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 128œ5000 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 20œ80 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 controlIt 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 structureœactivity 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 20œ80 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: Structureœactivity 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 Fougères, 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 Fougères, 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)