ASTM F2147-01

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Designation:F2147–01
Standard Practice for
Guinea Pig: Split Adjuvant and Closed Patch Testing for
Contact Allergens
This standard is issued under the fixed designation F 2147; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope liquid, extract, paste, or gel. The method includes four induc-
tion doses applied over ten days to the same shaved or
1.1 This practice is intended to determine the potential for a
depilated site on guinea pigs, followed by occlusive patching.
substance, or material extract, to elicit contact dermal allerge-
Freund’s Complete Adjuvant (FCA) is injected near the dose
nicity.
site on the fourth day (second induction dose). Following a rest
1.2 This practice is intended as an alternative to the Guinea
period, animals are challenged at a previously unexposed site,
Pig Maximization Test (GPMT), given the limitations on
and the reaction evaluated at 24, 48, and 72 h.
dosage form and tendency for false positives associated with
4.2 The closed patch method is used when topical applica-
the latter test. See Rationale and References.
tion is relevant, but the preferred dosage form does not permit
1.3 This standard does not purport to address all of the
injection under the skin or intradermally, and the discomfort
safety concerns, if any, associated with its use. It is the
involved with extended occlusive patching and adjuvant use is
responsibility of the user of this standard to establish appro-
tobeavoided.Itinvolvesrepeatedinductiondoses(3to6)over
priate safety and health practices and determine the applica-
14 days at the same shaved/depilated site, followed each time
bility of regulatory limitations prior to use.
by6hof occlusive wrapping. After a rest period, animals are
2. Referenced Documents
challenged at previously untreated sites, and their reactions
evaluated at least 24 and 48 h later.
2.1 ASTM Standards:
F 619 Practice for Extraction of Medical Plastics
5. Significance and Use
F 720 Practice for Testing Guinea Pigs for Contact Aller-
5.1 In selecting a material for human contact in medical
gens: Guinea Pig Maximization Test
applications, it is important to ensure the material will not
2.2 ISO Document:
stimulate the immune system to produce an allergic reaction
ISO 10993-10, 1995 Tests for Irritation and Sensitization
under relevant exposure conditions. Extractable chemicals
3. Terminology
produced by skin contact or during physiological exposures
may cause allergic reactions. Therefore, this practice provides
3.1 Definitions:
for evaluations of solid or semisolid dosage forms using
3.1.1 2,4 dinitrochlorobenzene (DNCB)—strong sensitizer,
material extracts or direct evaluation of the test article. The
used as a positive control.
rationale for this animal model is based on the fact that the
3.1.2 Freund’s Complete Adjuvant (FCA)—a
guinea pig has been shown to be an appropriate animal model
commercially-available mixture of oil and Mycobacterium that
for predicting human contact dermatitis; its tractable nature, its
is known to elicit an immune response.
availability from reputable suppliers, the historical database of
3.1.3 Guinea Pig Maximization Test (GPMT)—procedure
information already acquired using this species, and the corre-
described in Practice F 720 accepted as a “worst case” assay
lation of such results to data on known human allergens, all
for allergenic potential.
contribute to its widespread use for allergenicity studies (1-5).
4. Summary of Practice
5.2 The need for sensitization procedures other than the
maximization test (Practice F 720) is based on: (1) the need for
4.1 The split adjuvant method is used when topical appli-
a route of exposure more similar to use conditions, (2) concern
cation is considered relevant, and the dosage form is a solid,
over the use of adjuvant because of its recruitment of cell types
tothetestsitewhicharenottypicallyinvolvedinimmunologic
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
reactions, and because of the discomfort this causes in the
Surgical Materials and Devices and is the direct responsibility of Subcommittee
animals, (3) absence of a proper FCA-irritant control group in
F04.16 on Biocompatibility Test Methods.
Current edition approved Oct. 10, 2001. Published January 2002.
Annual Book of ASTM Standards, Vol 13.01.
3 4
Available from American National Standards Institute, 25 W. 43rd St., 4th The boldface numbers in parentheses refer to the list of references at the end of
Floor, New York, NY 10036. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2147
the traditional maximization design, and (4) the frequency of 8. Trial and Naive Challenge Tests
falsepositivesoftenencounteredwiththeGPMT.Bothofthese
8.1 It is recommended that at least two guinea pigs be used
tests are internationally accepted (1).
to assess the ability of the test article or undiluted extract to
irritate. Each flank of each animal can be used to patch two
6. Materials and Manufacturers
sites (upper and lower) of samples such as test article, 100 %
extract, 75 % extract, and 50 % extract. Animals should be
6.1 Hartley strain guinea pigs, either sex (but all in the test
of the same sex), 300 to 500 g at start of test, should be from shavedandwrappedasinthecompletetest(seeSection9),and
the sites evaluated after 24 to 72 h. Scoring should also be
the same shipment, same supplier, and should be healthy.
performed as in the complete test.
6.2 At least ten animals are used for each test material and
five for each control group. 8.2 It is also advisable to determine the difference between
irritation and sensitization under full test conditions for the
6.3 Freund’s Complete Adjuvant (FCA) (split adjuvant test
positive control by including in at least one test per laboratory
only).
a“naivechallenge”groupwhichisexposedtocontrolsonlyfor
6.4 Cotton gauze and occlusive bandage (examples, Elasto-
the challenge period. DNCB, for example, can be an irritant,
pore from 3M) or Hilltopt chambers (Hilltop, Cincinnati, OH)
and it is important that erythema and edema reactions seen
(optional for solid samples) and Vet wrap.
after challenge be true sensitization responses.
6.5 Positive control substance (0.1 to 1 % 2,4 DNCB is a
strong sensitizer; to test method sensitivity, it may be advisable
9. Procedure
to use cinnamaldehyde (10 % induction, 1 % challenge) as a
positive control (2)).
NOTE 3—This procedure is applicable to both methods except as noted.
9.1 Table 1 shows the timing of animal preparation, induc-
7. Preparation of Test Samples
tion dosing, challenge, and evaluation.
NOTE 1—All steps are applicable to both methods.
9.2 Animal Preparation:
7.1 Solid Samples—Cut flat sheet-like samples into 1- by 9.2.1 Weigh and shave or depilate animals within 24 h of
1-cm squares. These can be used for direct contact testing as test start. Depilatories should be used carefully and tested
long as the sample thickness does not exceed 1.0 mm. beforehand to understand proper use regimen so as not to
produce background irritation. Shave or depilate a site on the
NOTE 2—Pressureexertedbybandagingthicksamplescausesmechani-
left flank or shoulder area (use one or the other consistently)
cal irritation. The cotton pad may be removed from the Hilltop chamber
approximately a 2-in. square to expose bare skin, avoiding any
(or the chamber need not be used) to reduce pressure on thick solid test
abrasions or other abnormalities. Check animal health daily
articles. Further cutting should be considered if test articles are still
throughout the test.
causing pressure without the chamber or chamber pad.
9.2.2 Apply 0.3 mL of extract or semisolid (or less, if the
7.2 Gels, Pastes, Ointments—Semisolid test articles can be
amount has been validated, or 1 cm of a solid sample (less
used directly, applied at 0.2 mL/site.
than 1.0 mm thick) to the cotton pad of a Hilltop chamber. (A
7.3 Extracts—Prepare extracts in accordance with Practice
padless chamber can be used to dose gels or thicker samples).
F 619, at the highest temperature tolerated by the material
Stick the chamber to the skin and wrap with an appropriate
without physical melting or decomposition. Both aqueous and
elastic bandage. If a Hilltop chamber is not used, apply the test
nonaqueous extracts are recommended. Extracts should be
sample to gauze and cover with occlusive wrap. Follow the
decanted upon cooling, stored at room temperature (22 to
unwrap/evaluate schedule for the particular procedure as in
30°C), and used within 24 h. Extracts should be prepared fresh
Table 1.
for each treatment, preferably using a solvent which does not
9.2.3 After unwrapping, wait about 30 min before evalua-
give background reactions (ethanol is sometimes a problem in
tion. The test article may be removed by gentle wiping with
this regard), and is known to produce measurable extractables
gauze soaked with purified water or isopropyl alcohol (IPA)
(determined by a technique such as a nonvolatile residue test)
that has been diluted such that it will not dry the skin. Evaluate
without dissolving the test article.
the site using the criteria in Table 2. Rewrap if required (split
7.4 Negative Controls—Prepare solvent sham controls
adjuvant.)
(“blanks”) under the same conditions as test article extracts.
9.2.4 Repeat doses as outlined in Table 1. At the second
Saline controls may be eliminated if there are sufficient data
dose of the split adjuvant procedure, inject 0.05 mL of FCA
available to predict their results.
emulsified 1:1 with water for injection at four locations
7.5 Positive Controls—Positive controls should be prepared
bordering every test and control s
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