1、Designation: F 2148 07e1Standard Practice forEvaluation of Delayed Contact Hypersensitivity Using theMurine Local Lymph Node Assay (LLNA)1This standard is issued under the fixed designation F 2148; the number immediately following the designation indicates the year oforiginal adoption or, in the cas
2、e of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.e1NOTEFormatting and grammar were corrected editorially throughout in April 2007.1. Scope1.1 This prac
3、tice provides a methodology to use an in-situprocedure for the evaluation of delayed contact 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 re
4、ac-tions. This alternative is particularly applicable for materialsused in devices that 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
5、 skin but areused in devices that contact deep tissues or breached surfaces.The guinea pig maximization test should be used for thesesubstances.1.3 This practice consists of a protocol for assessing anincrease in lymphocyte proliferation within the nodes drainingthe site of administration on the ear
6、s of mice.1.4 The LLNA has been validated only for low molecularweight chemicals that can penetrate the skin. The absorbedchemical or metabolite must bind to macromolecules, such asproteins, to form immunogenic conjugates.1.5 This practice is one of several developed for theassessment of the biocomp
7、atibility of materials. Practice F 748may 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 convenience of the user and does not imply single source.Appropriate materials and r
8、eagents may be obtained frommany commercial supply houses.1.7 This standard does not purport to address 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-bil
9、ity of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F 619 Practice for Extraction of Medical PlasticsF 720 Practice for Testing Guinea Pigs for Contact Aller-gens: Guinea Pig Maximization TestF 748 Practice for Selecting Generic Biological Test Meth-ods for Material
10、s and DevicesF 750 Practice for Evaluating Material Extracts by SystemicInjection in the Mouse2.2 Other Document:ICCVAM NIH Publication No: 99-4494 The Murine LocalLymph Node Assay, 199933. Terminology3.1 Definitions:3.1.1 AOO, nacetone olive oil solution (4:1 v/v) is asuitable nonpolar solvent.3.1.
11、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 % formalde-hyde solution (formaldehyde) in PBS.3.1.6 ICCVAM, nInteragency Coordin
12、ating Committee 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 c
13、onsis-tently stimulating lymphocyte proliferation.3.1.10 saline, n0.9 % sodium chloride (aqueous, polarsolvent).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 M
14、ethods.Current edition approved Feb. 1, 2007. Published February 2007. Originallyapproved in 2001. Last previous edition approved in 2006 as F 2148 06a.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMSta
15、ndards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from NICEATM, NIEHS, 79 Alexander Dr., Mail Drop EC-17,Research Triangle Park, NC 27709.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United St
16、ates.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 solvent and anon-aqueous, nonpolar solvent.4. Summary of Practice4.1 Test and control substances or e
17、xtracts 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 identicalconditions.5. Significance and Use5.1 The propensity of a material to stimulate delayedcontact hype
18、rsensitivity 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 complex set of reactions andconsequences depending on the actual tissue/organ site ofreaction. Althoug
19、h 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 The standard historical test methods have involved theuse of guinea pigs with a cutaneous applicat
20、ion 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-mine dose responses.5.3 This practice may not be predictive of events occurringduring all types of im
21、plant 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 F 748.6. Preparation of Test Specimens6.1 Specimens should be prepared in accordance with Prac-tice
22、F 619. 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 gels shall be used directly if theyare not an irritant. A liquid that is an irritant shall be dilut
23、edwith an aqueous or nonaqueous solvent based on solubility ofthe liquid test article until the solution is nonirritating.6.3 Wholly aqueous solutions are not suitable for applica-tion to the ear. Therefore, for use in the assay, add 0.05 g ofhydroxyethyl cellulose4to each 10 mL of the aqueous vehic
24、lecontrol and test solutions to aid in holding the solution to theear.6.4 The final specimen to be extracted should be preparedwith a surface 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 th
25、e specimens do not becomecontaminated during preparation and aseptic technique isrecommended.7. Preparation of Positive Controls7.1 Nonaqueous Positive ControlWeigh 0.025 g ofDNCB and place in a flask. Add enough DMSO to dissolve allof the DNCB.Add more DMSO to bring the level up to 10 mL.Cap and sh
26、ake the flask until a homogeneous solution isobtained. The dose level of the positive control should notproduce 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 for
27、maldehyde in a 10-mL flask. Add enoughPBS to mix the two solutions. Add more PBS to bring the levelup to 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 hydroxyeth
28、ylcellulose4to each 10 mL of the aqueous positive control to aidin holding the solution to the ear until absorbed.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 document) f
29、ollowing the procedures described inPractice F 619.8. 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.8.2 Day OneUniquely identify each mouse (ear ta
30、gs orear notches may not be used). Weigh each mouse to the nearestwhole gram.8.3 A minimum of five mice shall be used for each 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
31、surface of both ears. Forthe aqueous groups only, the dorsal surface should be wipedwith acetone just before treating to aid in 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 nona
32、queous positive controls,aqueous and nonaqueous vehicle controls, aqueous extract oftest sample, and nonaqueous extract of test sample.8.3.2 For testing of liquid test articles, the groups shallinclude: aqueous and nonaqueous positive controls, liquid testsample, and either an aqueous or a nonaqueou
33、s vehicle controlappropriate for the nature of the liquid sample.8.3.3 The extract shall be used within 24 h of preparation.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
34、for eachday of the test.8.3.4 Observe each mouse daily for signs of local irritationat the application site and for signs of systemic toxicity (see4“Final Report on the Safety Assessment of Hydroxyethylcellulose, Hydrox-ypropylcellulose, Methylcellulose, Hydroxypropyl Methylcellulose, and CelluloseG
35、um,” J. Amer Coll Tox., Vol 5, No. 3, 1986, pp. 1-59.F214807e12Practices F 720 and F 750). 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
36、needs to be prepared toaccommodate this.8.4 Radiolabeled Tracer Preparation Prepare tritiatedthymidine to a working concentration of 80 Ci/mL (v/v). Theuse of I125I-UDR at 8 Ci/mL in PBS 10-5M fluorodeoxyuri-dine is also acceptable. Each mouse will receive 250 L of this.All standard precautions asso
37、ciated with using radioactivematerials must be adhered to. The laboratory must be licensedto use radioactive material and all personnel must be appro-priately trained 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 C
38、i/mM) to a stoppered flask.Add sterile 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 scintill
39、ation fluid toeach vial, mix so that 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 dpmF
40、or verification of the working tritiated 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 dpm71 040 dpm3 80 Ci/mLMake adju
41、stments to the solution as needed. Make 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
42、tritiated thymidine or 2 Ci I125IUDR 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
43、solutions from this point should be treatedas radioactive with appropriate precautions.NOTE 3If the investigators are not familiar with the location of thelymph nodes, refer to the diagram in the ICCVAM document, consult amouse anatomy book, or use a dye in trial mice to learn to locate theappropria
44、te nodes. One suggested procedure is 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 (auric
45、ular) lymph node of each ear.9.3 Prepare 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
46、 or transfer the lymph nodes directly 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
47、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 byvortexing. Beginning with the
48、centrifugation step identifiedimmediately above, in this subsection, repeat this washingprocedure two more times. After the final wash, remove thesupernatant above each pellet and resuspend in 3 mL cold 5 %TCA. Allow to precipitate at 2 to 8C for 18 6 1h.9.4 Measurement of Radioactivity (Tritiated T
49、hymidine):9.4.1 Centrifuge the precipitated cell suspension at 190 to200 xg for 10 min at 2 to 8C. Remove the supernatant aboveeach pellet and add 1-mL fresh 5 % TCA to the pellet and mixso that a vortex is formed to resuspend.9.4.2 Transfer the suspension to a labeled scintillation vialcontaining 10 mL of scintillation fluid, cap, and mix well byshaking vigorously.9.4.3 Prepare two reagent blanks to measure backgroundtritium levels. Add 10-mL of scintillation fluid and 1-mL 5 %TCA to the scintillation vial and mix well.9.4.4 Wipe the outside o
