ASTM F2148-07
(Practice)Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
SCOPE
1.1 This practice provides a methodology to use an in situ procedure for the evaluation of delayed contact hypersensitivity reactions.
1.2 This practice is intended to provide an alternative to the use of guinea pigs for evaluation of the ability of a device material to stimulate delayed contact hypersensitivity reactions. This alternative is particularly applicable for materials used in devices that contact only intact skin. However, the guinea pig maximization test is still the recommended method when assessing the delayed hypersensitivity response to metals or when testing substances that do not penetrate the skin but are used in devices that contact deep tissues or breached surfaces. The guinea pig maximization test should be used for these substances.
1.3 This practice consists of a protocol for assessing an increase in lymphocyte proliferation within the nodes draining the site of administration on the ears of mice.
1.4 The LLNA has been validated only for low molecular weight chemicals that can penetrate the skin. The absorbed chemical or metabolite must bind to macromolecules, such as proteins, to form immunogenic conjugates.
1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F 748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.
1.6 Identification of a supplier of materials or reagents is for the convenience of the user and does not imply single source. Appropriate materials and reagents may be obtained from many commercial supply houses.
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.
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Designation:F2148–07
Standard Practice for
Evaluation of Delayed Contact Hypersensitivity Using the
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Murine Local Lymph Node Assay (LLNA)
This standard is issued under the fixed designation F 2148; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
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1.1 This practice provides a methodology to use an in situ 2.1 ASTM Standards:
procedure for the evaluation of delayed contact hypersensitiv- F 619 Practice for Extraction of Medical Plastics
ity reactions. F 720 Practice for Testing Guinea Pigs for Contact Aller-
1.2 This practice is intended to provide an alternative to the gens: Guinea Pig Maximization Test
use of guinea pigs for evaluation of the ability of a device F 748 Practice for Selecting Generic Biological Test Meth-
material to stimulate delayed contact hypersensitivity reac- ods for Materials and Devices
tions. This alternative is particularly applicable for materials F 750 PracticeforEvaluatingMaterialExtractsbySystemic
used in devices that contact only intact skin. However, the Injection in the Mouse
guinea pig maximization test is still the recommended method 2.2 Other Document:
when assessing the delayed hypersensitivity response to metals ICCVAM NIH Publication No: 99-4494 The Murine Local
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orwhentestingsubstancesthatdonotpenetratetheskinbutare Lymph Node Assay, 1999
used in devices that contact deep tissues or breached surfaces.
3. Terminology
The guinea pig maximization test should be used for these
3.1 Definitions:
substances.
1.3 This practice consists of a protocol for assessing an 3.1.1 AOO—acetone olive oil solution (4:1 v/v) is a suitable
nonpolar solvent.
increase in lymphocyte proliferation within the nodes draining
the site of administration on the ears of mice. 3.1.2 aqueous solvent—in this assay refers to the polar
solvent, saline.
1.4 The LLNA has been validated only for low molecular
3.1.3 DMSO—dimethylsulfoxide (nonaqueous, suitable or-
weight chemicals that can penetrate the skin. The absorbed
chemical or metabolite must bind to macromolecules, such as ganic solvent).
3.1.4 DNCB—2,4-dinitrochlorobenzene.
proteins, to form immunogenic conjugates.
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1.5 This practice is one of several developed for the 3.1.5 formalin—a ⁄10 dilution of 37 to 39 % formaldehyde
solution (formaldehyde) in PBS.
assessment of the biocompatibility of materials. Practice F 748
may provide guidance for the selection of appropriate methods 3.1.6 ICCVAM—Interagency Coordinating Committee on
the Validation of Alternative Methods.
for testing materials for a specific application.
1.6 Identification of a supplier of materials or reagents is for 3.1.7 nonaqueous solvent—in this assay refers to the or-
ganic or nonpolar solvent, which shall be dimethylsulfoxide
the convenience of the user and does not imply single source.
Appropriate materials and reagents may be obtained from (DMSO) or acetone olive oil (AOO).
3.1.8 PBS—phosphate buffered saline pH 7.2.
many commercial supply houses.
1.7 This standard does not purport to address all of the 3.1.9 positive control—a substance capable of consistently
stimulating lymphocyte proliferation.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- 3.1.10 saline—0.9 % sodium chloride (aqueous, polar sol-
vent).
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. 3.1.11 TCA—5 % trichloroacetic acid.
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For referenced ASTM standards, visit the ASTM website, www.astm.org, or
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ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Surgical Materials and Devices and is the direct responsibility of Subcommittee Standards volume information, refer to the standard’s Document Summary page on
F04.16 on Biocompatibility Test Methods. the ASTM website.
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Current edition approved Feb. 1, 2007. Published February 2007. Originally Available from NICEATM, NIEHS, 79 Alexander Dr., Mail Drop EC-17,
approved in 2001. Last previous edition approved in 2006 as F 2148 – 06a. Research Triangle Park, NC 27709.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
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F2148–07
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3.1.12 tritiated thymidine— methyl thymidine, specific 6.6 Care should be taken that the specimens do not become
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activity 2 Ci/mM (in PBS) I IUDR-radioactive uridine. contaminated during preparation and aseptic technique is
3.1.13 vehicle controls—an aque
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