1、Designation: F2148 13Standard Practice forEvaluation of Delayed Contact Hypersensitivity Using theMurine Local Lymph Node Assay (LLNA)1This standard is issued under the fixed designation F2148; the number immediately following the designation indicates the year oforiginal adoption or, in the case of
2、 revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice provides a methodology to use an in-situprocedure for the evaluation of delayed c
3、ontact hypersensitiv-ity reactions.1.2 This practice is intended to provide an alternative to theuse of guinea pigs for evaluation of the ability of a devicematerial to stimulate delayed contact hypersensitivity reac-tions. This alternative is particularly applicable for materialsused in devices tha
4、t contact only intact skin. However, theguinea pig maximization test is still the recommended methodwhen assessing the delayed hypersensitivity response to metalsor when testing substances that do not penetrate the skin but areused in devices that contact deep tissues or breached surfaces.This pract
5、ice may be used for testing metals, with the excep-tion of nickel-containing metals, unless the unique physico-chemical properties of the materials may interfere with theability of LLNA to detect sensitizing substances.1.3 This practice consists of a protocol for assessing anincrease in lymphocyte p
6、roliferation within the nodes drainingthe 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 absorbedchemical or metabolite must bind to macromolecules, such asproteins, to form immunogenic conjugates.1.
7、5 This practice is one of several developed for theassessment of the biocompatibility of materials. Practice F748may provide guidance for the selection of appropriate methodsfor testing materials for a specific application.1.6 Identification of a supplier of materials or reagents is forthe convenien
8、ce of the user and does not imply a single source.Appropriate materials and reagents may be obtained frommany commercial supply houses.1.7 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.8 This standard does not purport to add
9、ress all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F619 Practice
10、 for Extraction of Medical PlasticsF720 Practice for Testing Guinea Pigs for ContactAllergens:Guinea Pig Maximization TestF748 Practice for Selecting Generic Biological Test Methodsfor Materials and DevicesF750 Practice for Evaluating Material Extracts by SystemicInjection in the Mouse2.2 Other Docu
11、ments:3ICCVAM NIH Publication No: 99-4494 The Murine LocalLymph Node Assay, 1999ICCVAM NIH Publication NO: 11-7709 Usefulness andLimitations of the Murine Local Lymph Node Assay forPotency Categorization of Chemicals Causing AllergicContact Dermatitis in Humans3. Terminology3.1 Definitions:3.1.1 AOO
12、, nacetone olive oil solution (4:1 v/v) is asuitable nonpolar solvent.3.1.2 aqueous solvent, nin this assay refers to the polarsolvent, saline.3.1.3 DMSO, ndimethylsulfoxide (nonaqueous, suitableorganic solvent).3.1.4 DNCB, n2,4-dinitrochlorobenzene.3.1.5 formalin, na110 dilution of 37 to 39 % forma
13、lde-hyde solution (formaldehyde) in PBS.1This practice is under the jurisdiction ofASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved June 1, 2013. Published August 2013. Ori
14、ginallyapproved in 2001. Last previous edition approved in 2012 as F2148 07 (2012).DOI: 10.1520/F2148-13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standar
15、ds Document Summary page onthe ASTM website.3Available 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 States13.1.6 ICCVAM, nInteragency Coordinating Com
16、mittee onthe Validation of Alternative Methods.3.1.7 nonaqueous solvent, nin this assay refers to theorganic or nonpolar solvent, which shall be dimethylsulfoxide(DMSO) or acetone olive oil (AOO).3.1.8 PBS, nphosphate buffered saline, pH 7.2.3.1.9 positive control, na substance capable of consis-ten
17、tly stimulating lymphocyte proliferation.3.1.10 saline, n0.9 % sodium chloride (aqueous, polarsolvent).3.1.11 TCA, n5 % trichloroacetic acid.3.1.12 tritiated thymidine, nH3methyl thymidine, specificactivity 2 Ci/mM (in PBS) I125IUDR-radioactive uridine.3.1.13 vehicle controls, nan aqueous, polar sol
18、vent and anon-aqueous, nonpolar solvent.4. Summary of Practice4.1 Test and control substances or extracts are applied to theears of test mice. The draining lymph nodes are harvested andlymphocyte proliferation evaluated. Comparisons are madewith the control and test specimens tested under identicalc
19、onditions.5. Significance and Use5.1 The propensity of a material to stimulate delayedcontact hypersensitivity must be assessed before clinical ap-plication of devices containing this material. Delayed hyper-sensitivity may occur anywhere in the body. Systemic delayedhypersensitivity may have a comp
20、lex set of reactions andconsequences depending on the actual tissue/organ site ofreaction. Although the reactions are seldom life-threatening,severe tissue and organ damage my result over time. Skin is theusual test site to determine the propensity of a material to causedelayed hypersensitivity.5.2
21、The standard historical test methods have involved theuse of guinea pigs with a cutaneous application and observa-tion of the reaction site. The use of the murine local lymphnode assay results in a numerical quantitation of stimulation,rather than subjective evaluation and could be used to deter-min
22、e dose responses.5.3 This practice may not be predictive of events occurringduring all types of implant applications. The user is cautionedto consider the appropriateness of the method in view of thematerials being tested, their potential applications, and therecommendations contained in Practice F7
23、48.6. Preparation of Test Specimens6.1 Specimens should be prepared in accordance with Prac-tice F619. All solid materials shall be extracted. Extractionsshall be done with an aqueous (polar) solvent and a nonaque-ous (nonpolar or organic) solvent, either DMSO or AOO.6.2 Liquid test articles and gel
24、s shall be used directly if theyare not irritants. A liquid that is an irritant shall be diluted withan aqueous or nonaqueous solvent based on solubility of theliquid test article until the solution is non-irritating.6.3 Wholly aqueous solutions are not suitable for applica-tion to the ear. Therefor
25、e, for use in the assay, add 0.05 g ofhydroxyethyl cellulose4to each 10 mL of the aqueous vehiclecontrol and test solutions to aid in holding the solution to theear. One percent Pluronic L92 may also be used as an aqeuousvehicle.6.4 The final specimen to be extracted should be preparedwith a surface
26、 finish consistent with end-use application.6.5 The specimen shall be sterilized by the method to beused for the final product.6.6 Care should be taken that the specimens do not becomecontaminated during preparation and aseptic technique isrecommended.7. Preparation of Positive Controls7.1 Nonaqueou
27、s Positive ControlThe use of a moderatepositive control as a substitute or in addition to a strongpositive control should be considered.7.1.1 Moderate Positive ControlPrepare a solution of25 % hexyl cinnamic aldehyde (HCA) in an acetone:olive oil(4:1 v/v) solvent. Shake the flask until a homogenous
28、solutionis obtained.7.1.2 Strong Positive ControlWeigh 0.025 g of DNCB andplace in a flask. Add enough DMSO to dissolve all of theDNCB. Add more DMSO to bring the level up to 10 mL. Capand shake the flask until a homogenous solution is obtained.7.1.3 The dose level of the positive control should not
29、produce systemic toxicity as evidenced by clinical observa-tions.7.2 Aqueous Positive ControlNeutral buffered formalin iscommercially available. (Or dilute formaldehyde110 in PBS.Place 1 mL of formaldehyde in a 10-mL flask. Add enoughPBS to mix the two solutions. Add more PBS to bring the levelup to
30、 10 mL. Cap and shake the flask until a homogeneoussolution is obtained.)7.3 Aqueous solutions are not suitable for application to theear. Therefore, for use in the assay, add 0.05 g of hydroxyethylcellulose4to each 10 mL of the aqueous positive control to aidin holding the solution to the ear until
31、 absorbed. One percentPluronic L92 may also be used as an aqueous vehicle.7.4 For all specimens requiring extractions, prepare anaqueous and non-aqueous extract (DMSO or AOO are recom-mended but other permissible extractants are listed in theICCVAM documents) following the procedures described inPra
32、ctice F619.8. Dosing of the Animals8.1 Healthy, non-pregnant female CBA/Ca or CBA/j micethat are seven to twelve weeks of age shall be used. House theanimals according to treatment group with five animals percage.4“Final Report on the Safety Assessment of Hydroxyethylcellulose,Hydroxypropylcellulose
33、, Methylcellulose, Hydroxypropyl Methylcellulose, andCellulose Gum,” J. Amer Coll Tox., Vol 5, No. 3, 1986, pp. 1-59.F2148 1328.2 Day OneUniquely identify each mouse (ear tags or earnotches may not be used). Weigh each mouse to the nearestwhole gram.8.3 A minimum of five mice shall be used for each
34、positiveand negative control and each test sample. They shall be treateddaily for three consecutive days by topical application of 25 Lof one of the solutions to the dorsal surface of both ears. Forthe aqueous groups only, the dorsal surface should be wipedwith acetone just before treating to aid in
35、 absorption of theaqueous solution, although it will not be completely absorbed.8.3.1 For testing, other than liquid test articles, the groupsshall include: aqueous and nonaqueous positive controls,aqueous and nonaqueous vehicle controls, aqueous extract ofthe test sample, and nonaqueous extract of
36、test sample.8.3.2 For testing of liquid test articles, the groups shallinclude: aqueous and nonaqueous positive controls, the liquidtest sample, and either an aqueous or a nonaqueous vehiclecontrol appropriate for the nature of the liquid sample.8.3.3 The extract shall be used within 24 h of prepara
37、tion.The extract should be stored in a stoppered container at roomtemperature. The applications shall be performed at 24 6 2hintervals on Days 2 and 3. Table 1 describes the events for eachday of the test.8.3.4 Observe each mouse daily for signs of local irritationat the application site and for sig
38、ns of systemic toxicity (seePractices F720 and F750). It may be advisable to pretest twomice if it is suspected that the material may be an irritant.NOTE 1The following steps through 9.3.3 until precipitation for 18 htake more than8htocomplete and the laboratory needs to be prepared toaccommodate th
39、is.8.4 Radiolabeled Tracer PreparationPrepare tritiated thy-midine to a working concentration of 80 Ci/mL (v/v). The useof I125I-UDR at 8 Ci/mL in PBS 10-5M fluorodeoxyuridineis also acceptable. Each mouse will receive 250 L of this. Allstandard precautions associated with using radioactive materi-a
40、ls shall be adhered to. The laboratory shall be licensed to useradioactive material and all personnel shall be appropriatelytrained and certified.8.4.1 To prepare the tritiated thymidine solution, add 0.8 mLof 1.0-mCi/mL tritiated thymidine (specific activity 2.0 Ci/mM) to a stoppered flask.Add ster
41、ile PBS to make 10 mL. Capand mix well.8.4.2 Confirm the concentration of this dilution. Dilute 0.08to 200 mL with water using a 200-mL flask. Cap and mix wellby inverting several times. Remove two 1-mL samples andplace in scintillation vials. Add 10 mL of scintillation fluid toeach vial, mix so tha
42、t a vortex is formed, and “count” in a betascintillation counter. Count each vial three times and calculatethe mean. Calculate the concentration. First convert counts perminute (cpm) to disintegrations per minute (dpm) as follows:cpmdecimal counter efficiency5 dpmFor verification of the working trit
43、iated thymidine solution,determine the closeness of the concentration to 80 Ci/mL. Thefinal diluted solution contains 0.032 Ci. Since 1.0Ci = 2 220 000 dpm, then 0.032 Ci = 71 040 dpm. There-fore:the Ci of the working solution 5mean dpm71040 dpm380 Ci/mLMake adjustments to the solution as needed. Ma
44、ke similarverifications if I125IUDR is used.8.5 In-Situ Labeling (Day 6)(72 6 3 h after the lasttreatment was applied to the ears).8.5.1 Record the weight of the mouse to the nearest gram.Inject the mouse intravenously with 250-L sterile PBS con-taining 20 Ci of tritiated thymidine or 2 Ci I125IUDR
45、via thelateral tail vein using a 1.0-mL syringe and a needle no largerthan 25 gage. The tail veins may be dilated for easierintravenous injection by placing the mice under a heat lamp.9. Lymph Node Collection and Lymph Node CellPreparationNOTE 2All equipment and solutions from this point should be t
46、reatedas radioactive with appropriate precautions.NOTE 3If the investigators are not familiar with the location of thelymph nodes, refer to the diagram in the ICCVAM documents, consult amouse anatomy book, or use a dye in trial mice to learn to locate theappropriate nodes. One suggested procedure is
47、 to inject 0.1 mL of 2 %Evans blue dye intradermally into the tissue of the ear and then euthanizethe mice after 5 to 10 min. Dissect the mice to expose the nodes.9.1 Euthanize the mice 5 h 6 45 min after injection of theradioisotope.9.2 Excise the draining (auricular) lymph node of each ear.9.3 Pre
48、pare a single-cell suspension of lymph node cells(LNC) for each mouse.9.3.1 Pool the nodes from both the left and right side of asingle mouse in a labeled test tube containing 1 to 3 mL ofPBS. Snip the nodes from a single mouse into small pieceswith small scissors or transfer the lymph nodes directl
49、y onto a200-mesh screen/cell dissociation cup. Gently mash the piecesthrough the screen directly into a 15-mL tube. Wash the meshwith 2 to 6 mL of PBS to facilitate the transfer of cell debrisinto the tube. Bring the volume in the centrifuge tube up to 10mL.9.3.2 Repeat this procedure for each animal.9.3.3 Centrifuge the samples for 10 min at 190 to 200 xg at2 to 8C. Remove each supernatant by aspiration, leaving 1 to2 mL of supernatant above each pellet. Gently agitate eachpellet then bring up to 10 mL with PBS and resuspend byTABLE 1 LLNA Ti
