1、Designation:E252608(Reapproved 2013)Standard Test Method forEvaluation of Cytotoxicity of Nanoparticulate Materials inPorcine Kidney Cells and Human Hepatocarcinoma Cells1This standard is issued under the fixed designation E2526;the number immediately following the designation indicates the year ofo
2、riginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This test method provides a methodology to assess thecytotoxicity o
3、f suspensions of nanoparticulate materials inporcine proximal tubule cells(LLC-PK1)and human hepato-carcinoma cells(Hep G2)which represents potential targetorgans following systemic administration1.2 This test method is part of the in vitro preclinicalcharacterization cascade.1.3 This test method co
4、nsists of a protocol utilizing twomethodsforestimationofcytotoxicity,3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)reduction and lactate dehydrogenase(LDH)release.1.4 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstand
5、ard.1.5 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 D
6、ocuments2.1 ASTM Standards:2F813 Practice for Direct Contact Cell Culture Evaluation ofMaterials for Medical DevicesF895 Test Method forAgar Diffusion Cell Culture Screeningfor CytotoxicityF1877 Practice for Characterization of ParticlesF1903 Practice for Testing For Biological Responses toParticles
7、 In Vitro2.2 ISO Standard:3ISO 10993-5 Biological Evaluation of Medical Devices:Part5 Tests for in vitro Cytotoxicity3.Terminology3.1 Abbreviations:3.1.1 APAPacetaminophen-positive control3.1.2 DMSOdimethyl sulfoxide3.1.3 DMEMDulbelccos modified eagles media3.1.4 Hep G2human hepatocarcinoma cells3.1
8、.5 LDHlactic dehydrogenase3.1.6 LLC-PK1porcine proximal tubule cells3.1.7 LPSlipopolysacchride,bacterial endotoxin3.1.8 MTT3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetra-zolium bromide3.1.9 Physiologic solutionisotonic with a pH 7.2 6 0.24.Summary of Test Method4.1 Nanoparticulate test materials in s
9、uspension in cellculture media and appropriate controls are added to cellcultures.The release of LDH indicates membrane damage andthe diminution of MTT reduction indicates loss of cell viability.These are quantitative indicators of cytotoxicity.Aseptic pro-cedures are required.5.Significance and Use
10、5.1 Assessing the propensity of a nanomaterial to causecytotoxicity to the cells of a target organ can assist inpreclinical development.5.2 The standard historical cytotoxicity testing of materialsand extracts of materials has used fibroblasts and is welldocumented in Practice F813,Test Method F895,
11、and ISO10993-5.The use of macrophages and micron size particles hasalso provided information on cytotoxicity and stimulationusing Practice F1903.1This test method is under the jurisdiction of ASTM Committee E56 onNanotechnology and is the direct responsibility of Subcommittee E56.03 onEnvironment,He
12、alth,and Safety.Current edition approved Sept.1,2013.Published September 2013.Originallyapproved in 2008.Last previous edition approved in 2008 as E2526 08.DOI:10.1520/E2526-08R13.2For referenced ASTM standards,visit the ASTM website,www.astm.org,orcontact ASTM Customer Service at serviceastm.org.Fo
13、r Annual Book of ASTMStandards volume information,refer to the standards Document Summary page onthe ASTM website.3Available from American National Standards Institute(ANSI),25 W.43rd St.,4th Floor,New York,NY 10036,http:/www.ansi.org.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,We
14、st Conshohocken,PA 19428-2959.United States1 5.3 This test method adds to the cytotoxicity test protocolsby using target organ cells.Two quantitative assays measuringLDH leakage and MTT reduction are used to estimate cyto-toxicity.5.4 This test method may not be predictive of eventsoccurring in all
15、types of nanomaterial applications and the useris cautioned to consider the appropriateness of the test forvarious types of nanomaterial applications.This procedureshould only be used to compare the cytoxicity of a series ofrelated nanomaterials.Meaningful comparison of unrelatednanomaterials is not
16、 possible without additional characteriza-tion of physicochemical properties of each individual nanoma-terial in the assay matrix.6.Reagents and Materials6.1 Purity of ReagentsReagent grade chemicals shall beused in all tests.Unless otherwise indicated,it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.4Other grades may be used,provided it is first ascertained that the reagen
