title: Mammalian genotoxicity assessment of methylene blue in plasma: implications for virus inactivation.
abstract: BACKGROUND
  The risk of adverse consequences of virus-inactivation procedures for plasma and cellular blood components must be less than the risk of transfusion-associated viral disease. Previous studies demonstrated that methylene blue, which is currently used in Europe for virus inactivation in fresh-frozen plasma, can elicit mutations in bacterial test systems. This study investigates the potential for methylene blue genotoxicity in two mammalian test systems.
  
  
  METHODS
  Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control consisting of a known genotoxic agent.
  
  
  RESULTS
  Intravenous administration to mice of 62 mg per kg of methylene blue did not increase the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methylene blue concentrations of 10 micrograms per mL (with S9 activation) and 30 micrograms per mL (without S9 activation) significantly increased the thymidine kinase mutation frequency of mouse lymphoma cells to approximately 110 x 10(-6), from a spontaneous frequency of 28 x 10(-6).
  
  
  CONCLUSIONS
  Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. Considerably more investigation will be required to assess the genotoxic potential of intravenously administered methylene blue used in virus-inactivation procedures, because of the likelihood of the formation of methylene blue photoproducts or the impact of metabolic conversion of methylene blue to leukomethylene blue in vivo.
authors.affiliation: Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Wagner
authors.forename: M A
authors.initials: MA
authors.lastname: Cifone
authors.forename: H
authors.initials: H
authors.lastname: Murli
authors.forename: R Y
authors.initials: RY
authors.lastname: Dodd
authors.forename: B
authors.initials: B
authors.lastname: Myhr
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects; D008223:Lymphoma / Q000201:enzymology / Q000473:pathology; D008751:Methylene Blue / Q000494:pharmacology*; D051379:Mice; D009153:Mutagens; D009154:Mutation; D013937:Thymidine Kinase / Q000235:genetics*
chemical_list: D009153:Mutagens; D013937:Thymidine Kinase; D008751:Methylene Blue