ASTM F720-24 豚鼠接触性过敏原测试的标准试验方法：豚鼠最大值测试

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Standard Test Method for

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the

Development of International Standards,Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade(TBT)Committee.

Designation:F720-24

INTERNATIONAL

Testing Guinea Pigs for Contact Allergens:Guinea Pig

Maximization Test

This standard is issued under the fixed designation F720;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

1.1 This test method is intended to determine the potential

for a test article to elicit contact dermal allergenicity.This is a

semi-quantitative method.There are no known limitations to

this method.

1.2 The values stated in SI units,including units officially

accepted for use with the SI,are to be regarded as standard.No

other systems of measurement are included in this standard.

1.3 This standard does not purport to address all of the

safety concerns,if any,associated with its use.It is the

responsibility 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 use.

1.4 This international standard was developed in accor-

dance with internationally recognized principles on standard-

ization established in the Decision on Principles for the

Development of International Standards,Guides and Recom-

mendations issued by the World Trade Organization Technical

Barriers to Trade(TBT)Committee.

2.Referenced Documents

2.1 ASTM Standards:²

F619 Practice for Extraction of Materials Used in Medical

Devices

2.2 ISO Standards:³

ISO 10993-10 Biological evaluation of medical devices—

Part 10:Tests for skin sensitization

3.Summary of Test Method

3.1 After a two-stage induction including intradermal injec-

tion of the test article with and without Freund's complete

!This test method is under the jurisdiction of ASTM Committee F04 on Medical

and Surgical Materials and Devices and is the direct responsibility of Subcommittee

F04.16 on Biocompatibility Test Methods.

Current edition approved Dec.1,2024.Published December 2024.Originally

approved in 1981.Last previous edition approved in 2017 as F720-17.DOI:

10.1520/F0720-24.

²For referenced ASTM standards,visit the ASTM website,www.astm.org,or

contact ASTM Customer Service at www.astm.org/contact.For Annual Book of

ASTM Standards volume information,refer to the standard's Document Summary

page on the ASTM website.

³Available from International Organization for Standardization (ISO),1,ch.de

la Voie-Creuse,CP56,CH-1211 Geneva 20,Switzerland,http://www.iso.org.

adjuvant(FCA)and occluded topical application of the test

article following pretreatment of the application site with

sodium lauryl sulfate(SLS),patches soaked in the test article

(for example,extract)are then applied on the skin of guinea

pigs at a previously untreated site.After 24 hours,the patches

are removed and the skin examined for allergic reaction,and

the intensity of the reaction is scored at 24 and 48 hours

subsequent to removal.

4.Significance and Use

4.1 In selecting a new material for human contact in medical

applications,it is important to ensure that the material will not

stimulate the immune system to produce an allergic reaction.

This practice provides recommendations for evaluating the

potential for chemicals to leach out of a material or device and

elicit an allergic reaction by applying the test article to the skin

of guinea pigs.The rationale for this practice is based on the

fact that the guinea pig has been shown to be a sensitive animal

model for predicting human allergic contact dermatitis.4 The

use of FCA and SLS tends to enhance the potential of a

material to cause an allergy.Therefore,this test,while not

guaranteeing that a material is nonallergenic,is one of the

sensitive animal tests in common use today.

5.Reagents and Materials

5.1 Hartley Strain Guinea Pigs,male or female (nulliparous

and non-pregnant),300 to 500 g.

5.1.1 Ten animals are used for each test article.Five animals

are used for each vehicle control article.For example,if both

polar and non-polar extracts of a device are tested,there will be

a total of 30 animals in the test(ten animals per polar/non-polar

test extract and five animals per polar/non-polar vehicle

control).

5.2 FCA.

5.3 Occlusive Surgical Tape,3.75 cm in width.

5.4 Elastic Bandage.

5.5 SLS(10 weight %)in USP petroleum jelly.

4Magnusson,B.,and Kligman,A.M.,"The identification of contact allergens by

anima assay.The guinea pig maximization test,”"Journal of Investigative

Dermatology,Vol 52,No.3,1969,pp.268-276.

F720-24

5.6 Positive Control Article—Five animals are used if the

positive control is run concurrently with the test article.

However,if the positive control is not run concurrently,testing

with the positive control article should be conducted at least

once every six months with ten test animals and five negative

control animals.

5.6.1 The following positive controls could be used:hexyl

cinnamic aldehyde(HCA),mercaptobenzothiazole,2,4-

dinitrochlorobenzene(DNCB),or formaldehyde(for water-

soluble test articles).

NoTE 1—Preferably,the testing laboratory should use a mild to

moderate sensitizer such as HCA or mercaptobenzothiazole as a positive

control to ensure the sensitivity of the test system for evaluating the

sensitization potential of a device.The concentrations chosen for induc-

tion and challenge should yield a moderate sensitization response.For

example,it has been reported in literature that a 50%sensitization rate

was observed with HCA at concentrations of 5000 ppm (that is,0.5%)at

induction and 1000 ppm(0.1%)at challenge.⁵If a strong sensitizer such

as 2,4-dinitrochlorobenzene(DNCB)is used as a positive control,it

should be used at a concentration of 0.1%to 0.5%for induction and

0.05 %to 0.1%for challenge and the sensitization rate should be at least

80%.

6.Preparation of Test and Control Articles

6.1 For the guinea pig maximization sensitization test,the

concentration of the test chemical (for example,test article

extract)for each induction exposure should be well tolerated

systemically.For a test article that is known or expected to be

an irritant,the highest concentration that causes mild to

moderate erythema but does not otherwise adversely affect the

animals should be used at the topical induction phase.For

non-irritant test articles such as most device extracts,pretreat-

ment of the application site with an irritant chemical such as

with 10%SLS is performed 24 hours before application of the

test patches at the topical induction phase.The concentration

for the challenge exposure should be the highest non-irritant

dose.

6.2 A preliminary test,when necessary,should be conducted

to determine the concentrations ofthe test article to be used for

the induction and challenge exposures for the main test.In

general,preliminary testing to determine the highest non-

irritating concentration is not necessary for the device extracts

prepared in saline or oil.The neat (that is,100 %)extracts are

used for testing.In cases where a preliminary test is needed,it

could be conducted by topically applying a range of dilutions

of the test article under occlusive dressings to the flanks of at

least two animals and evaluating the application sites for

erythema and edema using the Magnusson and Kligman

grading scale described in Table 1 after 24 hours of exposure.

6.3 Articles for Intradermal Injection:

6.3.1 Water-Soluble Constituents or Saline Extracts:

6.3.1.1 Dissolve the water-soluble constituent up to its

maximum solubility,or obtain a saline extraction liquid as

described in Practice F619 .

5Nakamura,Y.,et al.,“A quantitative comparison of induction and challenge

concentrations inducing a 50%positive response in three skin sensitization tests:

The guinea pig maximization test,adjuvant and patch test and Buehler test,"Journal

of Toxicological Sciences,Vol 24,No.2,1999,pp.123-131.

TABLE 1 Magnusson and Kligman Scale

Patch Test Reaction

Grading Scale

No visible change

Discrete or patchy erythema

Moderate and confluent erythema

Intense erythema and/or swelling

6.3.1.2 Combine equal volumes of the test article(s)de-

scribed in 6.3.1.1 and FCA.Homogenize by continuous and

vigorous vortex mixing for a minimum of 5 min.Similarly,for

the vehicle control combine equal volumes of the extraction

vehicle/solvent and FCA.Emulsification is complete when a

drop placed on the surface of a water-ice bath remains intact.

6.3.1.3 Also prepare the constituent in water or saline

extract to the same concentration without FCA.

6.3.2 Oil-Soluble Constituents:

6.3.2.1 Dissolve oil-soluble constituents in FCA.

6.3.2.2 Combine equal volumes of the oil-soluble constitu-

ent solution in FCA solution with an equal volume of water by

slowly adding the water to the adjuvant while homogenizing

with a rotating stirrer.Homogenize by continuous and vigorous

mixing for a minimum of 5 min.Emulsification is complete

when a drop placed on the surface of the water in a water-ice

bath remains intact

6.3.2.3 Also prepare the constituent to an equal concentra-

tion without FCA.

6.3.3 Vegetable Oil Extract Liquids:

6.3.3.1 Mix equal volumes of oil extract liquid obtained in

accordance with Practice F619 with an equal volume of FCA.

Similarly,for the vehicle control combine equal volumes ofthe

extraction vehicle/solvent and FCA.

6.3.3.2 Also prepare extract to an equal concentration in

water without FCA.

6.3.4 Alternative Article Preparation—As an alternative

method for sample extraction,the method described in ISO

10993-10:2021,Annex B,is recommended for consideration

for the extraction of polymeric materials.

6.3.5 Prepare control substances consistent with 6.3.1-

6.3.3.2 .

NoTE 2—If the final concentration of the suspected allergen results in

deleterious effects such as ulceration,necrosis,or systemic toxicity,use

the maximum tolerable concentration.

6.4 Articles for Topical Application:

6.4.1 Liquids—For all liquids,use the highest concentration

not causing excessive irritation or deleterious to the general

health of test animals.(See Note 2.)

6.4.1.1 For oil-miscible liquids,dilute with petroleum jelly

if necessary.

6.4.1.2 For water-miscible liquids,dilute with water if

necessary.

7.Procedure for Test and Control Articles

7.1 Induction:

7.1.1 Intradermal Injection:

7.1.1.1 Clip the shoulder region of each guinea pig free of

hair,exposing a 4 by 6cm area.

7.1.1.2 Three injection sites on each side of the spine should

be identified with at least 1.5 cm between sites.Intradermal

F720-24

injections are then made as indicated below,with Site 1 being the reaction(s)can be due to irritation rather than sensitization

closest to the animal's head. as the control animals have not been previously exposed to the

(1)Site 1—0.1 mL of 50:50(v/v)mixture of FCA and the test articles.

chosen vehicle 7.2.4.2 Per Option 2,the control animals are not dosed with

(2)Site 2—0.1 mL of test sample without adjuvant for the the test article and the test animals are not dosed with the

test animals;0.1 mL of extraction vehicle/solvent without control article.In the event of skin reactions observed in the

adjuvant for the control animals test animals where no preliminary testing was conducted to

(3)Site 3—0.1 mL of a 1:1 mixture of the 50:50(v/v) determine the highest non-irritating concentration of the test

FCA/chosen vehicle mixture (the solution used at Site 1)and articles (the use of highest non-irritating concentration is

test article (used at Site 2)for the test animals;0.1 mL of a 1:1 recommended for challenge phase application),additional

mixture of the 50:50(v/v)FCA/chosen vehicle mixture(solu- testing can be needed to rule out a false positive.For testing

tion used at Site 1)and extraction vehicle/solvent (used at Site with medical device extracts,preliminary testing to determine

2)for the control animals the highest non-irritating concentration of the extracts is not

generally conducted.

7.1.2.1 One week(7 ±1 days)after the intradermal 7.2.5 Apply the test(filter paper saturated with the test

injections,clip the test area (intradermal injection sites)of the article)and control (filter paper saturated with control vehicle)

guinea pigs. to the shaved sites which were not treated during the induction

7.1.2.2 If the test sample is non-irritating,treat each test area stage(such as the upper flank of each animal)and secure with

with 10%SLS in petroleum jelly,24 hours before applying the occlusive surgical tape and an elastic bandage as described in

test patches.Massage the SLS into the skin with a glass rod. 7.1.2.4.

Any remaining SLS should be removed prior to the topical 7.2.6 Leave the samples in place for 24±2 hours.

application of the test and control articles

7.1.2.3 Apply the test article to a 2 by 4 cm patch of

qualitative filter paper.For oil-miscible liquids,if they have 8.1 Read the challenge sites at 24 and 48 hours after

been diluted in accordance with 6.4.1.1,apply the diluted removing the patches.Grade each site for erythema and edema

liquid to the filter in a thick,even layer.Otherwise,apply the for each challenge site and at each time point in accordance

test liquid or the test extract neat to the test paper until with Table 1.

saturated. 8.2 Prepare a tabular listing of the reactions at 24 and 48

7.1.2.4 Apply filter paper to the injection site of the guinea hours.Magnusson and Kligman grades of 1 or greater in the

pigs.Cover with 3.75 cm occlusive surgical tape and wrap an test group generally indicate sensitization,provided grades of

elastic bandage around the torso to secure the tape. less than 1 are seen in control animals.If grades of 1 or greater

7.1.2.5 Leave in place for 48±2 hours. are noted in control animals,then the reactions of test animals

which exceed the most severe reaction in control animals are

7.2 Challenge: presumed to be due to sensitization.If the response is

7.2.1 Two weeks(14±1 days)following the induction equivocal,rechallenge is recommended to confirm the results

described in 7.1.2,perform the following: from the first challenge.A rechallenge shall be carried out one

7.2.2 Shave a 5 by 5cm area on the animal's flank. to two weeks after the first challenge following the method as

7.2.3 Delivery of Test and Control Articles: described for the first challenge,using a naive side on the

7.2.3.1 Test solids at a non-irritating concentration in petro- animal.

leum jelly by weight.Apply on a 2 by 2 cm piece of filter paper

as outlined in 7.1.2.3.

7.2.3.2 Test liquids at a non-irritating concentration.Apply

on a 2 by 2 cm piece of filter paper as outlined in 7.1.2.3.

7.2.3.3 Apply the control vehicle on a 2 by 2 cm piece of TABLE 2 Rating of Sensitization Response

filter paper. %Sensitized Grade Classification

7.2.4 All test and control animals shall be challenged. 0 0 none

(2)Control animals shall be dosed (topical application) 65 to 80 IV strong

with the control article and the test animals shall be dosed

(topical application)with the test article.This option is

applicable if a standard solvent such as saline or vegetable oil

is used as the control/extraction vehicle. 8.3.1 Based on the positive control used (for example,mild,

7.2.4.1 The use of Option 1 allows one to determine if any moderate,strong),the positive control should meet the sensi-

skin reactions observed in the test animals are due to irritation tization frequency specified in Table 2 for the test to be valid.

rather than sensitization.Because,both test and control animals

are dosed with both test and control articles,if any skin 9.Report

reactions to the test article are observed in the control animals, 9.1 The report shall include the following:

8.3 Rate the allergenicity of the test material in accordance

with Table 2.

81 to 100 V extreme

8.Interpretation of Results

7.1.2 Topical Application:

(1)Both test and control animals shall be dosed(topical 9 to 28 IⅡ mild

application)with both test and control articles;or 29 to 64 Ⅲ moderate

>0 to 8 weak

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1StandardTestMethodforThisinternationalstandardwasdevelopedinaccordancewithinternationallyrecognizedprinciplesonstandardizationestablishedintheDecisiononPrinciplesfortheDevelopmentofInternationalStandards,GuidesandRecommendationsissuedbytheWorldTradeOrganizationTechnicalBarrierstoTrade(TBT)Committee...

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