WHO by Inveresk Research,
    Tranent, Scotland, United Kingdom.

    Tennant, R.W. & Ashby, J. (1991) Classification according to chemical
    structure, mutagenicity to Salmonella and level of carcinogenicity of
    a further 39 chemicals tested for carcinogenicity by the US National
    Toxicology Program.  Mutat. Res., 257, 209-227.

    US Food and Drug Administration (1994)  General Principles for 
     Evaluating the Safety of Compounds Used in Food-producing Animals, 
    Rockville, Maryland, USA, Center for Veterinary Medicine.

    Won, H. (1996a) Evaluation of gentamicin for the induction of
    chromosomal aberrations using Chinese hamster lung cells (CHL)
     in vitro. Unpublished report from Department of Genetic Toxicology,
    Toxicology Research Institute, Korea Food and Drug Administration,
    Seoul, 122-020, Republic of Korea. Submitted to WHO by Dr G. Roberts,
    Chemical Product Assessment Section, Commonwealth Department of Health
    & Family Services, Woden, ACT, Australia.

    Won, H. (1996b) Evaluation of gentamicin for the induction of
    chromosomal aberrations using ddY male mouse  in vivo. Unpublished
    report from Department of Genetic Toxicology, Toxicology Research
    Institute, Korea Food and Drug Administration, Seoul, 122-020,
    Republic of Korea. Submitted to WHO by Dr G. Roberts, Chemical Product
    Assessment Section, Commonwealth Department of Health & Family
    Services, Woden, ACT, Australia.
    


    See Also:
       Toxicological Abbreviations
       Gentamicin (WHO Food Additives Series 34)
       GENTAMICIN (JECFA Evaluation)