WHO FOOD ADDITIVES SERIES: 51
First draft prepared by
Dr L. Ritter
Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada
Professor A.R. Boobis
Section on Clinical Pharmacology, Faculty of Medicine, Imperial College, London, England
Dr K. Greenlees
Office of New Animal Drug Evaluation, Center for Veterinary Medicine, Food and Drug Administration, Rockville, Maryland, USA
Dr K. Mitsumori
Laboratory of Veterinary Pathology, School of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan
Flumequine is a fluoroquinolone compound with antimicrobial activity against gram-negative organisms and is used in the treatment of enteric infections in food animals. It also has limited use in humans for the treatment of urinary-tract infections. Flumequine was evaluated by the Committee at its forty-second, forty-eighth and fifty-fourth meetings (Annex 1, references 110, 128 and 146). At its forty-eighth meeting, the Committee established an ADI of 0–30 µg/kg bw based on a toxicological end-point (hepatotoxiciy in male CD-1 mice in a 13-week study).
At its forty-eighth meeting, the Committee evaluated information related inter alia to a NOEL for hepatotoxicity and the mechanism of tumour induction. The present Committee, at the request of the Codex Committee on Residues of Veterinary Drugs in Foods at its Thirteenth Session (Codex Alimentarius Commission, 2001), evaluated new studies that had been carried out to elucidate further the mechanism of flumequine-induced hepatocarcinogenicity in mice.
In short-term and long-term studies of toxicity that had been evaluated by the Committee at its forty-second meeting, oral administration of flumequine caused dose-related hepatotoxic effects in rats and mice. Hypertrophy, degenerative changes and focal necrosis of hepatocytes were observed in rats at 400 and 800 mg/kg bw per day in a 2-year study, and in CD-1/ICR mice at 400 and 800 mg/kg bw per day in an 18-month study. The prevalence of hepatotoxic lesions increased with duration of treatment. At its forty-eighth meeting, the Committee noted that male mice were the most sensitive to flumequine-induced liver damage.
The results of long-term studies that had been evaluated previously by the Committee showed no carcinogenic effects in rats, but a dose-related increase in the incidence of liver tumours was observed in CD-1 mice at doses > 100 mg/kg bw per day. The tumour incidence paralleled hepatotoxic changes and was significantly higher in male than in female mice. As flumequine was inactive in a range of tests for genotoxicity, including assays for gene mutation in bacteria and mammalian cells in vitro and for chromosomal aberrations in vivo and in vitro, the mechanism of tumorigenicity was unclear. At its forty-eighth meeting, the Committee reviewed the available toxicological database in order to determine if the hepatocarcinogenicity of flumequine resulted from a genotoxic or a non-genotoxic mechanism. Genotoxic carcinogens act directly on DNA in the target tissue, inducing DNA or chromosomal damage such as strand breaks or mutations, which can typically be assessed in assays for genotoxicity and short-term assays in rodents. Non-genotoxic carcinogens do not have this activity, and non-genotoxic carcinogenicity can result from induction of cytotoxicity and cell proliferation, which probably cause tumorigenicity in target organs by sustained mitogenic stimulation. Non-genotoxic tumorigenesis in rodent liver can arise through several mechanisms, including hepatotoxicity. Flumequine produced consistently negative results when evaluated in various assays for genotoxicity in vitro and in vivo.
Flumequine was hepatotoxic, causing hepatocellular degeneration and focal necrosis in male and female mice. The dose-related severity of the lesions paralleled the incidence of liver tumours. The occurrence of foci of altered hepatocytes is an important intermediary step in hepatotoxicity-induced liver tumorigenicity.
The Committee at its forty-eighth meeting concluded that the liver tumorigenicity observed in mice exposed to flumequine was the result of a non-genotoxic mechanism, secondary to hepatotoxicity-induced necrosis–regeneration cycles. The Committee noted that, as the tumorigenicity was secondary to hepatotoxicity, the NOEL for both hepatotoxicity and carcinogenicity was 25 mg/kg bw per day. The Committee also noted that the NOEL for hepatotoxicity was determined from a 13-week study and extrapolated to the dose required for tumour formation observed at the end of the 18-month study in mice.
The Committee at its present meeting evaluated new information on the mechanism of action of flumequine-induced mouse liver tumorigenicity. Administration of flumequine in the diet of CD-1 mice at a concentration of 4000 ppm for 30 weeks (equivalent to the lowest dose in the 18-month study of carcinogenicity in mice) or after a single intraperitoneal injection of N-nitrosodiethylamine induced basophilic liver foci in males. Flumequine also increased the number of 8-hydroxydeoxy-guanosine adducts in liver. These responses are consistent with oxidative DNA damage and can be associated with carcinogenicity (Yoshida et al., 1999).
In another study, heterozygous p53-deficient mice (which have increased sensitivity to genotoxic carcinogens) that received a diet containing 4000 ppm of flumequine for 26 weeks developed basophilic liver foci at a time when there was no evidence of necrosis. The absence of cell death at the dose tested (which did not cause necrosis) showed that this would not confound interpretation of the significance of the altered liver foci (Takizawa et al., 2001).
In a 13-week study of two-stage hepatocarcinogenicity in mice, administration of a diet containing flumequine at a concentration of 4000 ppm induced altered liver foci in mice subsequently exposed to a mixed promoting regimen of D-galactosamine and phenobarbital, indicating that flumequine acted as a short-term initiator. A lower dose of flumequine (500 mg/kg bw) caused DNA strand breaks in a ‘comet’ assay at a time when liver damage was not evident. The absence of liver damage at the dose tested showed that this would not confound interpretation of the comet assay. The liver was more sensitive if it was undergoing cell proliferation due to regeneration or juvenile growth. Similarly, other tissues such as stomach, colon and urinary bladder showed more DNA breaks in response to various doses of flumequine, with dose-dependent DNA damage in these organs in adult mice 3 h, but not 24 h, after treatment. The results of these studies suggest that flumequine has initiating potential and that the hepatocarcinogenicity in mice might involve DNA strand breakage (Kashida et al., 2002).
Quinolones like flumequine exert their antibacterial activity by inhibiting bacterial topoisomerase II (DNA gyrase). Although there are some structural similarities between bacterial and mammalian topoisomerases, they differ substantially in overall structure. Fluoroquinolones in general have a much lower affinity for mammalian topoisomerases than for bacterial enzymes. Information on inhibition of mammalian topoisomerases by flumequine was not available to the Committee at its forty-eighth meeting.
Flumequine at doses that inhibit mammalian topoisomerase II might induce DNA damage, and the damage might be involved in mutagenic or other genotoxic steps in carcinogenicity. The Committee noted that there was inadequate evidence to confirm that the observed carcinogenicity of flumequine was secondary to inhibition of topoisomerase II or that this hypothesized mechanism is the sole basis for the tumour induction observed in the lifetime study of carcinogenicity in mice.
The Committee at its forty-second and forty-eighth meetings evaluated the tumorigenicity of flumequine. At its forty-second meeting, the Committee noted that there was evidence of compound-related hepatotumorigenic effects in male mice. As flumequine was inactive in a range of tests for genotoxicity, the mechanism of tumorigenesis was unclear. The Committee at its forty-eighth meeting considered that hepatocellular necrosis and regeneration subsequent to hepatotoxicity was the relevant mechanism for the induction of liver tumours by flumequine.
Flumequine has generally been considered to be a non-genotoxic carcinogen with only promoting activity. The Committee at its present meeting reviewed new studies on flumequine-induced tumorigenicity that were not available at the forty-eighth meeting, in which the mechanism of action in male mice was further investigated. Although the results of ‘comet’ assays indicated that flumequine caused double-strand DNA breaks, the Committee noted the limitations of this assay and considered that those results alone could not fully substantiate a genotoxic mechanism for the observed hepatocarcinogenicity of flumequine.
The Committee concluded that the new data raised further questions about the mechanism by which flumequine increases the incidence of liver tumours in male mice. The Committee evaluated evidence that supported the involvement of both genotoxic and non-genotoxic mechanisms. It noted that flumequine was not genotoxic in a comprehensive battery of assays in vitro and in vivo; however, in the absence of necrosis, it induced basophilic foci and DNA strand breaks in a ‘comet’ assay. The Committee therefore could not dismiss the possibility that flumequine induces tumours in mouse liver by a mechanism that includes genotoxic effects. It was, however, unable to identify the genotoxic effects involved in liver tumour formation or a threshold for those effects.
The Committee concluded that it could not support an ADI and withdrew the ADI that it had established at its forty-eighth meeting. Before establishment of an ADI can be considered, the Committee would wish to receive additional data on the genotoxic effects involved in tumour formation.
Codex Alimentarius Commission (2001) Report of the Thirteenth Session of the Codex Committee on Residues of Veterinary Drugs in Foods, Charleston, SC, USA, 4–7 December 200, Rome: Food and Agriculture Organization of the United Nations (unpublished document ALINORM 03/31).
Kashida, Y., Sasaki, Y.F., Ohsawa, K., Yokohama, N., Takahashi, A., Watanabe, T. & Mitsumori, K. (2002) Mechanistic study on flumequine hepatocarcinogenicity focusing on DNA damage in mice. Toxicol. Sci., 69, 317–321.
Takizawa, T., Mitsumori, K., Takagi, H., Onodera, H., Yasuhara, K., Tamura, T. & Hirose, M. (2001) Modifying effects of flumequine on dimethynotrosamine-induced hepatocarcinogenesis in heterozygous p53 deficient CBA mice. J. Toxicol. Pathol., 14, 135–143.
Yoshida, M., Miyajima, K., Shiraki, K., Ando, J., Kudoh, K., Nakae, D., Takahashi, M. & Maekawa, A. (1999) Hepatotoxicity and consequently increased cell proliferation are associated with flumequine hepatocarcinogenesis in mice. Cancer Lett., 141, 99–107.
See Also: Toxicological Abbreviations Flumequine (WHO Food Additives Series 53) Flumequine (WHO Food Additives Series 33) Flumequine (WHO Food Additives Series 39) FLUMEQUINE (JECFA Evaluation)