ASTM_F_895_-_11.pdf

Designation:F89511Standard Test Method forAgar Diffusion Cell Culture Screening for Cytotoxicity1This standard is issued under the fixed designation F895;the number immediately following the designation indicates the year of originaladoption or,in the case of revision,the year of last revision.Anumber in parentheses indicates the year of last reapproval.Asuperscriptepsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This test method is appropriate for materials in a varietyof shapes and for materials that are not necessarily sterile.Thistest method would be appropriate in situations in which theamount of material is limited.For example,small devices orpowders could be placed on the agar and the presence of a zoneof inhibition of cell growth could be examined.1.1.1 This test method is not appropriate for leachables thatdo not diffuse through agar or agarose.1.1.2 While the agar layer can act as a cushion to protect thecells from the specimen,there may be materials that aresufficiently heavy to compress the agar and prevent diffusion orto cause mechanical damage to the cells.This test methodwould not be appropriate for these materials.1.2 The L-929 cell line was chosen because it has asignificant history of use in assays of this type.This is notintended to imply that its use is preferred,only that the L-929is an established cell line,well characterized and readilyavailable,that has demonstrated reproducible results in severallaboratories.1.3 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Referenced Documents2.1 ASTM Standards:2F748 Practice for Selecting Generic Biological Test Methodsfor Materials and Devices2.2 ATCC Document:American Type Culture Collection,(ATCC)Catalogue ofStrains II3USP Negative Control Plastic Reference Standard43.Summary of Test Method3.1 Cell cultures are grown to a monolayer in culture dishes.The medium is aspirated and replaced with an agar-containingmedium that is allowed to solidify.Test control articles areplaced on the agar surface to evaluate the cytotoxic propertiesof a given material or device.Toxic components in the testarticle can diffuse into the culture medium,forming a concen-tration gradient and adversely affecting cells at varying dis-tances from the test article.This method is well suited forlow-density materials(film,paper,and so forth),powders,liquids,and high-density materials that could physically dam-age the cells if placed in direct contact with the cell monolayer.4.Significance and Use4.1 This test method is useful for assessing the cytotoxicpotential of new materials and formulations and as part of aquality control program for established medical devices andcomponents.4.2 This test method assumes that assessment of cytotoxic-ity provides useful information to aid in predicting the potentialclinical applications in humans.Cell culture methods haveshown good correlation with animal assays and are frequentlymore sensitive to cytotoxic agents.4.3 This cell culture test method is suitable for incorporationinto specifications and standards for materials to be used in theconstruction of medical devices that are to be implanted intothe human body or placed in contact with tissue fluids or bloodon a long-term basis.4.4 Some biomaterials with a history of safe clinical use inmedical devices are cytotoxic.This test method does not implythat all biomaterials must pass this assay to be considered safefor clinical use(Practice F748).5.Apparatus5.1 The following apparatus shall be used:1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Oct.1,2011.Published October 2011.Originallyapproved in 1984.Last previous edition approved in 2006 as F895 84(2006).DOI:10.1520/F0895-11.2For referenced ASTM standards,visit the ASTM website,www.astm.org,orcontact ASTM Customer Service at serviceastm.org.For Annual Book of ASTMStandards volume information,refer to the standards Document Summary page onthe ASTM website.3Fourth edition,1983,is available from American Type Culture Collection,12031 Parklawn Dr.,Rockville,MD 10892.Library of Congress No.76-640122.4U.S.Pharmacopeia,current edition,Rockville,MD.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 5.2 Incubator,which maintains the cultures at 37 6 2C,56 1%CO2,and greater than 90%relative humidity.5.3 Water Bath,capable of maintaining a temperature of 376 2C and 45 6 2C.5.4 Microscope,with inverted phase contrast optics andma