ASTM F2148-13
(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
5.1 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.
5.2 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.
5.3 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 F748.
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. This practice may be used for testing metals, with the exception of nickel-containing metals, unless the unique physicochemical properties of the materials may interfere with the ability of LLNA to detect sensitizing 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 F748 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 a single source. Appropriate materials and reagents may be obtained from many commercial supply houses.
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 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 − 13
Standard Practice for
Evaluation of Delayed Contact Hypersensitivity Using the
1
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.
1. Scope 1.8 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This practice provides a methodology to use an in-situ
responsibility of the user of this standard to establish appro-
procedure for the evaluation of delayed contact hypersensitiv-
priate safety and health practices and determine the applica-
ity reactions.
bility of regulatory limitations prior to use.
1.2 This practice is intended to provide an alternative to the
use of guinea pigs for evaluation of the ability of a device
2. Referenced Documents
material to stimulate delayed contact hypersensitivity reac-
2
2.1 ASTM Standards:
tions. This alternative is particularly applicable for materials
F619 Practice for Extraction of Medical Plastics
used in devices that contact only intact skin. However, the
F720 Practice forTesting Guinea Pigs for ContactAllergens:
guinea pig maximization test is still the recommended method
Guinea Pig Maximization Test
when assessing the delayed hypersensitivity response to metals
F748 PracticeforSelectingGenericBiologicalTestMethods
orwhentestingsubstancesthatdonotpenetratetheskinbutare
for Materials and Devices
used in devices that contact deep tissues or breached surfaces.
F750 Practice for Evaluating Material Extracts by Systemic
This practice may be used for testing metals, with the excep-
Injection in the Mouse
tion of nickel-containing metals, unless the unique physico-
3
2.2 Other Documents:
chemical properties of the materials may interfere with the
ability of LLNA to detect sensitizing substances. ICCVAM NIH Publication No: 99-4494 The Murine Local
Lymph Node Assay, 1999
1.3 This practice consists of a protocol for assessing an
ICCVAM NIH Publication NO: 11-7709 Usefulness and
increase in lymphocyte proliferation within the nodes draining
Limitations of the Murine Local Lymph Node Assay for
the site of administration on the ears of mice.
Potency Categorization of Chemicals Causing Allergic
1.4 The LLNA has been validated only for low-molecular-
Contact Dermatitis in Humans
weight chemicals that can penetrate the skin. The absorbed
chemical or metabolite must bind to macromolecules, such as
3. Terminology
proteins, to form immunogenic conjugates.
3.1 Definitions:
1.5 This practice is one of several developed for the
3.1.1 AOO, n—acetone olive oil solution (4:1 v/v) is a
assessment of the biocompatibility of materials. Practice F748
suitable nonpolar solvent.
may provide guidance for the selection of appropriate methods
3.1.2 aqueous solvent, n—in this assay refers to the polar
for testing materials for a specific application.
solvent, saline.
1.6 Identification of a supplier of materials or reagents is for
3.1.3 DMSO, n—dimethylsulfoxide (nonaqueous, suitable
the convenience of the user and does not imply a single source.
organic solvent).
Appropriate materials and reagents may be obtained from
3.1.4 DNCB, n—2,4-dinitrochlorobenzene.
many commercial supply houses.
1
3.1.5 formalin, n—a ⁄10 dilution of 37 to 39 % formalde-
1.7 The values stated in SI units are to be regarded as
hyde solution (formaldehyde) in PBS.
standard. No other units of measurement are included in this
standard.
1 2
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 June 1, 2013. Published August 2013. Originally the ASTM website.
3
approved in 2001. Last previous edition approved in 2012 as F2148 – 07 (2012). Available from NICEATM, NIEHS, 79 Alexander Dr., Mail Drop EC-17,
DOI: 10.1520/F2148-13. Research Triangle Park, NC 27709.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
F2148 − 13
3.1.6 ICCVAM, n—Interagency Coordinating Committee on 6.3 Wholly aqueous solutions are not suitable for applica-
the Validation of Alternative Methods. tion to the ear. Therefore, for use in the assay, add 0.05 g of
4
hydroxyethyl ce
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM 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: F2148 − 07 (Reapproved 2012) F2148 − 13
Standard Practice for
Evaluation of Delayed Contact Hypersensitivity Using the
1
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.
1. 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 This practice may be used for these testing metals, with
the exception of nickel-containing metals, unless the unique physicochemical properties of the materials may interfere with the
ability of LLNA to detect sensitizing 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 F748 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 a single source.
Appropriate materials and reagents may be obtained from many commercial supply houses.
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 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:
F619 Practice for Extraction of Medical Plastics
F720 Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F750 Practice for Evaluating Material Extracts by Systemic Injection in the Mouse
3
2.2 Other Document:Documents:
ICCVAM NIH Publication No: 99-4494 The Murine Local Lymph Node Assay, 1999
ICCVAM NIH Publication NO: 11-7709 Usefulness and Limitations of the Murine Local Lymph Node Assay for Potency
Categorization of Chemicals Causing Allergic Contact Dermatitis in Humans
1
This practice 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.
Current edition approved Oct. 1, 2012June 1, 2013. Published October 2012August 2013. Originally approved in 2001. Last previous edition approved in 20072012 as
ε1
F2148 – 07 (2012). . DOI: 10.1520/F2148-07R12.10.1520/F2148-13.
2
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 the ASTM website.
3
Available from NICEATM, NIEHS, 79 Alexander Dr., Mail Drop EC-17, Research Triangle Park, NC 27709.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
F2148 − 13
3. Terminology
3.1 Definitions:
3.1.1 AOO, n—acetone olive oil solution (4:1 v/v) is a suitable nonpolar solvent.
3.1.2 aqueous solvent, n—in this assay refers to the polar solvent, saline.
3.1.3 D
...
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