1、Designation: F 2147 01 (Reapproved 2006)Standard Practice forGuinea Pig: Split Adjuvant and Closed Patch Testing forContact Allergens1This standard is issued under the fixed designation F 2147; 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 (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice is intended to determine the potential for asubstance, or material extract, to e
3、licit contact dermal allerge-nicity.1.2 This practice is intended as an alternative to the GuineaPig Maximization Test (GPMT), given the limitations ondosage form and tendency for false positives associated withthe latter test. See Rationale and References.1.3 This standard does not purport to addre
4、ss 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:2F 619 Practice
5、for Extraction of Medical PlasticsF 720 Practice for Testing Guinea Pigs for Contact Aller-gens: Guinea Pig Maximization Test2.2 ISO Document:ISO 10993-10, 1995 Tests for Irritation and Sensitization33. Terminology3.1 Definitions:3.1.1 2,4 dinitrochlorobenzene (DNCB)strong sensitizer,used as a posit
6、ive control.3.1.2 Freunds Complete Adjuvant (FCA)acommercially-available mixture of oil and Mycobacterium thatis known to elicit an immune response.3.1.3 Guinea Pig Maximization Test (GPMT)proceduredescribed in Practice F 720 accepted as a “worst case” assayfor allergenic potential.4. Summary of Pra
7、ctice4.1 The split adjuvant method is used when topical appli-cation is considered relevant, and the dosage form is a solid,liquid, extract, paste, or gel. The method includes four induc-tion doses applied over ten days to the same shaved ordepilated site on guinea pigs, followed by occlusive patchi
8、ng.Freunds Complete Adjuvant (FCA) is injected near the dosesite on the fourth day (second induction dose). Following a restperiod, animals are challenged at a previously unexposed site,and the reaction evaluated at 24, 48, and 72 h.4.2 The closed patch method is used when topical applica-tion is re
9、levant, but the preferred dosage form does not permitinjection under the skin or intradermally, and the discomfortinvolved with extended occlusive patching and adjuvant use isto be avoided. It involves repeated induction doses (3 to 6) over14 days at the same shaved/depilated site, followed each tim
10、eby6hofocclusive wrapping. After a rest period, animals arechallenged at previously untreated sites, and their reactionsevaluated at least 24 and 48 h later.5. Significance and Use5.1 In selecting a material for human contact in medicalapplications, it is important to ensure the material will notsti
11、mulate the immune system to produce an allergic reactionunder relevant exposure conditions. Extractable chemicalsproduced by skin contact or during physiological exposuresmay cause allergic reactions. Therefore, this practice providesfor evaluations of solid or semisolid dosage forms usingmaterial e
12、xtracts or direct evaluation of the test article. Therationale for this animal model is based on the fact that theguinea pig has been shown to be an appropriate animal modelfor predicting human contact dermatitis; its tractable nature, itsavailability from reputable suppliers, the historical databas
13、e ofinformation already acquired using this species, and the corre-lation of such results to data on known human allergens, allcontribute to its widespread use for allergenicity studies (1-5).45.2 The need for sensitization procedures other than themaximization test (Practice F 720) is based on: (1)
14、 the need fora route of exposure more similar to use conditions, (2) concern1This 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 Marc
15、h 1, 2006. Published April 2006. Originallyapproved in 2001. Last previous edition approved in 2001 as F 2147 01.2For 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
16、 standards Document Summary page onthe ASTM website.3Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036.4The boldface numbers in parentheses refer to the list of references at the end ofthis standard.1Copyright ASTM International, 100 Barr Harbo
17、r Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.over the use of adjuvant because of its recruitment of cell typesto the test site which are not typically involved in immunologicreactions, and because of the discomfort this causes in theanimals, (3) absence of a proper FCA-irrit
18、ant control group inthe traditional maximization design, and (4) the frequency offalse positives often encountered with the GPMT. Both of thesetests are internationally accepted (1).6. Materials and Manufacturers6.1 Hartley strain guinea pigs, either sex (but all in the testof the same sex), 300 to
19、500 g at start of test, should be fromthe same shipment, same supplier, and should be healthy.6.2 At least ten animals are used for each test material andfive for each control group.6.3 Freunds Complete Adjuvant (FCA) (split adjuvant testonly).6.4 Cotton gauze and occlusive bandage (examples, Elasto
20、-pore from 3M) or Hilltop chambers (Hilltop, Cincinnati, OH)(optional for solid samples) and Vet wrap.6.5 Positive control substance (0.1 to 1 % 2,4 DNCB is astrong sensitizer; to test method sensitivity, it may be advisableto use cinnamaldehyde (10 % induction, 1 % challenge) as apositive control (
21、2).7. Preparation of Test SamplesNOTE 1All steps are applicable to both methods.7.1 Solid SamplesCut flat sheet-like samples into 1- by1-cm squares. These can be used for direct contact testing aslong as the sample thickness does not exceed 1.0 mm.NOTE 2Pressure exerted by bandaging thick samples ca
22、uses mechani-cal irritation. The cotton pad may be removed from the Hilltop chamber(or the chamber need not be used) to reduce pressure on thick solid testarticles. Further cutting should be considered if test articles are stillcausing pressure without the chamber or chamber pad.7.2 Gels, Pastes, Oi
23、ntmentsSemisolid test articles can beused directly, applied at 0.2 mL/site.7.3 ExtractsPrepare extracts in accordance with PracticeF 619, at the highest temperature tolerated by the materialwithout physical melting or decomposition. Both aqueous andnonaqueous extracts are recommended. Extracts shoul
24、d bedecanted upon cooling, stored at room temperature (22 to30C), and used within 24 h. Extracts should be prepared freshfor each treatment, preferably using a solvent which does notgive background reactions (ethanol is sometimes a problem inthis regard), and is known to produce measurable extractab
25、les(determined by a technique such as a nonvolatile residue test)without dissolving the test article.7.4 Negative ControlsPrepare solvent sham controls(“blanks”) under the same conditions as test article extracts.Saline controls may be eliminated if there are sufficient dataavailable to predict thei
26、r results.7.5 Positive ControlsPositive controls should be preparedfresh before induction in the same solvent used for extractionif possible. If the solvent is volatile, a fresh solution may beneeded for challenge. The use of amber bottles with minimumheadspace should also be considered. Alternative
27、ly, positivecontrol testing may be performed quarterly or at anotherreasonable frequency if the laboratory performs significantnumbers of these tests and results are consistent. The latterpractice reduces animal usage.8. Trial and Naive Challenge Tests8.1 It is recommended that at least two guinea p
28、igs be usedto assess the ability of the test article or undiluted extract toirritate. Each flank of each animal can be used to patch twosites (upper and lower) of samples such as test article, 100 %extract, 75 % extract, and 50 % extract. Animals should beshaved and wrapped as in the complete test (
29、see Section 9), andthe sites evaluated after 24 to 72 h. Scoring should also beperformed as in the complete test.8.2 It is also advisable to determine the difference betweenirritation and sensitization under full test conditions for thepositive control by including in at least one test per laborator
30、ya “naive challenge” group which is exposed to controls only forthe challenge period. DNCB, for example, can be an irritant,and it is important that erythema and edema reactions seenafter challenge be true sensitization responses.9. ProcedureNOTE 3This procedure is applicable to both methods except
31、as noted.9.1 Table 1 shows the timing of animal preparation, induc-tion dosing, challenge, and evaluation.9.2 Animal Preparation:9.2.1 Weigh and shave or depilate animals within 24 h oftest start. Depilatories should be used carefully and testedbeforehand to understand proper use regimen so as not t
32、oproduce background irritation. Shave or depilate a site on theleft flank or shoulder area (use one or the other consistently)approximately a 2-in. square to expose bare skin, avoiding anyabrasions or other abnormalities. Check animal health dailythroughout the test.9.2.2 Apply 0.3 mL of extract or
33、semisolid (or less, if theamount has been validated, or 1 cm2of a solid sample (lessthan 1.0 mm thick) to the cotton pad of a Hilltop chamber. (Apadless chamber can be used to dose gels or thicker samples).Stick the chamber to the skin and wrap with an appropriateelastic bandage. If a Hilltop chambe
34、r is not used, apply the testsample to gauze and cover with occlusive wrap. Follow theunwrap/evaluate schedule for the particular procedure as inTable 1.9.2.3 After unwrapping, wait about 30 min before evalua-tion. The test article may be removed by gentle wiping withgauze soaked with purified water
35、 or isopropyl alcohol (IPA)that has been diluted such that it will not dry the skin. Evaluatethe site using the criteria in Table 2. Rewrap if required (splitadjuvant.)9.2.4 Repeat doses as outlined in Table 1. At the seconddose of the split adjuvant procedure, inject 0.05 mL of FCAemulsified 1:1 wi
36、th water for injection at four locationsbordering every test and control site (0.2 mL total).9.2.5 At the end of the induction period, allow the animalsto rest unwrapped for 10 to 14 days.9.2.6 Challenge using the same procedures as for induction,but at a site on the right shaved flank/shoulder.F 21
37、47 01 (2006)29.2.7 Unwrap and evaluate as described in Table 2.Itisrecommended the reader be experienced, and unfamiliar withthe site treatment during reading.10. Interpretation and Results10.1 At same-day post-challenge, all of the positive controlanimals must have scores $1 (one level above the hi
38、ghestnegative control score), or at least 60 % of these animals musthave scores $2 (at least one level higher than the highestnegative control score). A majority of the negative controlgroup should have scores of 0, and no score should be above 1.10.2 Response frequency is calculated by dividing the
39、number of animals in each group with a positive response(scores at least one level higher than the highest negativecontrol score) by the total number of animals treated in thatgroup.10.3 For a material to be considered a sensitizer, a majorityof the animals in a treatment group (50 %) must be consid
40、-ered sensitized. The level and frequency of scores determinethe degree of the sensitization, with frequency being the moreimportant. A low frequency of high scores is unusual and maysuggest a retest or another type of evaluation/investigation isneeded. A high frequency of low scores may also requir
41、e areassay for clarification. Classification of materials by assayresults is not provided, as it is up to the device manufacturer todetermine the acceptability of test results.10.4 If there is any question about the frequency, relevance,or reproducibility of scores, rechallenge the questionable grou
42、p(along with appropriate controls) at new sites seven to ninedays after the last challenge observation.TABLE 1 Timing of Animal Preparation, Induction Dosing, Challenge, and EvaluationDay(s) of Study Test Dose(s)ActivityModified Split Adjuvant Closed Patch1 NA randomize/shave randomize/shave1 0.3 mL
43、 of liquid or a 1-cm2solidpiece (thickness 1 mm)apply dose to upper flank; bandageocclusivelyapply dose to upper flank; bandageocclusively for 6 h, then evaluate3 0.3 mL of liquid or a 1-cm2solidpiece (thickness 1 mm)unwrapA; evaluate after stabilizationperiod (30 min). apply new samplesNA5 0.3 mL o
44、f liquid or a 1-cm2solidpiece (thickness 1 mm)unwrap and evaluate. apply samples and wrap;inject 1:1 FCA in water around test sites(0.05 mL per injection; 0.2-mL total)NA78 0.3 mL of liquid or a 1-cm2solidpiece (thickness 1 mm)unwrap; evaluate. apply samples apply samples; wrap occlusively for 6 h,t
45、hen unwrap and evaluate910 0.3 mL of liquid or a 1-cm2solidpiece (thickness 1 mm)unwrap; evaluate NA923 or1024NA rest period NA14 0.3 mL of liquid or a 1-cm2solidpiece (thickness 1 mm)NA apply samples; wrap for 6 h, then unwrapand evaluate1428 0.3 mL of liquid or a 1-cm2solidpiece (thickness 1 mm)NA
46、 rest period23 or 24 0.3 mL of liquid or a 1-cm2solidpiece (thickness 1 mm)Shave new site on opposite upper flank 2 hbefore treatment; apply sample; wrapNA24/25 NA unwrap; evaluate NA25/26 NA evaluate NA26/27 NA evaluate NA28 0.3 mL of liquid or a 1-cm2solidpiece (thickness 1 mm)evaluate; do not red
47、ose shave new site on opposite flank 2 h beforetreatment; apply sample, wrap for 6 h29 NA NA evaluate30 NA NA evaluate31 NA NA evaluateAWrapping of a shorter duration may be used if validated.TABLE 2 Evaluation CriteriaErythema and Eschar Formation ValueNo erythema 0Very slight erythema (barely perc
48、eptible and patchy) 1Well-defined erythema (slight but confluent, or moderate patchy) 2Moderate to severe erythema 3Severe erythema (beet redness) to slight eschar formation (injuriesin depth)4Necrosis NScab SEdema Formation ValueNo edema 0Very slight edema (barely perceptible) 1Slight edema (edges
49、of area well-defined by definite raising) 2Moderate edema (raised approximately 1 mm) 3Severe edema (raised more than 1 mm and extending beyond thearea of exposure)4F 2147 01 (2006)311. Report11.1 Report the following information:11.1.1 Test and control material descriptions, genericnames, product names, manufacturers names and addresses,and lot numbers,11.1.2 Method of preparation of each extract,11.1.3 General conditions of animal health,11.1.4 Scoring of erythema and edema for each animal ateach scoring period (see Tables 1 and 2).11.1.5 Overall assessm
