ASTM F2148-2018 Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA).pdf

1、Designation: F2148 18Standard 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 a combi-nation of in vivio and in situ procedures f

3、or the evaluation ofdelayed contact hypersensitivity 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

4、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 skin but areused in devices that contact deep tissues or

5、breached surfaces.This practice 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 assessin

6、g anincrease in lymphocyte proliferation in the lymph nodesdraining the site of test article administration on the ears ofmice.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 asp

7、roteins, 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 selection of appropriate methodsfor testing materials for a specific application.1.6 Identification of a supplier of

8、 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 values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard

9、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 safety, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to us

10、e.1.9 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers

11、to Trade (TBT) Committee.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 Selecting Generic Biological Test Methodsfor Materials and DevicesF750 Practice

12、 for Evaluating Material Extracts by SystemicInjection in the Mouse2.2 Other Documents:3ICCVAM NIH Publication No: 99-4494 The Murine LocalLymph Node Assay, 1999ICCVAM NIH Publication No: 10-7512 Test Method Evalu-ation Report on Using the Murine Local Lymph NodeAssay for Testing Pesticide Formulati

13、ons, Metals, Sub-stances in Aqueous Solutions, and Other Products, 2010ICCVAM NIH Publication NO: 11-7709 Usefulness andLimitations of the Murine Local Lymph Node Assay forPotency Categorization of Chemicals Causing AllergicContact Dermatitis in Humans1This practice is under the jurisdiction ofASTM

14、Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Dec. 1, 2018. Published February 2019. Originallyapproved in 2001. Last previous edition approved in 2013 as F2148 13. DOI:10.15

15、20/F2148-18.2For 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 Dr.,

16、 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 StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the

17、Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.13. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 AOO, nacetone olive oil solution (4:1 v/v) is as

18、uitable 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 (formaldehyde) in PBS.3.1.6

19、 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).3.1.8 PBS, nphosphate buffered saline, pH 7.2.3.1.9 positive contr

20、ol, na substance capable of consis-tently 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 ve

21、hicle controls, nan aqueous, polar solvent 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

22、test specimens tested under identicalconditions.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 d

23、elayedhypersensitivity may have a complex 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

24、 to causedelayed hypersensitivity.5.2 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 eva

25、luation and could be used to deter-mine 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 there

26、commendations contained in Practice F748.6. 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 o

27、r AOO.6.2 Liquid test articles and gels 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

28、 for applica-tion to the ear. Therefore, 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 aqueousvehicle.6.4 The final specimen to be extr

29、acted should be preparedwith a surface finish consistent with its 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. Pre

30、paration of Positive Controls7.1 Nonaqueous 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) so

31、lvent. Shake the flask until a homogenous 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 dos

32、e 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 formaldehyde in a 10-mL flask. Add enoughPBS to mix the two solut

33、ions.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 hydroxyethylcellulose4to each 10 mL of the aqueous positive control to ai

34、din holding the solution to the ear until absorbed. One percentPluronic L92 may also be used as an aqueous vehicle.4“Final Report on the Safety Assessment of Hydroxyethylcellulose,Hydroxypropylcellulose, Methylcellulose, Hydroxypropyl Methylcellulose, andCellulose Gum,” J. Amer Coll Tox., Vol 5, No.

35、 3, 1986, pp. 1-59.F2148 1827.4 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 inPractice F619.8. Dosing of the Animals8.1 Healthy

36、 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 tags or earnotches may not be used). Weigh each mouse to the nearestwhole gram.8.3 A min

37、imum 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 surface of both ears. Forthe aqueous groups only, the dorsal surface should be wipedwi

38、th 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 nonaqueous positive controls,aqueous and nonaqueous vehicle controls, aqueous extract ofth

39、e 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 vehiclecontrol appropriate for the nature of the liquid sample.8.3.3 The ext

40、ract 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 eachday of the test.8.3.4 Observe each mouse daily for signs of local irri

41、tationat 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 9.3.3 until precipitation for 18 htake more than8htocomplete and the laborat

42、ory needs to be prepared toaccommodate this.8.4 Radiolabeled Tracer PreparationPrepare tritiated thy-midine to a working approximate concentration of 80 Ci/mL(v/v). The use of I125I-UDR at 8 Ci/mL in PBS 10-5Mfluorodeoxyuridine is also acceptable. Each mouse will receive250 L radiolabeled tracer sol

43、ution intravenously. All standardprecautions associated with using radioactive materials shall beadhered to. The laboratory shall be licensed to use radioactivematerial and all personnel shall be appropriately trained andcertified.8.4.1 To prepare the tritiated thymidine solution, add 0.8 mLof 1.0-m

44、Ci/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 0.08mL to 200 mL with water using a 200-mL flask. Cap and mixwell by inverting several times. Remove two 1-mL samplesan

45、d place in scintillation vials. Add 10 mL of scintillation fluidto each vial, mix so that a vortex is formed, and “count” in abeta scintillation counter. Count each vial three times andcalculate the mean. Calculate the concentration. First convertcounts per minute (cpm) to disintegrations per minute

46、 (dpm) asfollows: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. There-fore:the Ci of the wor

47、king 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 gram.Inject the mouse intraven

48、ously 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 gauge. The tail veins may be dilated for easierintravenous injection by placing the mice under a heat lamp.Care should be taken with th

49、e heat lamps to prevent unneces-sary pain and distress in animals resulting from overheating.9. 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 documents (Annex, I.ICCVAM-Recommended Test Method Protocol: The Murine LocalLymph Node Assay: 2-Bromodeoxyuridine-ELISA Test Method (LLNA:BrdU-ELISA), a Nonradioactive Alterna