ASTM F2148-07e1
(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)
SIGNIFICANCE AND USE
The propensity of a material to stimulate delayed contact hypersensitivity must be assessed before clinical application of devices containing this material. Delayed hypersensitivity may occur anywhere in the body. Systemic delayed hypersensitivity may have a complex set of reactions and consequences depending on the actual tissue/organ site of reaction. Although the reactions are seldom life-threatening, severe tissue and organ damage my result over time. Skin is the usual test site to determine the propensity of a material to cause delayed hypersensitivity.
The standard historical test methods have involved the use of guinea pigs with a cutaneous application and observation of the reaction site. The use of the murine local lymph node assay results in a numerical quantitation of stimulation, rather than subjective evaluation and could be used to determine dose responses.
This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F 748.
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.
1.7 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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Standards Content (Sample)
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Designation: F2148 − 07
StandardPractice 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 F2148; 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 (´) indicates an editorial change since the last revision or reapproval.
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´ NOTE—Formatting and grammar were corrected editorially throughout in April 2007.
1. Scope responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
1.1 This practice provides a methodology to use an in-situ
bility of regulatory limitations prior to use.
procedure for the evaluation of delayed contact hypersensitiv-
ity reactions.
2. Referenced Documents
1.2 This practice is intended to provide an alternative to the 2
2.1 ASTM Standards:
use of guinea pigs for evaluation of the ability of a device
F619 Practice for Extraction of Medical Plastics
material to stimulate delayed contact hypersensitivity reac-
F720 PracticeforTestingGuineaPigsforContactAllergens:
tions. This alternative is particularly applicable for materials
Guinea Pig Maximization Test
used in devices that contact only intact skin. However, the
F748 PracticeforSelectingGenericBiologicalTestMethods
guinea pig maximization test is still the recommended method
for Materials and Devices
when assessing the delayed hypersensitivity response to metals
F750 Practice for Evaluating Material Extracts by Systemic
orwhentestingsubstancesthatdonotpenetratetheskinbutare
Injection in the Mouse
used in devices that contact deep tissues or breached surfaces.
2.2 Other Document:
The guinea pig maximization test should be used for these
ICCVAM NIH Publication No: 99-4494 The Murine Local
substances. 3
Lymph Node Assay, 1999
1.3 This practice consists of a protocol for assessing an
3. Terminology
increase in lymphocyte proliferation within the nodes draining
the site of administration on the ears of mice.
3.1 Definitions:
3.1.1 AOO, n—acetone olive oil solution (4:1 v/v) is a
1.4 The LLNA has been validated only for low molecular
suitable nonpolar solvent.
weight chemicals that can penetrate the skin. The absorbed
chemical or metabolite must bind to macromolecules, such as
3.1.2 aqueous solvent, n—in this assay refers to the polar
proteins, to form immunogenic conjugates.
solvent, saline.
1.5 This practice is one of several developed for the 3.1.3 DMSO, n—dimethylsulfoxide (nonaqueous, suitable
assessment of the biocompatibility of materials. Practice F748 organic solvent).
may provide guidance for the selection of appropriate methods
3.1.4 DNCB, n—2,4-dinitrochlorobenzene.
for testing materials for a specific application.
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3.1.5 formalin, n—a ⁄10 dilution of 37 to 39 % formalde-
1.6 Identification of a supplier of materials or reagents is for
hyde solution (formaldehyde) in PBS.
the convenience of the user and does not imply single source.
3.1.6 ICCVAM, n—Interagency Coordinating Committee on
Appropriate materials and reagents may be obtained from
the Validation of Alternative Methods.
many commercial supply houses.
3.1.7 nonaqueous solvent, n—in this assay refers to the
1.7 This standard does not purport to address all of the
organic or nonpolar solvent, which shall be dimethylsulfoxide
safety concerns, if any, associated with its use. It is the
(DMSO) or acetone olive oil (AOO).
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ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Surgical Materials and Devices and is the direct responsibility of Subcommittee contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
F04.16 on Biocompatibility Test Methods. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Feb. 1, 2007. Published February 2007. Originally the ASTM website.
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approved in 2001. Last previous edition approved in 2006 as F2148 – 06a. DOI: Available from NICEATM, NIEHS, 79 Alexander Dr., Mail Drop EC-17,
10.1520/F2148-07E01. 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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3.1.8 PBS, n—phosphate buffered saline, pH 7.2. hydroxyethyl cellulose to each 10 mL of the aqueous vehicle
control and test solutions to aid in holding the solution to the
3.1.9 positive control, n—a substance capable of consis-
ear.
tently stimulating lymphocyte proliferation.
6.4 The final specimen to be extracted should be prepared
3.1.10 saline, n—0.9 % sodium chloride (aqu
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