ASTM F2148-18

Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)

SIGNIFICANCE AND USE
5.1 The propensity of a material to stimulate delayed contact hypersensitivity must be assessed before clinical application of devices containing this material. Delayed hypersensitivity may occur anywhere in the body. Systemic delayed hypersensitivity may have a complex set of reactions and consequences depending on the actual tissue/organ site of reaction. Although the reactions are seldom life-threatening, severe tissue and organ damage my result over time. Skin is the usual test site to determine the propensity of a material to cause delayed hypersensitivity.  
5.2 The standard historical test methods have involved the use of guinea pigs with a cutaneous application and observation of the reaction site. The use of the murine local lymph node assay results in a numerical quantitation of stimulation, rather than subjective evaluation and could be used to determine dose responses.  
5.3 This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
SCOPE
1.1 This practice provides a methodology to use a combination of in vivio and in situ procedures for the evaluation of delayed contact hypersensitivity reactions.  
1.2 This practice is intended to provide an alternative to the use of guinea pigs for evaluation of the ability of a device material to stimulate delayed contact hypersensitivity reactions. This alternative is particularly applicable for materials used in devices that contact only intact skin. However, the guinea pig maximization test is still the recommended method when assessing the delayed hypersensitivity response to metals or when testing substances that do not penetrate the skin but are used in devices that contact deep tissues or breached surfaces. This practice may be used for testing metals, with the exception of nickel-containing metals, unless the unique physicochemical properties of the materials may interfere with the ability of LLNA to detect sensitizing substances.  
1.3 This practice consists of a protocol for assessing an increase in lymphocyte proliferation in the lymph nodes draining the site of test article administration on the ears of mice.  
1.4 The LLNA has been validated only for low-molecular-weight chemicals that can penetrate the skin. The absorbed chemical or metabolite must bind to macromolecules, such as proteins, to form immunogenic conjugates.  
1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.  
1.6 Identification of a supplier of materials or reagents is for the convenience of the user and does not imply a single source. Appropriate materials and reagents may be obtained from many commercial supply houses.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 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 appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.9 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.

General Information

• Status: Published
• Publication Date: 30-Nov-2018
• ICS: 07.100.10 - Medical microbiology
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.16 - Biocompatibility Test Methods

Relations

• Replaces: ASTM F2148-13 - Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
• Effective Date: 01-Dec-2018
• Refers: ASTM F750-20 - Standard Practice for Evaluating Acute Systemic Toxicity of Material Extracts by Systemic Injection in the Mouse
• Effective Date: 01-Jun-2020
• Refers: ASTM F720-17 - Standard Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
• Effective Date: 01-Sep-2017
• Refers: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Apr-2016
• Refers: ASTM F720-13 - Standard Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
• Effective Date: 01-Jun-2013
• Refers: ASTM F750-87(2012) - Standard Practice for Evaluating Material Extracts by Systemic Injection in the Mouse
• Effective Date: 01-Oct-2012
• Refers: ASTM F720-81(2012) - Standard Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
• Effective Date: 01-Oct-2012
• Refers: ASTM F748-06(2010) - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Jun-2010
• Refers: ASTM F619-03(2008) - Standard Practice for Extraction of Medical Plastics
• Effective Date: 01-Aug-2008
• Refers: ASTM F750-87(2007) - Standard Practice for Evaluating Material Extracts by Systemic Injection in the Mouse
• Effective Date: 01-Feb-2007
• Refers: ASTM F720-81(2007)e1 - Standard Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
• Effective Date: 01-Feb-2007
• Refers: ASTM F750-87(2007)e1 - Standard Practice for Evaluating Material Extracts by Systemic Injection in the Mouse
• Effective Date: 01-Feb-2007
• Refers: ASTM F720-81(2007) - Standard Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
• Effective Date: 01-Feb-2007
• Refers: ASTM F748-06 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Dec-2006
• Refers: ASTM F748-04 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-May-2004

Overview

ASTM F2148-18, "Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)," establishes a methodology for assessing the potential of materials, especially those used in medical devices, to cause delayed contact hypersensitivity reactions. This international standard provides an alternative to traditional guinea pig testing, utilizing a murine (mouse-based) local lymph node assay to deliver objective, quantitative results. The LLNA is primarily intended for materials that contact intact skin but may be adaptable to other applications with appropriate caution.

The standard is essential for evaluating the biocompatibility of device materials before clinical use, helping identify materials that might induce allergic responses upon skin contact.

Key Topics

• Purpose and Significance

  • Assesses the propensity of materials to stimulate delayed contact hypersensitivity, which may lead to tissue or organ damage over time.
  • Focuses on testing prior to clinical application of devices to safeguard patient health.
  • Provides quantitative data, improving upon the subjective evaluation of traditional methods such as guinea pig maximization tests.

• LLNA Methodology

  • Utilizes a combination of in vivo and in situ procedures.
  • Involves application of test substances to mouse ears and assessment of lymphocyte proliferation in the draining lymph nodes.
  • Valid for low-molecular-weight chemicals capable of penetrating the skin.
  • Employs statistical analysis to determine sensitization potential (e.g., stimulation index, significance testing).

• Test Specimen Preparation

  • Extracts of test materials are prepared in both aqueous and nonaqueous solvents according to standardized procedures.
  • Controls are used to ensure reliability, including both negative and positive controls.

• Limitations and Precautions

  • Not universally predictive for all implant or deep tissue device applications.
  • Not validated for certain materials, especially those that do not penetrate the skin or nickel-containing metals.
  • Use of LLNA must be considered in context with the material’s end use and relevant guidelines, such as ASTM F748.

• Safety and Compliance

  • Requires adherence to safety, health, and regulatory practices.
  • Users are responsible for determining regulatory applicability and ensuring laboratory accreditation, especially due to the use of radioactive materials in the assay.

Applications

• Medical Device Material Testing

  • Critical in evaluating the biocompatibility of polymers, additives, and coatings intended for medical devices that will contact skin.
  • Used as a screening tool to determine if a material may cause sensitization or allergic reactions.

• Biocompatibility Assessment

  • Supports regulatory submissions by providing objective data for risk assessment.
  • Can inform product development and material selection to avoid delays related to adverse sensitization findings.

• Alternative to Animal Testing

  • Offers an ethical and statistically robust alternative to guinea pig-based assays, reducing animal use and subjectivity in results.
  • Can provide dose-response data, enabling refined safety evaluations.

Related Standards

• ASTM F619: Practice for Extraction of Medical Plastics
• ASTM F720: Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
• ASTM F748: Practice for Selecting Generic Biological Test Methods for Materials and Devices
• ASTM F750: Practice for Evaluating Material Extracts by Systemic Injection in the Mouse
• ICCVAM Guidelines: Validated protocols and evaluation reports for LLNA and similar assays

For comprehensive biocompatibility assessments and regulatory acceptance, consult these related ASTM standards and follow relevant guidance on method selection and interpretation.

---

By implementing ASTM F2148-18, laboratories and manufacturers can ensure standardized, reliable evaluation of delayed contact hypersensitivity, aiding the development and approval of safer medical devices and materials.

Standards Content (Sample)

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: F2148 − 18
Standard Practice for
Evaluation of Delayed Contact Hypersensitivity Using the
Murine Local Lymph Node Assay (LLNA)
This standard is issued under the fixed designation F2148; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.7 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
1.1 This practice provides a methodology to use a combi-
standard.
nation of in vivio and in situ procedures for the evaluation of
1.8 This standard does not purport to address all of the
delayed contact hypersensitivity reactions.
safety concerns, if any, associated with its use. It is the
1.2 This practice is intended to provide an alternative to the
responsibility of the user of this standard to establish appro-
use of guinea pigs for evaluation of the ability of a device
priate safety, health, and environmental practices and deter-
material to stimulate delayed contact hypersensitivity reac-
mine the applicability of regulatory limitations prior to use.
tions. This alternative is particularly applicable for materials
1.9 This international standard was developed in accor-
used in devices that contact only intact skin. However, the
dance with internationally recognized principles on standard-
guinea pig maximization test is still the recommended method
ization established in the Decision on Principles for the
when assessing the delayed hypersensitivity response to metals
Development of International Standards, Guides and Recom-
orwhentestingsubstancesthatdonotpenetratetheskinbutare
mendations issued by the World Trade Organization Technical
used in devices that contact deep tissues or breached surfaces.
Barriers to Trade (TBT) Committee.
This practice may be used for testing metals, with the excep-
tion of nickel-containing metals, unless the unique physico-
2. Referenced Documents
chemical properties of the materials may interfere with the
2.1 ASTM Standards:
ability of LLNA to detect sensitizing substances.
F619 Practice for Extraction of Medical Plastics
1.3 This practice consists of a protocol for assessing an
F720 PracticeforTestingGuineaPigsforContactAllergens:
increase in lymphocyte proliferation in the lymph nodes
Guinea Pig Maximization Test
draining the site of test article administration on the ears of
F748 PracticeforSelectingGenericBiologicalTestMethods
mice.
for Materials and Devices
F750 Practice for Evaluating Material Extracts by Systemic
1.4 The LLNA has been validated only for low-molecular-
Injection in the Mouse
weight chemicals that can penetrate the skin. The absorbed
chemical or metabolite must bind to macromolecules, such as
2.2 Other Documents:
proteins, to form immunogenic conjugates.
ICCVAM NIH Publication No: 99-4494 The Murine Local
Lymph Node Assay, 1999
1.5 This practice is one of several developed for the
ICCVAM NIH Publication No: 10-7512 Test Method Evalu-
assessment of the biocompatibility of materials. Practice F748
ation Report on Using the Murine Local Lymph Node
may provide guidance for the selection of appropriate methods
Assay for Testing Pesticide Formulations, Metals, Sub-
for testing materials for a specific application.
stances in Aqueous Solutions, and Other Products, 2010
1.6 Identification of a supplier of materials or reagents is for
ICCVAM NIH Publication NO: 11-7709 Usefulness and
the convenience of the user and does not imply a single source.
Limitations of the Murine Local Lymph Node Assay for
Appropriate materials and reagents may be obtained from
Potency Categorization of Chemicals Causing Allergic
many commercial supply houses.
Contact Dermatitis in Humans
1 2
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Surgical Materials and Devices and is the direct responsibility of Subcommittee contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
F04.16 on Biocompatibility Test Methods. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Dec. 1, 2018. Published February 2019. Originally the ASTM website.
approved in 2001. Last previous edition approved in 2013 as F2148 – 13. DOI: Available from NICEATM, NIEHS, 79 Alexander Dr., Mail Drop EC-17,
10.1520/F2148-18. Research Triangle Park, NC 27709.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2148 − 18
3. Terminology materials being tested, their potential applications, and the
recommendations contained in Practice F748.
3.1 Definitions of Terms Specific to This Standard:
3.1.1 AOO, n—acetone olive oil solution (4:1 v/v) is a
6. Preparation of Test Specimens
suitable nonpolar solvent.
6.1 Specimens should be prepared in accordance with Prac-
3.1.2 aqueous solvent, n—in this assay refers to the polar
tice F619. All solid materials shall be extracted. Extractions
solvent, saline.
shall be done with an aqueous (polar) solvent and a nonaque-
3.1.3 DMSO, n—dimethylsulfoxide (nonaqueous, suitable
ous (nonpolar or organic) solvent, either DMSO or AOO.
organic solvent).
6.2 Liquid test articles and gels shall be used directly if they
3.1.4 DNCB, n—2,4-dinitrochlorobenzene.
are not irritants.Aliquid that is an irritant shall be diluted with
3.1.5 formalin, n—a ⁄10 dilution of 37 to 39 % formalde- an aqueous or nonaqueous solvent based on solubility of the
hyde solution (formaldehyde) in PBS.
liquid test article until the solution is non-irritating.
3.1.6 ICCVAM, n—Interagency Coordinating Committee on
6.3 Wholly aqueous solutions are not suitable for applica-
the Validation of Alternative Methods.
tion to the ear. Therefore, for use in the assay, add 0.05 g of
hydroxyethyl cellulose to each 10 mL of the aqueous vehicle
3.1.7 nonaqueous solvent, n—in this assay refers to the
control and test solutions to aid in holding the solution to the
organic or nonpolar solvent, which shall be dimethylsulfoxide
ear. One percent Pluronic L92 may also be used as an aqueous
(DMSO) or acetone olive oil (AOO).
vehicle.
3.1.8 PBS, n—phosphate buffered saline, pH 7.2.
6.4 The final specimen to be extracted should be prepared
3.1.9 positive control, n—a substance capable of consis-
with a surface finish consistent with its end-use application.
tently stimulating lymphocyte proliferation.
6.5 The specimen shall be sterilized by the method to be
3.1.10 saline, n—0.9 % sodium chloride (aqueous, polar
used for the final product.
solvent).
6.6 Care should be taken that the specimens do not become
3.1.11 TCA, n—5 % trichloroacetic acid.
H3
contaminated during preparation and aseptic technique is
3.1.12 tritiated thymidine, n— methyl thymidine, specific
125 recommended.
activity 2 Ci/mM (in PBS) I IUDR-radioactive uridine.
3.1.13 vehicle controls, n—an aqueous, polar solvent and a
7. Preparation of Positive Controls
non-aqueous, nonpolar solvent.
7.1 Nonaqueous Positive Control—The use of a moderate
positive control as a substitute or in addition to a strong
4. Summary of Practice
positive control should be considered.
4.1 Test and control substances or extracts are applied to the
7.1.1 Moderate Positive Control—Prepare a solution of
ears of test mice. The draining lymph nodes are harvested and
25 % hexyl cinnamic aldehyde (HCA) in an acetone:olive oil
lymphocyte proliferation evaluated. Comparisons are made
(4:1 v/v) solvent. Shake the flask until a homogenous solution
with the control and test specimens tested under identical
is obtained.
conditions.
7.1.2 StrongPositiveControl—Weigh0.025gofDNCBand
place in a flask. Add enough DMSO to dissolve all of the
5. Significance and Use
DNCB.Add more DMSO to bring the level up to 10 mL. Cap
and shake the flask until a homogenous solution is obtained.
5.1 The propensity of a material to stimulate delayed
7.1.3 The dose level of the positive control should not
contact hypersensitivity must be assessed before clinical ap-
produce systemic toxicity as evidenced by clinical observa-
plication of devices containing this material. Delayed hyper-
tions.
sensitivity may occur anywhere in the body. Systemic delayed
hypersensitivity may have a complex set of reactions and
7.2 Aqueous Positive Control—Neutral buffered formalin is
consequences depending on the actual tissue/organ site of
commercially available. (Or dilute formaldehyde ⁄10 in PBS.
reaction. Although the reactions are seldom life-threatening,
Place 1 mL of formaldehyde in a 10-mL flask. Add enough
severetissueandorgandamagemyresultovertime.Skinisthe
PBS to mix the two solutions.Add more PBS to bring the level
usualtestsitetodeterminethepropensityofamaterialtocause
up to 10 mL. Cap and shake the flask until a homogeneous
delayed hypersensitivity.
solution is obtained.)
5.2 The standard historical test methods have involved the
7.3 Aqueous solutions are not suitable for application to the
use of guinea pigs with a cutaneous application and observa-
ear. Therefore, for use in the assay, add 0.05 g of hydroxyethyl
tion of the reaction site. The use of the murine local lymph
cellulose to each 10 mLof the aqueous positive control to aid
node assay results in a numerical quantitation of stimulation,
in holding the solution to the ear until absorbed. One percent
rather than subjective evaluation and could be used to deter-
Pluronic L92 may also be used as an aqueous vehicle.
mine dose responses.
5.3 This practice may not be predictive of events occurring
“Final Report on the Safety Assessment of Hydroxyethylcellulose,
during all types of implant applications. The user is cautioned
Hydroxypropylcellulose, Methylcellulose, Hydroxypropyl Methylcellulose, and
to consider the appropriateness of the method in view of the Cellulose Gum,” J. Amer Coll Tox., Vol 5, No. 3, 1986, pp. 1-59.
F2148 − 18
7.4 For all specimens requiring extractions, prepare an 8.4 Radiolabeled Tracer Preparation—Prepare tritiated thy-
aqueous and non-aqueous extract (DMSO or AOO are recom- midine to a working approximate concentration of 80 µCi/mL
125 -5
mended but other permissible extractants are listed in the (v/v). The use of I I-UDR at 8 µCi/mL in PBS 10 M
ICCVAM documents) following the procedures described in fluorodeoxyuridine is also acceptable. Each mouse will receive
Practice F619. 250 µL radiolabeled tracer solution intravenously.All standard
precautionsassociatedwithusingradioactivematerialsshallbe
8. Dosing of the Animals
adhered to. The laboratory shall be licensed to use radioactive
8.1 Healthy, non-pregnant female CBA/Ca or CBA/j mice material and all personnel shall be appropriately trained and
certified.
that are seven to twelve weeks of age shall be used. House the
animals according to treatment group with five animals per
8.4.1 Topreparethetritiatedthymidinesolution,add0.8mL
cage. of 1.0-mCi/mL tritiated thymidine (specific activity 2.0 Ci/
mM)toastopperedflask.AddsterilePBStomake10mL.Cap
8.2 Day One—Uniquelyidentifyeachmouse(eartagsorear
and mix well.
notches may not be used). Weigh each mouse to the nearest
8.4.2 Confirm the concentration of this dilution. Dilute 0.08
whole gram.
mL to 200 mL with water using a 200-mL flask. Cap and mix
8.3 Aminimum of five mice shall be used for each positive
well by inverting several times. Remove two 1-mL samples
andnegativecontrolandeachtestsample.Theyshallbetreated
and place in scintillation vials.Add 10 mLof scintillation fluid
dailyforthreeconsecutivedaysbytopicalapplicationof25µL
to each vial, mix so that a vortex is formed, and “count” in a
of one of the solutions to the dorsal surface of both ears. For
beta scintillation counter. Count each vial three times and
the aqueous groups only, the dorsal surface should be wiped
calculate the mean. Calculate the concentration.
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F2148 − 13 F2148 − 18
Standard Practice for
Evaluation of Delayed Contact Hypersensitivity Using the
Murine Local Lymph Node Assay (LLNA)
This standard is issued under the fixed designation F2148; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This practice provides a methodology to use an in-situ procedure a combination of in vivio and in situ procedures for the
evaluation of delayed contact hypersensitivity reactions.
1.2 This practice is intended to provide an alternative to the use of guinea pigs for evaluation of the ability of a device material
to stimulate delayed contact hypersensitivity reactions. This alternative is particularly applicable for materials used in devices that
contact only intact skin. However, the guinea pig maximization test is still the recommended method when assessing the delayed
hypersensitivity response to metals or when testing substances that do not penetrate the skin but are used in devices that contact
deep tissues or breached surfaces. This practice may be used for testing metals, with the exception of nickel-containing metals,
unless the unique physicochemical properties of the materials may interfere with the ability of LLNA to detect sensitizing
substances.
1.3 This practice consists of a protocol for assessing an increase in lymphocyte proliferation withinin the lymph nodes draining
the site of test article administration on the ears of mice.
1.4 The LLNA has been validated only for low-molecular-weight chemicals that can penetrate the skin. The absorbed chemical
or metabolite must bind to macromolecules, such as proteins, to form immunogenic conjugates.
1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide
guidance for the selection of appropriate methods for testing materials for a specific application.
1.6 Identification of a supplier of materials or reagents is for the convenience of the user and does not imply a single source.
Appropriate materials and reagents may be obtained from many commercial supply houses.
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 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 appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.9 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.
2. Referenced Documents
2.1 ASTM Standards:
F619 Practice for Extraction of Medical Plastics
F720 Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F750 Practice for Evaluating Material Extracts by Systemic Injection in the Mouse
2.2 Other Documents:
ICCVAM NIH Publication No: 99-4494 The Murine Local Lymph Node Assay, 1999
This practice 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 June 1, 2013Dec. 1, 2018. Published August 2013February 2019. Originally approved in 2001. Last previous edition approved in 20122013 as
F2148 – 07 (2012).F2148 – 13. DOI: 10.1520/F2148-13.10.1520/F2148-18.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from NICEATM, NIEHS, 79 Alexander Dr., 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 States
F2148 − 18
ICCVAM NIH Publication No: 10-7512 Test Method Evaluation Report on Using the Murine Local Lymph Node Assay for
Testing Pesticide Formulations, Metals, Substances in Aqueous Solutions, and Other Products, 2010
ICCVAM NIH Publication NO: 11-7709 Usefulness and Limitations of the Murine Local Lymph Node Assay for Potency
Categorization of Chemicals Causing Allergic Contact Dermatitis in Humans
3. Terminology
3.1 Definitions:Definitions of Terms Specific to This Standard:
3.1.1 AOO, n—acetone olive oil solution (4:1 v/v) is a suitable nonpolar solvent.
3.1.2 aqueous solvent, n—in this assay refers to the polar solvent, saline.
3.1.3 DMSO, n—dimethylsulfoxide (nonaqueous, suitable organic solvent).
3.1.4 DNCB, n—2,4-dinitrochlorobenzene.
3.1.5 formalin, n—a ⁄10 dilution of 37 to 39 % formaldehyde solution (formaldehyde) in PBS.
3.1.6 ICCVAM, n—Interagency Coordinating Committee on the Validation of Alternative Methods.
3.1.7 nonaqueous solvent, n—in this assay refers to the organic or nonpolar solvent, which shall be dimethylsulfoxide (DMSO)
or acetone olive oil (AOO).
3.1.8 PBS, n—phosphate buffered saline, pH 7.2.
3.1.9 positive control, n—a substance capable of consistently stimulating lymphocyte proliferation.
3.1.10 saline, n—0.9 % sodium chloride (aqueous, polar solvent).
3.1.11 TCA, n—5 % trichloroacetic acid.
H3 125
3.1.12 tritiated thymidine, n— methyl thymidine, specific activity 2 Ci/mM (in PBS) I IUDR-radioactive uridine.
3.1.13 vehicle controls, n—an aqueous, polar solvent and a non-aqueous, nonpolar solvent.
4. Summary of Practice
4.1 Test and control substances or extracts are applied to the ears of test mice. The draining lymph nodes are harvested and
lymphocyte proliferation evaluated. Comparisons are made with the control and test specimens tested under identical conditions.
5. Significance and Use
5.1 The propensity of a material to stimulate delayed contact hypersensitivity must be assessed before clinical application of
devices containing this material. Delayed hypersensitivity may occur anywhere in the body. Systemic delayed hypersensitivity may
have a complex set of reactions and consequences depending on the actual tissue/organ site of reaction. Although the reactions are
seldom life-threatening, severe tissue and organ damage my result over time. Skin is the usual test site to determine the propensity
of a material to cause delayed hypersensitivity.
5.2 The standard historical test methods have involved the use of guinea pigs with a cutaneous application and observation of
the reaction site. The use of the murine local lymph node assay results in a numerical quantitation of stimulation, rather than
subjective evaluation and could be used to determine dose responses.
5.3 This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to
consider the appropriateness of the method in view of the materials being tested, their potential applications, and the
recommendations contained in Practice F748.
6. Preparation of Test Specimens
6.1 Specimens should be prepared in accordance with Practice F619. All solid materials shall be extracted. Extractions shall be
done with an aqueous (polar) solvent and a nonaqueous (nonpolar or organic) solvent, either DMSO or AOO.
6.2 Liquid test articles and gels shall be used directly if they are not irritants. A liquid that is an irritant shall be diluted with
an aqueous or nonaqueous solvent based on solubility of the liquid test article until the solution is non-irritating.
6.3 Wholly aqueous solutions are not suitable for application to the ear. Therefore, for use in the assay, add 0.05 g of
hydroxyethyl cellulose to each 10 mL of the aqueous vehicle control and test solutions to aid in holding the solution to the ear.
One percent Pluronic L92 may also be used as an aqeuousaqueous vehicle.
6.4 The final specimen to be extracted should be prepared with a surface finish consistent with its end-use application.
6.5 The specimen shall be sterilized by the method to be used for the final product.
“Final Report on the Safety Assessment of Hydroxyethylcellulose, Hydroxypropylcellulose, Methylcellulose, Hydroxypropyl Methylcellulose, and Cellulose Gum,” J.
Amer Coll Tox., Vol 5, No. 3, 1986, pp. 1-59.
F2148 − 18
6.6 Care should be taken that the specimens do not become contaminated during preparation and aseptic technique is
recommended.
7. Preparation of Positive Controls
7.1 Nonaqueous Positive Control—The use of a moderate positive control as a substitute or in addition to a strong positive
control should be considered.
7.1.1 Moderate Positive Control—Prepare a solution of 25 % hexyl cinnamic aldehyde (HCA) in an acetone:olive oil (4:1 v/v)
solvent. Shake the flask until a homogenous solution is obtained.
7.1.2 Strong Positive Control—Weigh 0.025 g of DNCB and place in a flask. Add enough DMSO to dissolve all of the DNCB.
Add more DMSO to bring the level up to 10 mL. Cap and shake the flask until a homogenous solution is obtained.
7.1.3 The dose level of the positive control should not produce systemic toxicity as evidenced by clinical observations.
7.2 Aqueous Positive Control—Neutral buffered formalin is commercially available. (Or dilute formaldehyde ⁄10 in PBS. Place
1 mL of formaldehyde in a 10-mL flask. Add enough PBS to mix the two solutions. Add more PBS to bring the level up to 10
mL. Cap and shake the flask until a homogeneous solution is obtained.)
7.3 Aqueous solutions are not suitable for application to the ear. Therefore, for use in the assay, add 0.05 g of hydroxyethyl
cellulose to each 10 mL of the aqueous positive control to aid in holding the solution to the ear until absorbed. One percent
Pluronic L92 may also be used as an aqueous vehicle.
7.4 For all specimens requiring extractions, prepare an aqueous and non-aqueous extract (DMSO or AOO are recommended but
other permissible extractants are listed in the ICCVAM documents) following the procedures described in Practice F619.
8. Dosing of the Animals
8.1 Healthy, non-pregnant female CBA/Ca or CBA/j mice that are seven to twelve weeks of age shall be used. House the
animals according to treatment group with five animals per cage.
8.2 Day One—Uniquely identify each mouse (ear tags or ear notches may not be used). Weigh each mouse to the nearest whole
gram.
8.3 A minimum of five mice shall be used for each positive and negative control and each test sample. They shall be treated
daily for three consecutive days by topical application of 25 μL of one of the solutions to the dorsal surface of both ears. For the
aqueous groups only, the dorsal surface should be wiped with acetone just before treating to aid in absorption of the aqueous
solution, although it will not be completely absorbed.
8.3.1 For testing, other than liquid test articles, the groups shall include: aqueous and nonaqueous positive controls, aqueous and
nonaqueous vehicle controls, aqueous extract of the test sample, and nonaqueous extract of test sample.
8.3.2 For testing of liquid test articles, the groups shall include: aqueous and nonaqueous positive controls, the liquid test
sample, and either an aqueous or a nonaqueous vehicle control appropriate for the nature of the liquid sample.
8.3.3 The extract shall be used within 24 h of preparation. The extract should be stored in a stoppered container at room
temperature. The applications shall be performed at 24 6 2 h intervals on Days 2 and 3. Table 1 describes the events for each day
of the test.
8.3.4 Observe each mouse daily for signs of local irritation at the application site and for signs of systemic toxicity (see Practices
F720 and F750). It may be advisable to pretest two mice if it is suspected that the material may be a
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