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Background to COM review 1. Flunixin in the form of the meglumine salt is a non-steroidal anti-inflammatory drug (NSAID) and a non-narcotic analgesic drug with antipyretic activities. It is used in veterinary medicine (including food-producing animals) but is not used in human medicine. Flunixin meglumine dissociates in vivo to Flunixin and meglumine. In 1997, the European Medicine Evaluation Agency's (EMEA) Committee on Veterinary Medicinal Products (CVMP) considered the safety of Flunixin meglumine as part of its review of old veterinary medicinal products, operating under Council Regulation 2377/90, which covers the marketing authorisation of pharmacologically active substances used in veterinary medicines. 2. With regard to meglumine, the CVMP considered that it could be regarded as an excipient that had the purpose of increasing the solubility of Flunixin. The CVMP concluded that "meglumine used as an excipient at up to 1.5 mg/kg bw does not fall within the scope of Council Regulation (EEC) number 2377/90". 3. The Food Standards Agency is concerned about the possible mutagenicity of Flunixin meglumine, meglumine and Flunixin. The Food Standards Agency considered that it would be useful to seek the opinion of the COM. 4. The COM undertook an initial review of the mutagenicity data on Flunixin meglumine, Flunixin and meglumine during 2002/2003. A statement was published in March 2003 (www.advisorybodies.doh.gov.uk/com/flunmeg). The COM requested additional in-vitro mutagenicity data on meglumine, which is considered in this statement. Assessment of Mutagenicity data
5. In the statement COM/03/S2, the COM agreed that there was limited evidence that Flunixin was mutaganic in-vitro, as data were inadequate, but there was no evidence to suggest that Flunixin had mutagenic potential in vivo, as a bone-marrow assay was carried out in mice that was considered adequate by the COM. Members, however, considered that the issue of the inadequate in-vitro data should be raised with the CVMP. Meglumine 6. In the statement COM/03/S2, negative results were reported in an old plate incorporation assay in a limited number of Salmonella typhimurium strains. A positive result had been reported in a bone-marrow micronucleus assay in BSI mice using intraperitoneal administration of 500 or 1000 mg/kg bw suspended in 0.25% methylcellulose (two doses given 24 hours apart and harvest 6 hours after last dose). A repeat test using 1000 mg/kg bw was also positive. Members queried how the repeat test could have been undertaken on the same day as the initial test. It was noted that negative results were obtained in a separate in-vivo micronucleus assay using intraperitoneal administration of two doses given 24 hours apart at up to 600 mg/kg bw of meglumine to CD1 mice. Members noted that a clear positive control had only been seen in males and not females in this latter assay and that the sampling regimen (24 and 48 hours after last dose) differed from the study in BS1 mice. A number of in-vivo bone marrow micronucleus assays were undertaken for the Committee using a variety of single/double intraperitoneal dosing regimens at dose levels of 500 mg/kg bw and 1000 mg/kg bw and sampling for micronuclei at 6 or 24 h after the last dose. There was some evidence for an effect 6 h after the second dose in one study using Alpk:ApfCD-1 mice, but no definite conclusions could be reached as the result was not repeatable, there was considerable individual animal variation and the observed effect could have been complicated by toxicity. Flunixin meglumine 7. In the statement COM/03/2 the Committee agreed that there was limited evidence that Flunixin meglumine was an in-vitro mutagen. However, although negative results had been documented in an in-vivo micronucleus assay in BS1 mice, members were concerned about adequacy of the selection of dose levels. Therefore the inconclusive in-vivo data on meglumine suggested that a definite conclusion regarding Flunixin meglumine could not be reached. COM Evaluation 8. The Committee noted the poor quality of mutagenicity data on flunixin,
meglumine and Flunixin meglumine. Members noted that while negative results
obtained in carcinogenicity bioassays with Flunixin meglumine in the rat
and the mouse provided some reassurance with regard to flunixin, they
were not informative with regard to meglumine. 9. The Committee was aware of several in-vivo bone-marrow micronucleus
assays using meglumine but noted that the results were inconsistent. Two
studies using BS1 and Alpk:ApfCD-1 mice reported a mutagenic effect following
a treatment regime of two doses of meglumine (24 hour apart) with a sampling
time of 6 hours after the second dose (ie 30 hours after the first dose).
However this positive result was not repeated in two bone marrow micronucleus
assays in mice using an equivalent treatment regime. 10. The Committee requested additional in-vitro assays with meglumine
to properly assess whether meglumine had mutagenic potential in vitro
before considering the need for any further in-vivo mutagenicity
studies. Thus data were required from an in-vitro chromosomal aberration
assay and a mouse lymphoma assay, which together with the data currently
available for a test in bacteria using Salmonella, would complete the
in-vitro package to modern standards given in the COM guidance. If these
were negative, there would be no concerns regarding meglumine. Data submitted in February 2005 Mouse Lymphoma Assay 11. A Mouse Lymphoma Assay was undertaken that conformed to the OECD Guideline 4761 and ICH S2B Guideline.2,3 12. Meglumine was not mutagenic, at the concentrations tested in L5178Y
mouse lymphoma cells, either with or without exogenous metabolic activation. Chromosome Aberration Assay 13. A cytogenetics test was undertaken which conformed to OECD Guideline
473.4,5 14. Meglumine, at the concentrations tested, did not significantly increase chromosome aberrations or decrease the mitotic index in human blood lymphocytes in vitro, either with or without exogenous metabolic activation. COM conclusion and recommendations 15. The COM confirmed the following general conclusions on the mutagenicity
of flunixin, Flunixin meglumine and meglumine
16. The COM reaffirmed that it is difficult to draw any definite conclusions
on the mutagenicity of these chemicals. The prudent conclusions reached
for flunixin and Flunixin meglumine were reaffirmed.
17. The COM agreed a revised conclusion for meglumine using the new information
from the in-vitro mutagenicity studies considered in this statement.
March 2005 References 2. ICH Harmonised Tripartite Guideline (S2B): Genotoxicity: A Standard
Battery for Genotoxicity Testing of Pharmaceutics. Federal Register 1997:
62 FR. 624721. 3. Cifone MA. Mouse lymphoma cell (L5178Y TK+/-) forward mutation assay
of SCH 3537.Covance Laboratories Inc Virginia, USA. Schering-Plough study
Number 03402. 28 September 2004. 4. OECD. 1997. Test guideline 473. In Vitro Mammalian Chromosome
Aberration Test. In: OECD Guideline for Testing of Chemicals. Paris, Organisation
for Economic Cooperation and Development. 5. Murli H. Chromosome aberration study of SCH 3537 in human peripheral
blood lymphocytes. Covance Laboratories Inc Virginia, USA. Schering-Plough
study number 03403, 16 September 2004.
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