1、Designation: F2148 07 (Reapproved 2012)Standard 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
2、or, in the case of 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 evalu
3、ation 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 reac-tions. This alternative is particularly applicable for materialsu
4、sed 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 skin but areused in devices that contact deep tissues or breached s
5、urfaces.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 ears of mice.1.4 The LLNA has been validated only for low-molecular-wei
6、ght 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 biocompatibility of materials. Practice F748may provide guidance for the s
7、election 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 a single source.Appropriate materials and reagents may be obtained frommany commercial supply houses.1.7 The
8、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 address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate
9、 safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F619 Practice for Extraction of Medical PlasticsF720 Practice for Testing Guinea Pigs for ContactAllergens:Guinea Pig Maximization TestF748 Practice for Sel
10、ecting Generic Biological Test Methodsfor Materials and DevicesF750 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 solu
11、tion (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 % formalde-hyde solution (formal
12、dehyde) in PBS.3.1.6 ICCVAM, nInteragency Coordinating 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).1This practice is under the jurisdiction ofAST
13、M Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Oct. 1, 2012. Published October 2012. Originallyapproved in 2001. Last previous edition approved in 2007 as F2148 071. DOI:10.
14、1520/F2148-07R12.2For 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 standards Document Summary page onthe ASTM website.3Available from NICEATM, NIEHS, 79 Alexander
15、 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 States13.1.8 PBS, nphosphate buffered saline, pH 7.2.3.1.9 positive control, na substance capable of consis-tently stimulating lymphocyte
16、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 solvent and anon-aqueous, nonp
17、olar 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 identicalconditions.5. Significance a
18、nd 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 complex set of reactions andcon
19、sequences 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 The standard historical tes
20、t 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-mine dose responses.5.3 This p
21、ractice 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 F748.6. Preparation of Test S
22、pecimens6.1 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 gels shall be used directly if
23、 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. Therefore, for use in the assay, ad
24、d 0.05 g ofhydroxyethyl cellulose4to each 10 mL of the aqueous vehiclecontrol 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 met
25、hod 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 Nonaqueous Positive ControlWeigh 0.025 g of DNCBand place in a flask. Add enough DMSO to dissolve al
26、l of theDNCB. Add more DMSO to bring the level up to 10 mL. Capand shake the flask until a homogeneous solution is obtained.The dose level of the positive control should not producesystemic toxicity as evidenced by clinical observations.7.2 Aqueous Positive ControlNeutral buffered formalin iscommerc
27、ially 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 10 mL. Cap and shake the flask until a homogeneoussolution is obtained.)7.3 Aqueous solutions are not suitable for application
28、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 absorbed.7.4 For all specimens requiring extractions, prepare anaqueous and non-aqueous extract (DMSO or AOO are recom-mended b
29、ut other permissible extractants are listed in theICCVAM document) following the procedures described inPractice 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 f
30、ive animals percage.8.2 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 positiveand negative control and each test sample. They shall be treateddaily for three consecutive days b
31、y 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 absorption of theaqueous solution, although it will not be completely absorbed.4“Final Report on the Safe
32、ty Assessment of Hydroxyethylcellulose,Hydroxypropylcellulose, Methylcellulose, Hydroxypropyl Methylcellulose, andCellulose Gum,” J. Amer Coll Tox., Vol 5, No. 3, 1986, pp. 1-59.F2148 07 (2012)28.3.1 For testing, other than liquid test articles, the groupsshall include: aqueous and nonaqueous positi
33、ve controls,aqueous and nonaqueous vehicle controls, aqueous extract ofthe test sample, and nonaqueous extract of 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 veh
34、iclecontrol appropriate 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 for e
35、achday of the test.8.3.4 Observe each mouse daily for signs of local irritationat the application site and for signs 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
36、.3 until precipitation for 18 htake more than8htocomplete and the laboratory needs to be prepared toaccommodate this.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 acc
37、eptable. Each mouse will receive 250 L of this. Allstandard precautions associated with using radioactive materi-als 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
38、 solution, add 0.8 mLof 1.0-mCi/mL tritiated thymidine (specific activity 2.0 Ci/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.
39、 Remove two 1-mL samples andplace in scintillation vials. Add 10 mL of scintillation 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
40、disintegrations per minute (dpm) as follows:cpmdecimal counter efficiency5 dpmFor 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. T
41、here-fore:the Ci of the working solution 5mean dpm71040 dpm380 Ci/mLMake adjustments 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 gra
42、m.Inject the mouse intravenously with 250-L sterile PBS con-taining 20 Ci of 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.
43、 Lymph Node Collection and Lymph Node CellPreparationNOTE 2All equipment and 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 am
44、ouse anatomy book, or use a dye in trial mice to learn to locate theappropriate 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
45、 h 6 45 min after injection of theradioisotope.9.2 Excise the draining (auricular) 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 o
46、fPBS. Snip the nodes from a single mouse into small pieceswith small scissors 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 d
47、ebrisinto 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 t
48、hen bring up to 10 mL with PBS and resuspend byvortexing. Beginning with the 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 pre
49、cipitate at 2 to 8C for 18 6 1h.9.4 Measurement of Radioactivity (Tritiated Thymidine):9.4.1 Centrifuge the precipitated cell suspension at 190 to200 xg for 10 min at 2 to 8C. Remove the supernatant aboveTABLE 1 LLNA TimetableDay Activity1 Prepare extracts to be 24 h old on Day 1 and plan forsuitableextracts for Days 2 and 31 Weigh mouse, mark, and treat ears with samples2 Observe mice and record any toxicity or irritation, treat ears3 Observe mice and record any toxicity or irritation, treat ears4 Observe mice and record any toxicity or irri
