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WHO FOOD ADDITIVES SERIES: 53

FLUMEQUINE (addendum)

First draft prepared by

Mrs M.E.J. Pronk
Centre for Substances and Integrated Risk Assessment
National Institute for Public Health and the Environment Bilthoven, The Netherlands

Explanation

Biological data

Hepatotoxicity and carcinogenicity

Genotoxicity

Comments

Evaluation

References

1. EXPLANATION

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 for the treatment of urinary tract infections in humans. Flumequine was evaluated by the Committee at its forty-second, forty-eighth, fifty-fourth and sixtieth meetings (Annex 1, references 110, 128, 146 and 162). At its forty-eighth meeting, the Committee established an acceptable daily intake (ADI) of 0-30 g/kg bw on the basis of hepatotoxicity in male CD-1 mice in a 13-week study. The Committee at that meeting concluded that flumequine was a non-genotoxic hepatocarcinogen, and that the induction of hepatocellular necrosis-regeneration cycles by hepatotoxicity was considered to be the relevant mechanism for induction of liver tumours in mice.

At its sixtieth meeting, the Committee evaluated new studies that had been carried out to further elucidate the mechanism of flumequine-induced hepatocarcinogenicity in mice. On the basis of these new studies, the Committee could not dismiss the possibility that flumequine induces tumours in the mouse liver by a mechanism that includes genotoxic effects. The Committee therefore concluded that it could not support an ADI and withdrew the ADI that had been established at its forty-eighth meeting. The Committee expressed the wish to receive additional data on the mechanism involved in tumour formation before re-establishment of an ADI could be considered. The present Committee evaluated a new study investigating the genotoxic potential of flumequine in a test for unscheduled DNA synthesis in the liver in vivo.

2. BIOLOGICAL DATA

2.1 Hepatotoxicity and carcinogenicity

In short-term and long-term studies of toxicity that were evaluated by the Committee at its forty-second and forty-eighth meetings, oral administration of flumequine caused dose-related hepatotoxic effects in rats and CD-1 mice. The liver damage was most pronounced in male mice, and included degenerative changes with hypertrophy, fatty vacuolation, focal necrosis and increased mitotic activity. After cessation of treatment with flumequine, the liver damage was reversed. Treatment with flumequine had little or no effect on P450-dependent hepatic drug-metabolizing enzymes or on glucuronyl transferase. Flumequine increased the plasma activities of alanine and aspartate aminotransferases, alkaline phosphatase and lactate dehydrogenase. The overall no-observed-effect level (NOEL) for hepatotoxic effects in mice was 25 mg/kg bw per day.

The results of long-term studies of toxicity that were evaluated by the Committee at its forty-second meeting showed that flumequine had no carcinogenic effects in rats, whereas in CD-1 mice an increase in the incidence of liver tumours was observed at oral doses of flumequine of >400 mg/kg bw per day (the lowest dose tested) in an 18-month study. The incidence of tumours in male mice was significantly higher than that in female mice. In male mice, the incidence of liver tumours increased in a dose-related and time-dependent manner, and was paralleled by an increase in the incidence of hepatotoxic changes.

The present Committee re-evaluated the three short-term studies in mice, which used a two-stage hepatocarcinogenesis protocol, that were presented to the Committee at its sixtieth meeting. In these studies, treatment with flumequine caused the development of basophilic liver foci, which could suggest that flumequine has tumour initiating potential. However, the Committee also noted that concurrent hepatotoxicity (evidenced by pale, vacuolated hepatocytes with fatty droplets, inflammatory cell infiltration, increased mitotic figures and/or necrosis) was observed, as well as a regenerative response to these toxic changes and indications of oxidative stress.

2.2 Genotoxicity

Flumequine gave negative results in various assays for genotoxicity that were evaluated by the Committee at its forty-second meeting. These assays included assays in bacteria in vitro (reverse mutation in Salmonella typhimurium) and mammalian cells (gene mutation at the Hprt locus in mouse lymphoma cells and gene mutation in Chinese hamster ovary cells), and an assay for chromosome aberration in bone-marrow cells in rats in vivo.

At its sixtieth meeting, the Committee evaluated a "comet" assay in which flumequine sporadically caused DNA strand breaks in the liver in vivo. Although this could indicate that flumequine has genotoxic activity, the Committee also noted the limitations of this assay and that the effect in the liver was only marginal.

The Committee at its present meeting evaluated new information on the genotoxic potential of flumequine. Flumequine was investigated in a test for unscheduled DNA synthesis in liver cells in rats in vivo. Groups of three young adult male Fischer rats were given flumequine by gavage in a single dose of 156.25, 312.5 or 625 mg/kg bw. The doses given were based on the results of a preliminary test of toxicity, and the vehicle used was 0.5% (w/v) carboxymethyl-cellulose. Liver cells were prepared 2-4 h or 12-16 h after treatment of the animals, and were subsequently incubated with tritium-labelled thymidine (3H-TdR) for 3-8h. Using autoradiography, DNA damage and subsequent repair were determined by measuring the incorporation of 3H-TdR into the DNA of the liver cells. Three control groups of three male rats received either vehicle only (negative control group), 2-acetamidofluorene (positive control group for the 12-16 h sampling time) or dimethylhydrazine (positive control group for the 2-4 h sampling time). Additional groups of three male rats were treated with flumequine in the same doses. Plasma samples were taken from these animals at 2, 4 and 12 h after treatment, and the samples were analysed for flumequine and its metabolites. The study complied with OECD test guideline 486, and was certified for good laboratory practice and quality assurance.

Under these test conditions, in the presence of high concentrations of flumequine in plasma, flumequine did not induce unscheduled DNA synthesis, whereas the positive controls clearly did (Nesslany, 2003). This indicates that flumequine does not interact directly with liver DNA.

3. COMMENTS

In short-term and long-term studies of toxicity that were evaluated by the Committee at its forty-second and forty-eighth meetings, oral administration of flumequine caused dose-related hepatotoxic effects in rats and CD-1 mice. The liver damage was most pronounced in male mice, and included degenerative changes with hypertrophy, fatty vacuolation, focal necrosis and increased mitotic activity. After cessation of treatment with flumequine, the liver damage was reversed. Treatment with flumequine had little or no effect on P450-dependent hepatic drug-metabolizing enzymes or on glucuronyl transferase. Flumequine increased the plasma activities of alanine and aspartate aminotransferases, alkaline phosphatase and lactate dehydrogenase. The overall NOEL for hepatotoxic effects in mice was 25 mg/kg bw per day.

The results of long-term studies of toxicity that were evaluated by the Committee at its forty-second meeting showed that flumequine had no carcinogenic effects in rats, whereas in CD-1 mice an increase in the incidence of liver tumours was observed at oral doses of flumequine of > 400 mg/kg bw per day (the lowest dose tested) in an 18-month study. The incidence of tumours in male mice was significantly higher than that in female mice. In male mice, the incidence of liver tumours increased in a dose-related and time-dependent manner, and was paralleled by an increase in the incidence of hepatotoxic changes.

The present Committee re-evaluated the three short-term studies that used a two-stage hepatocarcinogenesis protocol in mice. In these studies, which were presented to the Committee at its sixtieth meeting, treatment with flumequine caused the development of basophilic liver foci, which could suggest that flumequine has tumour-initiating potential. The Committee also noted, however, that concurrent hepatotoxicity (evidenced by pale, vacuolated hepatocytes with fatty droplets, inflammatory cell infiltration, increased mitotic figures and/or necrosis) was observed, and a regenerative response to these toxic changes and indications of oxidative stress.

Flumequine gave negative results in various assays for genotoxicity that were evaluated by the Committee at its forty-second meeting. These included assays in vitro in bacteria (reverse mutation in Salmonella typhimurium) and mammalian cells (gene mutation at the Hprt locus in lymphoma cells in mice and gene mutation in Chinese hamster ovary cells), and an assay for chromosome aberration in rat bone marrow in vivo. At its sixtieth meeting, the Committee evaluated a comet assay in which flumequine sporadically caused DNA strand breaks in the liver in vivo. Although this could indicate that flumequine has genotoxic activity, the Committee also noted the limitations of this assay and that the effect in the liver was only marginal.

The Committee at its present meeting also evaluated a new, adequately conducted test for unscheduled DNA synthesis with flumequine in rat liver cells in vivo. The result of this test was negative, indicating that flumequine does not interact directly with liver DNA.

4. EVALUATION

The Committee concluded that the available data support a non-genotoxic, threshold-based mechanism for tumour formation by flumequine in the mouse liver. The Committee therefore re-established the ADI of 0-30 g/kg bw that it had originally established for flumequine at its forty-eighth meeting. This ADI was based on the overall NOEL for hepatotoxicity of 25 mg/kg bw per day, observed in a 13-week study in mice, and a safety factor of 1000. A safety factor of 1000 was chosen to reflect the short duration of the study and the lack of histochemical characterization of the foci of altered hepatocytes.

5. REFERENCE

Nesslany, F. (2003) Measurement of unscheduled DNA synthesis (UDS) in rat hepatocytes using an in vivo procedure with flumequine. Unpublished draft report No. IPL-R 031017 from Institut Pasteur de Lille, Lille, France. Submitted to WHO by CEVA Santé Animale, Libourne, France.



    See Also:
       Toxicological Abbreviations
       Flumequine (JECFA Food Additives Series 51)
       Flumequine (WHO Food Additives Series 33)
       Flumequine (WHO Food Additives Series 39)
       FLUMEQUINE (JECFA Evaluation)