ASTM E2526-08(2013)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: E2526 − 08 (Reapproved 2013)
Standard Test Method for
Evaluation of Cytotoxicity of Nanoparticulate Materials in
Porcine Kidney Cells and Human Hepatocarcinoma Cells
This standard is issued under the fixed designation E2526; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2.2 ISO Standard:
ISO10993-5BiologicalEvaluationofMedicalDevices:Part
1.1 This test method provides a methodology to assess the
5 Tests for in vitro Cytotoxicity
cytotoxicity of suspensions of nanoparticulate materials in
porcine proximal tubule cells (LLC-PK1) and human hepato-
3. Terminology
carcinoma cells (Hep G2) which represents potential target
organs following systemic administration 3.1 Abbreviations:
3.1.1 APAP—acetaminophen- positive control
1.2 This test method is part of the in vitro preclinical
3.1.2 DMSO—dimethyl sulfoxide
characterization cascade.
3.1.3 DMEM—Dulbelcco’s modified eagles media
1.3 This test method consists of a protocol utilizing two
3.1.4 Hep G2—human hepatocarcinoma cells
methods for estimation of cytotoxicity, 3-(4,5-
Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT)
3.1.5 LDH—lactic dehydrogenase
reduction and lactate dehydrogenase (LDH) release.
3.1.6 LLC-PK1—porcine proximal tubule cells
1.4 The values stated in SI units are to be regarded as
3.1.7 LPS—lipopolysacchride, bacterial endotoxin
standard. No other units of measurement are included in this
3.1.8 MTT—3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetra-
standard.
zolium bromide
1.5 This standard does not purport to address all of the
3.1.9 Physiologic solution—isotonic with a pH 7.2 6 0.2
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
4. Summary of Test Method
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. 4.1 Nanoparticulate test materials in suspension in cell
culture media and appropriate controls are added to cell
cultures.The release of LDH indicates membrane damage and
2. Referenced Documents
thediminutionofMTTreductionindicateslossofcellviability.
2.1 ASTM Standards:
These are quantitative indicators of cytotoxicity. Aseptic pro-
F813Practice for Direct Contact Cell Culture Evaluation of
cedures are required.
Materials for Medical Devices
F895TestMethodforAgarDiffusionCellCultureScreening
5. Significance and Use
for Cytotoxicity
5.1 Assessing the propensity of a nanomaterial to cause
F1877Practice for Characterization of Particles
cytotoxicity to the cells of a target organ can assist in
F1903Practice for Testing For Biological Responses to
preclinical development.
Particles In Vitro
5.2 The standard historical cytotoxicity testing of materials
and extracts of materials has used fibroblasts and is well
documented in Practice F813, Test Method F895, and ISO
This test method is under the jurisdiction of ASTM Committee E56 on
Nanotechnology and is the direct responsibility of Subcommittee E56.03 on
10993-5.Theuseofmacrophagesandmicronsizeparticleshas
Environment, Health, and Safety.
also provided information on cytotoxicity and stimulation
Current edition approved Sept. 1, 2013. Published September 2013. Originally
using Practice F1903.
approved in 2008. Last previous edition approved in 2008 as E2526 – 08. DOI:
10.1520/E2526-08R13.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
the ASTM website. 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2526 − 08 (2013)
only to aid laboratories initiating these test procedures. This does not
5.3 This test method adds to the cytotoxicity test protocols
indicate endorsement by ASTM. Other equivalent sources may be
by using target organ cells.Two quantitative assays measuring
available.
LDH leakage and MTT reduction are used to estimate cyto-
toxicity.
7. Experimental Procedure
5.4 This test method may not be predictive of events
7.1 Aseptic precautions are required.
occurringinalltypesofnanomaterialapplicationsandtheuser
7.2 Positive Control Preparation:
is cautioned to consider the appropriateness of the test for
7.2.1 LLC-PK1 Acetaminophen (APAP) positive control:
various types of nanomaterial applications. This procedure
25 mM APAP in M199 cell culture media.
should only be used to compare the cytoxicity of a series of
7.2.2 Hepatocyte Acetaminophen (APAP) positive control:
related nanomaterials. Meaningful comparison of unrelated
20 mM APAP in RPMI 1640 cell culture media.
nanomaterials is not possible without additional characteriza-
7.2.3 Triton X100 is diluted to 1% in cell culture medium.
tion of physicochemical properties of each individual nanoma-
This is the positive control for the LDH assay.
terial in the assay matrix.
7.3 MTT Assay Reagents:
6. Reagents and Materials 7.3.1 MTT solution-5mg/mL MTT in PBS, store for up to
one month at 4°C in the dark.
6.1 Purity of Reagents—Reagent grade chemicals shall be
7.3.2 Glycine Buffer-0.1M glycine (MW 75.07), 0.1 M
used in all tests. Unless otherwise indicated, it is intended that
NaCl (MW 58.44), pH 10.5, store at room temperature.
all reagents conform to the specifications of the Committee on
Analytical Reagents of theAmerican Chemical Society where 7.4 Biovision LDH-Cytotoxicity Assay Kit Reagents:
7.4.1 Reconstitute catalyst in 1 mL dH 0 for 10 min and
such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently vortex (stable for 2 weeks at 4°C).
7.4.2 Reactionmixture(forone96-wellplate):Add250mL
high purity to permit its use without lessening the accuracy of
the determination. of reconstituted, catalyst solution to 11.25 mL of dye solution
(stable for 2 weeks at 4°C).
6.2 Reagents and supplies (aseptic procedures are needed
7.4.3 For other LDH Cytotoxicity assay kits, follow their
andcareshouldbetakentousesterilereagentsandsuppliesas
instructions.
necessary).
7.5 Cell Culture:
6.2.1 MTT (3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetra-
7.5.1 LLC-PK1 Cell Preparation:
zolium bromide).
7.5.1.1 Harvest cells from flasks prepared from cryopre-
6.2.2 Acetaminophen.
served cells according to the instructions from the supplier
6.2.3 Dimethyl sulfoxide.
(limit passages to 20). An example of the appearance of the
6.2.4 Glycine.
cells is in Fig. 1.
6.2.5 Sodium Chloride.
7.5.1.2 Count cell concentration using a Coulter type coun-
6.2.6 Medium 199 Cell Culture Media.
6.2.7 Triton X 100. ter or hemocytometer.
7.5.1.3 Dilute cells to a density of 2.5 × 10 cells/mol in
6.2.8 LDH-CytotoxicityAssay Kit (Biovision Cat. # K311-
400 was used in developing this test method)*. M199 (3% FBS) cell culture media.
7.5.1.4 4 Plate 100 µL cells/well as per plate format de-
6.2.9 96 well flat bottom cell culture plates.
6.2.10 RPMI 1640. scribed in Fig. 3 for 4 plates (time zero, 6 hour sample
exposure,24hoursampleexposure,48hoursampleexposure).
6.2.11 L-glutamine.
6.2.12 Fetal bovine serum (FBS). The format indicates no cells in rows D&E and they serve as
particle controls. Each plate accommodates two samples
6.3 Cell Lines:
(RowsA-CandF-H).Eachnanoparticulatematerialistestedat
6.3.1 LLC-PK1(porcineproximaltubulecell)(ATCC#CL-
9 dilutions. Column 11 receives the positive control and
101)*.
column 12 receives Triton X 100.
6.3.2 Hep G2 (human hepatocarcinoma)(ATCC # HB-
7.5.1.5 Incubate plates for 24 hours at 5% CO , 37°C and
8065)*.
95%humidity(cellsshouldbeapproximately80%confluent).
6.4 Equipment:
7.5.2 Hep G2 Cell Preparation:
6.4.1 Plate reader.
7.5.2.1 Harvest cells from flasks prepared from cryopre-
6.4.2 Plate Centrifuge set at 700-800 g.
served cells according to the instructions from the supplier.
6.4.3 Cell Culture Microscope.
(limit passages to 20). An example of the appearance of the
cells is in Fig. 2.
NOTE 1—Commercial sources are indicated for informational purposes
7.5.2.2 Count cell concentration using a Coulter type coun-
ter or hemocytometer.
7.5.2.3 Dilute cells to a density of 5.0 × 10 cells/mol in
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For Suggestions on the testing of reagents not
RPMI 1640 (2 mM L-glutamine, 1
...