ASTM F895-11

Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity

SIGNIFICANCE AND USE
This test method is useful for assessing the cytotoxic potential of new materials and formulations and as part of a quality control program for established medical devices and components.
This test method assumes that assessment of cytotoxicity provides useful information to aid in predicting the potential clinical applications in humans. Cell culture methods have shown good correlation with animal assays and are frequently more sensitive to cytotoxic agents.
This cell culture test method is suitable for incorporation into specifications and standards for materials to be used in the construction of medical devices that are to be implanted into the human body or placed in contact with tissue fluids or blood on a long-term basis.
Some biomaterials with a history of safe clinical use in medical devices are cytotoxic. This test method does not imply that all biomaterials must pass this assay to be considered safe for clinical use (Practice F748).
SCOPE
1.1 This test method is appropriate for materials in a variety of shapes and for materials that are not necessarily sterile. This test method would be appropriate in situations in which the amount of material is limited. For example, small devices or powders could be placed on the agar and the presence of a zone of inhibition of cell growth could be examined.
1.1.1 This test method is not appropriate for leachables that do not diffuse through agar or agarose.
1.1.2 While the agar layer can act as a cushion to protect the cells from the specimen, there may be materials that are sufficiently heavy to compress the agar and prevent diffusion or to cause mechanical damage to the cells. This test method would not be appropriate for these materials.
1.2 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply that its use is preferred, only that the L-929 is an established cell line, well characterized and readily available, that has demonstrated reproducible results in several laboratories.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

General Information

• Status: Historical
• Publication Date: 30-Sep-2011
• ICS: 07.100.10 - Medical microbiology
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.16 - Biocompatibility Test Methods

Relations

• Replaces: ASTM F895-84(2006) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Oct-2011
• Replaced By: ASTM F895-11(2016) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Apr-2016
• Referred By: ASTM F2759-19 - Standard Guide for Assessment of the Ultra-High Molecular Weight Polyethylene (UHMWPE) Used in Orthopedic and Spinal Devices
• Effective Date: 01-Oct-2011
• Referred By: ASTM F2529-13(2021) - Standard Guide for <emph type="ital"> in vivo</emph> Evaluation of Osteoinductive Potential for Materials Containing Demineralized Bone (DBM)
• Effective Date: 01-Oct-2011
• Referred By: ASTM F813-20 - Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices
• Effective Date: 01-Oct-2011
• Referred By: ASTM F2695-12(2020) - Standard Specification for Ultra-High Molecular Weight Polyethylene Powder Blended With Alpha-Tocopherol (Vitamin E) and Fabricated Forms for Surgical Implant Applications
• Effective Date: 01-Oct-2011
• Referred By: ASTM F648-21 - Standard Specification for Ultra-High-Molecular-Weight Polyethylene Powder and Fabricated Form for Surgical Implants
• Effective Date: 01-Oct-2011
• Referred By: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Oct-2011
• Referred By: ASTM F2097-20 - Standard Guide for Design and Evaluation of Primary Flexible Packaging for Medical Products
• Effective Date: 01-Oct-2011
• Referred By: ASTM E1299-96(2023) - Standard Specification for Reusable Phase-Change-Type Fever Thermometer for Intermittent Determination of Human Temperature
• Effective Date: 01-Oct-2011
• Referred By: ASTM F2103-18 - Standard Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications
• Effective Date: 01-Oct-2011
• Referred By: ASTM F451-21 - Standard Specification for Acrylic Bone Cement
• Effective Date: 01-Oct-2011
• Referred By: ASTM F2721-09(2023) - Standard Guide for Preclinical <emph type="ital">in vivo</emph> Evaluation in Critical-Size Segmental Bone Defects
• Effective Date: 01-Oct-2011
• Referred By: ASTM F2224-09(2020) - Standard Specification for High Purity Calcium Sulfate Hemihydrate or Dihydrate for Surgical Implants
• Effective Date: 01-Oct-2011
• Referred By: ASTM E3219-20 - Standard Guide for Derivation of Health-Based Exposure Limits (HBELs)
• Effective Date: 01-Oct-2011

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: F895 − 11
StandardTest Method for
1
Agar Diffusion Cell Culture Screening for Cytotoxicity
ThisstandardisissuedunderthefixeddesignationF895;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginal
adoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2.2 ATCC Document:
American Type Culture Collection, (ATCC) Catalogue of
1.1 Thistestmethodisappropriateformaterialsinavariety
3
Strains II
ofshapesandformaterialsthatarenotnecessarilysterile.This
4
USP Negative Control Plastic Reference Standard
test method would be appropriate in situations in which the
amount of material is limited. For example, small devices or
3. Summary of Test Method
powderscouldbeplacedontheagarandthepresenceofazone
3.1 Cellculturesaregrowntoamonolayerinculturedishes.
of inhibition of cell growth could be examined.
The medium is aspirated and replaced with an agar-containing
1.1.1 This test method is not appropriate for leachables that
medium that is allowed to solidify. Test control articles are
do not diffuse through agar or agarose.
placed on the agar surface to evaluate the cytotoxic properties
1.1.2 Whiletheagarlayercanactasacushiontoprotectthe
of a given material or device. Toxic components in the test
cells from the specimen, there may be materials that are
article can diffuse into the culture medium, forming a concen-
sufficientlyheavytocompresstheagarandpreventdiffusionor
tration gradient and adversely affecting cells at varying dis-
to cause mechanical damage to the cells. This test method
tances from the test article. This method is well suited for
would not be appropriate for these materials.
low-density materials (film, paper, and so forth), powders,
1.2 The L-929 cell line was chosen because it has a
liquids, and high-density materials that could physically dam-
significant history of use in assays of this type. This is not
agethecellsifplacedindirectcontactwiththecellmonolayer.
intended to imply that its use is preferred, only that the L-929
4. Significance and Use
is an established cell line, well characterized and readily
available, that has demonstrated reproducible results in several
4.1 This test method is useful for assessing the cytotoxic
laboratories.
potential of new materials and formulations and as part of a
quality control program for established medical devices and
1.3 The values stated in SI units are to be regarded as
components.
standard. No other units of measurement are included in this
standard.
4.2 This test method assumes that assessment of cytotoxic-
ityprovidesusefulinformationtoaidinpredictingthepotential
1.4 This standard does not purport to address all of the
clinical applications in humans. Cell culture methods have
safety concerns, if any, associated with its use. It is the
shown good correlation with animal assays and are frequently
responsibility of the user of this standard to establish appro-
more sensitive to cytotoxic agents.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
4.3 Thiscellculturetestmethodissuitableforincorporation
intospecificationsandstandardsformaterialstobeusedinthe
2. Referenced Documents
construction of medical devices that are to be implanted into
2
2.1 ASTM Standards: thehumanbodyorplacedincontactwithtissuefluidsorblood
F748PracticeforSelectingGenericBiologicalTestMethods on a long-term basis.
for Materials and Devices
4.4 Some biomaterials with a history of safe clinical use in
medicaldevicesarecytotoxic.Thistestmethoddoesnotimply
that all biomaterials must pass this assay to be considered safe
1
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
for clinical use (Practice F748).
andSurgicalMaterialsandDevicesandisthedirectresponsibilityofSubcommittee
F04.16 on Biocompatibility Test Methods.
5. Apparatus
Current edition approved Oct. 1, 2011. Published October 2011. Originally
approved in 1984. Last previous edition approved in 2006 as F895–84 (2006).
5.1 The following apparatus shall be used:
DOI: 10.1520/F0895-11.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
3
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Fourth edition, 1983, is available from American Type Culture Collection,
Standards volume information, refer to the standard’s Document Summary page on 12031 Parklawn Dr., Rockville, MD 10892. Library of Congress No. 76-640122.
4
the ASTM website. U.S. Pharmacopeia, current edition, Rockville, MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
F895 − 11
5.2 Incubator, which m
...

This document is not anASTM standard and is intended only to provide the user of anASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation:F895–84 (Reapproved 2006) Designation:F895–11
Standard Test Method for
1
Agar Diffusion Cell Culture Screening for Cytotoxicity
ThisstandardisissuedunderthefixeddesignationF895;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginal
adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.Asuperscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
This standard has been approved for use by agencies of the Department of Defense.
1. Scope
1.1 This test method is appropriate for materials in a variety of shapes and for materials that are not necessarily sterile. This
test method would be appropriate in situations in which the amount of material is limited. For example, small devices or powders
could be placed on the agar and the presence of a zone of inhibition of cell growth could be examined.
1.1.1 This test method is not appropriate for leachables that do not diffuse through agar or agarose.
1.1.2 Whiletheagarlayercanactasacushiontoprotectthecellsfromthespecimen,theremaybematerialsthataresufficiently
heavy to compress the agar and prevent diffusion or to cause mechanical damage to the cells. This test method would not be
appropriate for these materials.
1.2 The L-929 cell line was chosen because it has a significant history of use in assays of this type.This is not intended to imply
that its use is preferred, only that the L-929 is an established cell line, well characterized and readily available, that has
demonstrated reproducible results in several laboratories.
1.3
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2
2.1 ASTM Standards:
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
2.2 ATCC Document:
3
American Type Culture Collection, (ATCC) Catalogue of Strains II
4
USP Negative Control Plastic Reference Standard
3. Summary of Test Method
3.1 Cell cultures are grown to a monolayer in culture dishes. The medium is aspirated and replaced with an agar-containing
mediumthatisallowedtosolidify.Testcontrolarticlesareplacedontheagarsurfacetoevaluatethecytotoxicpropertiesofagiven
material or device. Toxic components in the test article can diffuse into the culture medium, forming a concentration gradient and
adversely affecting cells at varying distances from the test article.This method is well suited for low-density materials (film, paper,
and so forth), powders, liquids, and high-density materials that could physically damage the cells if placed in direct contact with
the cell monolayer.
4. Significance and Use
4.1 This test method is useful for assessing the cytotoxic potential of new materials and formulations and as part of a quality
control program for established medical devices and components.
1
This test method is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods.
´1
Current edition approved March 1, 2006. Published April 2006. Originally approved in 1984. Last previous edition approved in 2001 as F895–84 (2001) . DOI:
10.1520/F0895-84R06.
Current edition approved Oct. 1, 2011. Published October 2011. Originally approved in 1984. Last previous edition approved in 2006 as F895 – 84 (2006). DOI:
10.1520/F0895-11.
2
For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
3
Fourth edition, 1983, is available from American Type Culture Collection, 12031 Parklawn Dr., Rockville, MD 10892. Library of Congress No. 76-640122.
4
U.S. Pharmacopeia, current edition, Rockville, MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
1

---------------------- Page: 1 ----------------------
F895–11
4.2 This test method assumes that assessment of cytotoxicity provides useful information to aid in predicting the potential
clinical applications in humans. Cell culture me
...