1、Designation: F2147 01 (Reapproved 2016)Standard Practice forGuinea Pig: Split Adjuvant and Closed Patch Testing forContact Allergens1This standard is issued under the fixed designation F2147; the number immediately following the designation indicates the year oforiginal adoption or, in the case of r
2、evision, 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 is intended to determine the potential for asubstance, or material extract, to elic
3、it 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 The values stated in SI units are to be re
4、garded asstandard. No other units of measurement are included in thisstandard.1.4 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 deter
5、mine 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 Test2.2 ISO Document:ISO 10993-10, 1995 Tests for Irritation an
6、d Sensitization33. Terminology3.1 Definitions:3.1.1 2,4 dinitrochlorobenzene (DNCB)strong sensitizer,used as a positive control.3.1.2 Freunds Complete Adjuvant (FCA)a commercially-available mixture of oil and Mycobacterium that is known toelicit an immune response.3.1.3 Guinea Pig Maximization Test
7、(GPMT)proceduredescribed in Practice F720 accepted as a “worst case” assay forallergenic potential.4. Summary of Practice4.1 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 ind
8、uc-tion doses applied over ten days to the same shaved ordepilated site on guinea pigs, followed by occlusive patching.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 sit
9、e,and the reaction evaluated at 24, 48, and 72 h.4.2 The closed patch method is used when topical applica-tion is relevant, 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 avo
10、ided. It involves repeated induction doses (3 to 6) over14 days at the same shaved/depilated site, followed each timeby6hofocclusive 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
11、In selecting a material for human contact in medicalapplications, it is important to ensure that the material will notstimulate the immune system to produce an allergic reactionunder relevant exposure conditions. Extractable chemicalsproduced by skin contact or during physiological exposuresmay caus
12、e allergic reactions. Therefore, this practice providesfor evaluations of solid or semisolid dosage forms usingmaterial extracts 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 pr
13、edicting human contact dermatitis. Its tractable nature, itsavailability from reputable suppliers, the historical database of1This practice is under the jurisdiction ofASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompati
14、bility Test Methods.Current edition approved April 1, 2016. Published June 2016. Originallyapproved in 2001. Last previous edition approved in 2010 as F2147 01 (2010).DOI: 10.1520/F2147-01R16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at ser
15、viceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.Copyright ASTM International, 100 Barr Har
16、bor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1information 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 procedur
17、es other than themaximization test (Practice F720) is based on: (1) the need fora route of exposure more similar to use conditions; (2) concernover the use of adjuvant because of its recruitment of cell typesto the test site which are not typically involved in immunologicreactions, and because of th
18、e discomfort this causes in theanimals; (3) absence of a proper FCA-irritant 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 str
19、ain guinea pigs, either sex (but all in the testof the same sex), 300 to 500 g at the start of the test, should befrom the same shipment and 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) (sp
20、lit adjuvant testonly).6.4 Cotton gauze and occlusive bandage (examples, Elasto-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 advisa
21、bleto use cinnamaldehyde (10 % induction, 1 % challenge) as apositive control. (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 thickn
22、ess does not exceed 1.0 mm.NOTE 2Pressure exerted by bandaging thick samples causes 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 ar
23、e stillcausing pressure without the chamber or chamber pad.7.2 Gels, Pastes, OintmentsSemisolid test articles can beused directly, applied at 0.2 mL/site.7.3 ExtractsPrepare extracts in accordance with PracticeF619, at the highest temperature tolerated by the materialwithout physical melting or deco
24、mposition. Both aqueous andnonaqueous extracts are recommended. Extracts should 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 so
25、metimes a problem inthis regard), and is known to produce measurable extractables(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 co
26、ntrols may be eliminated if sufficient data to predicttheir results are available.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 am
27、ber bottles with minimumheadspace should also be considered. Alternatively, 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 a
28、nd Naive Challenge Tests8.1 It is recommended that at least two guinea pigs 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 the test article,100 % extract, 75 % extract, and
29、 50 % extract. Animals shouldbe shaved and wrapped as in the complete test (see Section 9),and the 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 condit
30、ions for thepositive control by including in at least one test per laboratorya “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 resp
31、onses.9. ProcedureNOTE 3This procedure is applicable to both methods except 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
32、 carefully and testedbeforehand to understand proper use regimen so as not toproduce 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. Ch
33、eck animal health dailythroughout the test.9.2.2 Apply 0.3 mL of extract or 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
34、to the skin and wrap with an appropriateelastic bandage. If a Hilltop chamber 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
35、article may be removed by gentle wiping withgauze soaked with purified water or isopropyl alcohol (IPA)4The boldface numbers in parentheses refer to the list of references at the end ofthis standard.F2147 01 (2016)2that has been diluted such that it will not dry the skin. Evaluatethe site using the
36、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 with water for injection at four locationsbordering every test and control site (0.2 mL total).9.2.5 At the end of
37、 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.9.2.7 Unwrap and evaluate as described in Table 2.Itisrecommended that the reader be experienced, and unfamiliarwith
38、 the site treatment during reading.10. Interpretation and Results10.1 On the same day post-challenge all of the positivecontrol animals shall have scores 1 (one level above thehighest negative control score), or at least 60 % of theseanimals must have scores 2 (at least one level higher than thehigh
39、est negative control score). A majority of the negativecontrol group should have scores of 0, and no score should beabove 1.10.2 Response frequency is calculated by dividing thenumber of animals in each group with a positive responseTABLE 1 Timing of Animal Preparation, Induction Dosing, Challenge,
40、and EvaluationDay(s) of Study Test Dose(s)ActivityModified Split Adjuvant Closed Patch1 NA randomize/shave randomize/shave1 0.3 mL of liquid or a 1-cm2solidpiece (thickness 50 %) of the animals in a treatment group must be consid-ered sensitized. The level and frequency of scores determinethe degree
41、 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 require areassay for clarification. Classification of materials by assayresul
42、ts 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 group(along with appropriate controls) at new sites seven to ninedays after
43、 the last challenge observation.11. 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
44、 Scoring of erythema and edema for each animal ateach scoring period (see Tables 1 and 2);11.1.5 Overall assessment of sensitization response.12. Precision and Bias12.1 Precision and bias for this practice has not beendetermined because the required studies would be time-consuming, expensive, and an
45、 inappropriate use of animals.13. Keywords13.1 acute toxicity; allergenicity; biocompatibility; guineapigs; immunotoxicity; sensitizationAPPENDIX(Nonmandatory Information)X1. RATIONALEX1.1 The maximization test is a “worst case” evaluation ofpotential allergenicity under exposure and physiological c
46、on-ditions which do not mimic most medical applications. Mate-rials are injected into the skin, and therefore, dosage forms arelimited to liquids, or extracts prepared in physiologicallyacceptable solvents, for example, saline, which often is a poorpolymer solvent. The use of Freunds Adjuvant, altho
47、ughincluded here for a modified split adjuvant test, can becriticized as recruiting types of immune cells to the exposuresite that would not be involved under typical exposure condi-tions. The FCA and extensive wrapping can cause significantdiscomfort to the animals; FCA use requires the animals ber
48、eported in the U.S. Department of Agriculture Category E forpain and suffering.X1.2 The original GPMT design has been criticized by itsown originators (6) as lacking a necessary control group, andcareful interpretation is required because of its higher fre-quency of false positives. Although the clo
49、sed patch techniquehas been criticized by some, it is accepted internationally (1),and can be tested using cinamaldehyde as a positive control.The 5/6/99 FDA immunotoxicity guidance allows for GPMTand other sensitization test procedures, as long as the choice ofprocedure can be justified.X1.3 Table X1.1 has been provided to compare the variousaspects of the methods here, along with the GPMT. See detailsof procedures in Tables 1 and 2.F2147 01 (2016)4REFERENCES(1) Immunotoxicity Testing Guidance 5/6/99 http:/www.fda.gov/cdrh/ost/ostggp/immunotox.html.(2) Ritz,
