ASTM F2148-01
(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 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.
1.7 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.
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Designation:F2148–01
Standard Practice for
Evaluation of Delayed Contact Hypersensitivity Using the
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
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
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.
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 This standard does not purport to address all of the 3.1.7 nonaqueous solvent—in this assay refers to the or-
ganic or nonpolar solvent, which shall be dimethylsulfoxide
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- (DMSO) or acetone olive oil (AOO).
3.1.8 PBS—phosphate buffered saline pH 7.2.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. 3.1.9 positive control—a substance capable of consistently
stimulating lymphocyte proliferation.
1.7 Identification of a supplier of materials or reagents is for
the convenience of the user and does not imply single source. 3.1.10 saline—0.9 % sodium chloride (aqueous, polar sol-
vent).
Appropriate materials and reagents may be obtained from
many commercial supply houses. 3.1.11 TCA—5 % trichloroacetic acid.
H3
3.1.12 tritiated thymidine— methyl thymidine, specific
activity 2 Ci/mM (in PBS) I IUDR-radioactive uridine.
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Surgical Materials and Devices and is the direct responsibility of Subcommittee Annual Book of ASTM Standards, Vol 13.01.
F04.16 on Biocompatibility Test Methods. Available from NICEATM, NIEHS, 79 Alexander Dr., Mail Drop EC-17,
Current edition approved Oct. 10, 2001. Published January 2002. Research Triangle Park, NC 27709.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2148
3.1.13 vehicle controls—an aqueous, polar solvent and a 7. Preparation of Positive Controls
non-aqueous, nonpolar solvent.
7.1 Nonaqueous Positive Control—Weigh 0.025 g of
DNCB and place in a flask.Add enough DMSO to dissolve all
4. Summary of Practice
oftheDNCB.AddmoreDMSOtobringthelevelupto10mL.
4.1 Test and control substances or extracts are applied to the
Cap and shake the flask until a homogeneous solution is
ears of test mice. The draining lymph nodes are harvested and
obtained. The dose level of the positive control should not
lymphocyte proliferation evaluated. Comparisons are made
produce systemic toxicity as evidenced by clinical observa-
with the control and test specimens tested under identical
tions.
conditions.
7.2 Aqueous Positive Control—Neutral buffered formalin is
commercially available. (Or dilute formaldehyde ⁄10 in PBS.
5. Significance and Use
Place 1 mL of formaldehyde in a 10-mL flask. Add enough
5.1 The propensity of a material to stimulate delayed
PBS to mix the two solutions.Add more PBS to bring the level
contact hypersensitivity must be assessed before clinical ap-
up to 10 mL. Cap and shake the flask until a homogeneous
plication of devices containing this material. Delayed hyper-
solution is obtained.)
sensitivity may occur anywhere in the body. Systemic delayed
7.3 Aqueous solutions are not suitable for application to the
hypersensitivity may have a complex set of reactions and
ear. Therefore, for use in the assay, add 0.05 g of hydroxyethyl
consequences depending on the actual tissue/organ site of
cellulose to each 10 mLof the aqueous positive control to aid
reaction. Although the reactions are seldom life-threatening,
in holding the solution to the ear until absorbed.
severetissueandorgandamagemyresultovertime.Skinisthe
usualtestsitetodeterminethepropensityofamaterialtocause 7.4 For all specimens requiring extractions, prepare an
aqueous and non-aqueous extract (DMSO or AOO are recom-
delayed hypersensitivity.
5.2 The standard historical test methods have involved the mended but other permissible extractants are listed in the
ICCVAM document) following the procedures described in
use of guinea pigs with a cutaneous application and observa-
tion of the reaction site. The use of the murine local lymph Practice F 619.
node assay results in a numerical quantitation of stimulation,
rather than subjective evaluation and could be used to deter- 8. Dosing of the Animals
mine dose responses.
8.1 Female CBA/Ca or CBA/j mice that are healthy and
5.3 This practice may not be predictive of events occurring
nonpregnant and seven to twelve weeks of age shall be used.
during all types of implant applications. The user is cautioned
House the animals according to treatment group with five
to consider the appropriateness of the method in view of the
animals per cage.
materials being tested, their potential applications, and the
8.2 Day One—Uniquely identify each mouse (ear tags or
recommendations contained in Practice F 748.
ear notches may not be used).Weigh each mouse to the nearest
whole gram.
6. Preparation of Test Specimens
8.3 Aminimum of five mice shall be used for each positive
6.1 Specimens should be prepared in accordance with Prac-
and negative control and each test sample. They will be treated
tice F 619. All solid materials and gels shall be extracted.
daily for three consecutive days by topical application of 25 µL
Extractions shall be done with an aqueous (polar) solvent and
of one of the solutions to the dorsal surface of both ears. For
a nonaqueous (nonpolar or organic) solvent either DMSO or
the aqueous groups only, the dorsal surface should be wiped
AOO.
with acetone just before treating to aid in absorption of the
6.2 Liquid test articles shall be used directly if they are not
aqueous solution, although it will not be completely absorbed.
an irritant. Liquids that are an irritant shall be diluted with an
8.3.1 For testing, other than liquid test articles, the groups
aqueousornonaqueoussolventbasedonsolubilityoftheliquid
shall include: aqueous and nonaqueous positive controls,
test article until the solution is nonirritating.
aqueous and nonaqueous vehicle controls, aqueous extract of
6.3 Wholly aqueous solutions are not suitable for applica-
test sample, and nonaqueous extract of test sample. To control
tion to the ear. Therefore, for use in the assay, add 0.05 g of
for inhibitory or toxic effects of the extract, test with the
hydroxyethyl cellulose to each 10 mL of the aqueous vehicle
positive control added to the aqueous and the nonaqueous
control and test solutions to aid in holding the solution to the
extract.
ear.
6.4 The final specimen to be extracted should be prepared 8.3.2 For testing of liquid test articles, the groups shall
with a surface finish consistent with end-use application. include: aqueous and nonaqueous positive controls, liquid test
6.5 The specimen shall be sterilized by the method to be sample, and either an aqueous or a nonaqueous vehicle control
used for the final product. appropriate for the nature of the liquid sample. To control for
6.6 Care should be taken that the specimens do not become inhibitory or toxic effects of the extract, test with the positive
contaminated during preparation and aseptic technique is control added to the liquid.
recommended.
8.3.3 The extract shall be used within 24 h of preparation.
The extract should be stored in a stoppered container at room
temperature. The applications shall be performed at 24 62h
“Final Report on the Safety Assessment of Hydroxyethylcellulose, Hydrox-
intervals on Days 2 and 3.Table 1 describes the events for each
ypropylcellulose, Methylcellulose, Hydroxypropyl Methylcellulose, and Cellulose
Gum,” J. Amer Coll Tox., Vol 5, No. 3, 1986, pp. 1-59. day of the test.
F2148
TABLE 1 LLNA Timetable
8.5.1 Record the weight of the mouse to the nearest gram.
Day Activity Inject the mouse intravenously with 250-µL sterile PBS con-
taining 20 µCi of tritiated thymidine or 2 µCi I IUDR via the
<1 Prepare extracts to be <24 h old on Day 1 and plan for suitable
extracts for Days 2 and 3
lateral tail vein using a 1.0-mL syringe and a needle no larger
1 Weigh mouse, mark, and treat ears with samples
than 25 gage. The tail veins may be dilated for easi
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