ASTM F749-20

Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit

SIGNIFICANCE AND USE
4.1 This practice is to be used to help assess the biocompatibility of materials used in medical devices. It is an acute toxicological test designed to evaluate any irritation caused by device materials by gross assessment.  
4.2 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the appropriateness of this practice in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
Note 1: Some materials (e.g., absorbables) may result in an extract pH (e.g., ≤2.0 or ≥11.5) that cannot be used with this practice.  
4.3 The only applicable limitation is the extract preparation. Refer to Section 4.3 of Practice F619 for a description of this limitation.
SCOPE
1.1 This practice is an intracutaneous reactivity test used to assess the potential of the material under test to produce irritation following intradermal injections of extracts of the material.  
1.2 The liquids injected into the rabbits are those obtained by Practice F619 where the extraction vehicles are saline, vegetable oil, or other liquids simulating human body fluids.  
1.3 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.  
1.4 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.  
1.5 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: 31-Jan-2020
• ICS: 11.220 - Veterinary medicine
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.16 - Biocompatibility Test Methods

Relations

• Replaces: ASTM F749-13 - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Feb-2020
• Refers: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Apr-2016
• 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 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
• Refers: ASTM F619-03 - Standard Practice for Extraction of Medical Plastics
• Effective Date: 01-Nov-2003
• Refers: ASTM F619-02 - Standard Practice for Extraction of Medical Plastics
• Effective Date: 10-Apr-2002
• Refers: ASTM F748-98 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 10-Aug-1998
• Refers: ASTM F619-79(1997)e1 - Standard Practice for Extraction of Medical Plastics
• Effective Date: 10-Dec-1997
• Referred By: ASTM F2212-20 - Standard Guide for Characterization of Type I Collagen as Starting Material for Surgical Implants and Substrates for Tissue Engineered Medical Products (TEMPs)
• Effective Date: 01-Feb-2020
• Referred By: ASTM F2347-15 - Standard Guide for Characterization and Testing of Hyaluronan as Starting Materials Intended for Use in Biomedical and Tissue Engineered Medical Product Applications
• Effective Date: 01-Feb-2020
• Referred By: ASTM F648-21 - Standard Specification for Ultra-High-Molecular-Weight Polyethylene Powder and Fabricated Form for Surgical Implants
• Effective Date: 01-Feb-2020
• Referred By: ASTM F2759-19 - Standard Guide for Assessment of the Ultra-High Molecular Weight Polyethylene (UHMWPE) Used in Orthopedic and Spinal Devices
• Effective Date: 01-Feb-2020
• Referred By: ASTM F2565-21 - Standard Guide for Extensively Irradiation-Crosslinked Ultra-High Molecular Weight Polyethylene Fabricated Forms for Surgical Implant Applications
• Effective Date: 01-Feb-2020

Overview

ASTM F749-20 is the Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit, developed by ASTM International. This standard provides a methodology for assessing the biocompatibility of materials used in medical devices by means of acute toxicological testing. The test specifically evaluates the potential of a material to cause irritation following the intradermal (intracutaneous) injection of its extracts into rabbit skin. This approach helps identify the presence of injurious or irritating leachable substances, contributing to the overall safety assessment of medical devices intended for human use.

Key Topics

• Biocompatibility Testing: The standard focuses on the evaluation of medical device materials’ compatibility with biological tissues by detecting irritation potential.
• Test Procedure: Material extracts are prepared with extraction vehicles such as saline or vegetable oil (as per ASTM F619) to simulate human body fluids, then injected into albino rabbits.
• Assessment Criteria: The reaction at injection sites is monitored over 72 hours for signs of erythema (redness), edema (swelling), eschar, or necrosis.
• Reporting: Severity of tissue reactions is recorded using standardized rating scales. Pass/fail criteria are based on statistical comparison of test and control sites.
• Limitations: Not all material types (e.g., highly acidic or alkaline extracts) are appropriate for this test. The extraction process and vehicle selection are key to valid results.
• Ethical Considerations: Animal care and use must conform to established guidelines and ethical standards.

Applications

The practical value of ASTM F749-20 lies in its broad applicability within the medical device industry, where it serves as a key part of material safety assessments prior to regulatory submissions and market release. Common applications include:

• Preclinical Product Evaluation: Used for initial screening of new or modified materials to ensure no harmful leachable substances before proceeding to more extensive animal or clinical testing.
• Regulatory Submissions: Results from ASTM F749-20 support compliance with domestic and international medical device regulations that require evidence of biocompatibility.
• Quality Control: Manufacturers apply this standard as part of their quality management systems to routinely verify the safety of manufacturing lots or new suppliers.
• Material Selection: R&D teams utilize findings from intracutaneous injection testing to make informed choices about biomaterials suited for specific medical device applications.
• Support for Implantable Devices: Though used for a variety of products, the method is generally suited for devices or components with tissue or temporary body contact, as more complex or absorbable implantables may require alternative tests.

Related Standards

Implementing ASTM F749-20 often involves coordination with other standards for a comprehensive biocompatibility assessment. Key related standards include:

• ASTM F619 - Practice for Extraction of Medical Plastics: Provides the detailed procedure for preparing extracts from test materials using various vehicles.
• ASTM F748 - Practice for Selecting Generic Biological Test Methods for Materials and Devices: Offers guidance on choosing appropriate biological evaluation methods depending on the application and material.
• ISO 10993 Series - Biological Evaluation of Medical Devices: While ASTM F749 focuses on irritancy in rabbits, ISO 10993 covers a broader range of biocompatibility evaluations and is frequently referenced for global regulatory compliance.
• U.S. Pharmacopeia <88> Biological Reactivity Tests, In Vivo: Complements information from ASTM F749 regarding animal testing protocols.

By following the protocols outlined in ASTM F749-20, medical device professionals can systematically assess the irritation potential of material extracts, bolstering both patient safety and regulatory compliance in the dynamic field of medical device development.

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: F749 − 20
Standard Practice for
Evaluating Material Extracts by Intracutaneous Injection in
the Rabbit
ThisstandardisissuedunderthefixeddesignationF749;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginal
adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.Asuperscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope or other extraction vehicles can be used, as described in
PracticeF619.Theextractliquidisinjectedintorabbitsandthe
1.1 This practice is an intracutaneous reactivity test used to
animals are observed at regular intervals for 72 h for erythema,
assess the potential of the material under test to produce
edema, eschar formation or necrosis.
irritation following intradermal injections of extracts of the
material.
4. Significance and Use
1.2 The liquids injected into the rabbits are those obtained
4.1 This practice is to be used to help assess the biocom-
by Practice F619 where the extraction vehicles are saline,
patibility of materials used in medical devices. It is an acute
vegetable oil, or other liquids simulating human body fluids.
toxicological test designed to evaluate any irritation caused by
1.3 This practice is one of several developed for the
device materials by gross assessment.
assessment of the biocompatibility of materials. Practice F748
4.2 This practice may not be appropriate for all types of
may provide guidance for the selection of appropriate methods
implant applications. The user is cautioned to consider the
for testing materials for a specific application.
appropriateness of this practice in view of the materials being
1.4 The values stated in SI units, including units officially
tested, their potential applications, and the recommendations
accepted for use with the SI, are to be regarded as standard. No
contained in Practice F748.
other systems of measurement are included in this standard.
NOTE 1—Some materials (e.g., absorbables) may result in an extract pH
1.5 This international standard was developed in accor-
(e.g., ≤2.0 or ≥11.5) that cannot be used with this practice.
dance with internationally recognized principles on standard-
4.3 The only applicable limitation is the extract preparation.
ization established in the Decision on Principles for the
Refer to Section 4.3 of Practice F619 for a description of this
Development of International Standards, Guides and Recom-
limitation.
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
5. Apparatus
2. Referenced Documents
5.1 Cages—There shall be one cage for each rabbit exposed
to one extract liquid. Each rabbit shall be uniquely identified
2.1 ASTM Standards:
with this identity recorded.
F619 Practice for Extraction of Medical Plastics
F748 PracticeforSelectingGenericBiologicalTestMethods
5.2 Syringes—Sterile syringes, not greater than 2 mL in
for Materials and Devices volume, with a precision of no less than 60.10 mL shall be
used. Sterile needles of 21 to 26 gauge shall be used.
3. Summary of Practice
6. Test Animals
3.1 The extract liquid is prepared in accordance with Prac-
tice F619. The extraction vehicles are saline and vegetable oil,
6.1 Rabbits—The rabbits shall be healthy thin-skinned al-
binotype,notpreviouslyusedforanytest.Animalcareshallbe
in accordance with the Guide for Care and Use of Laboratory
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Animals. Rabbits with significant scars or wounds are not
Surgical Materials and Devices and is the direct responsibility of Subcommittee
suitableforthistest.Aminimumofthreerabbitsareusedinthe
F04.16 on Biocompatibility Test Methods.
Current edition approved Feb. 1, 2020. Published March 2020. Originally
approved in 1982. Last previous edition approved in 2013 as F749 – 13. DOI:
10.1520/F0749-20.
2 3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or National Research Council Guide for the Care and Use of LaboratoryAnimals,
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM 8th ed. (2011), Institute of LaboratoryAnimal Research Division on Earth and Life
Standards volume information, refer to the standard’s Document Summary page on Sciences, Washington, D.C., National Academies of Science Press (http://
the ASTM website. www.nap.edu/catalog.php?record_id=12910).
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F749 − 20
test. If the results of the first test are inconclusive, three more cause skin irritation in place of or in addition to clipping may
rabbits will be needed to complete the test. bedesirable.Swabtheskingentlywithdilutedalcohol,anddry
6.1.1 During the test, the rabbits shall be fed appropriate the skin prior to injection.
laboratory animal diet and potable, uncontaminated drinking
9.2 Agitate each extract liquid vigorously prior to with-
water.
drawal of each injection dose to ensure even distribution of the
extracted matter. If the extract liquid appears to contain
7. Sampling
particulates, record this and consider it when reporting the
7.1 Sample in accordance with Practice F619.
results.
8. Sample and Test Specimen 9.3 Inject intracutaneously 0.2 mL of the sample extract
liquid per site at five sites on one side of the dorsal midline of
8.1 The sample is the extract of the test article (that is,
each of three rabbits. Similarly, at five other sites on the other
plastic or other material) exposed to the extraction procedure.
side of the dorsal midline of each rabbit, inject 0.2 mL per site
As a result of the extraction in Practice F619, for each
of the corresponding blank extract liquid. See Fig. 1.
extraction vehicle there are available:(1) sample extract liquid,
9.4 Examine the injected sites 24, 48, and 72 h after the
and (2) a blank extract liquid. These extract liquids are to be
injected into the test animals within 24 h of the end of the injection for gross evidence of tissue reaction, such as
e
...

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: F749 − 13 F749 − 20
Standard Practice for
Evaluating Material Extracts by Intracutaneous Injection in
the Rabbit
This standard is issued under the fixed designation F749; 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 is a nonspecific, acute toxicity an intracutaneous reactivity test used to help determine assess the
biocompatibility of materials used in medical devices.potential of the material under test to produce irritation following intradermal
injections of extracts of the material.
1.2 The liquids injected into the rabbits are those obtained by Practice F619 where the extraction vehicles are saline, vegetable
oil, or other liquids simulating human body fluids.
1.3 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.
1.4 The values stated in SI units units, including units officially accepted for use with the SI, are to be regarded as standard.
No other unitssystems of measurement are included in this standard.
1.5 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
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
3. Summary of Practice
3.1 The extract liquid is prepared in accordance with Practice F619. The extraction vehicles are saline and vegetable oil, or other
extraction vehicles can be used, as described in Practice F619. The extract liquid is injected into rabbits and the animals are
observed at regular intervals for 72 h for erythema, edema, eschar formation or necrosis.
4. Significance and Use
4.1 This practice is to be used to help assess the biocompatibility of materials used in medical devices. It is an acute
toxicological test designed to detect the presence of injurious leachable substances.evaluate any irritation caused by device
materials by gross assessment.
4.2 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the
appropriateness of the methodthis practice in view of the materials being tested, their potential applications, and the
recommendations contained in Practice F748.
NOTE 1—Some materials (e.g., absorbables) may result in an extract pH (e.g., ≤2.0 or ≥11.5) that cannot be used with this practice.
4.3 The only applicable limitation is the extract preparation. Refer to SectionsSection 4.3 and 4.4 of Practice F619 for a
description of this limitation.
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 March 1, 2013Feb. 1, 2020. Published March 2013March 2020. Originally approved in 1982. Last previous edition approved in 20122013 as
F749 – 98 (2012).F749 – 13. DOI: 10.1520/F0749-13.10.1520/F0749-20.
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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F749 − 20
5. Apparatus
5.1 Cages—There shall be one cage for each rabbit exposed to one extract liquid. Each rabbit shall be uniquely identified with
this identity recorded.
5.2 Syringes—Sterile syringes, not greater than 2 mL in volume, with a precision of no less than 60.10 mL shall be used. Sterile
needles of 21 to 26 gauge shall be used.
6. Test Animals
6.1 Rabbits—The rabbits shall be healthy thin-skinned albino type, not previously used for any test. Animal care shall be in
accordance with the Guide for Care and Use of Laboratory Animals. Rabbits with significant scars or wounds are not suitable for
this test. For each extraction vehicle, a A minimum of three rabbits are used in the test. If the results of the first test are inconclusive,
three more rabbits will be needed to complete the test with that extraction vehicle for one material.test.
6.1.1 During the test, the rabbits shall be fed normally, with commercially available feed and tap appropriate laboratory animal
diet and potable, uncontaminated drinking water.
7. Sampling
7.1 Sample in accordance with Practice F619.
8. Sample and Test Specimen
8.1 The sample is the extract of the test article (that is, plastic or other material) exposed to the extraction procedure. As a result
of the extraction in Practice F619, for each extraction vehicle there are available: (1(1)) sample extract liquid, and (2(2)) a blank
extract liquid. These extract liquids are to be injected into the test animals within 24 h of the end of the extraction procedure.
Record storage conditions if not used immediately after preparation.
8.1.1 There are usually four extract liquids prepared from two extraction vehicles available for test, those based on saline and
vegetable oil. Samples based on other extraction vehicles may be available, as described in Practice F619, or as required by the
standard for the medical device. Both extracts and blank controls shall be subjected to the same extraction temperature and time
prior to use in the test animals.
8.2 The test specimen is the combination of the test site and 0.2 mL of the injected extract liquid. A total of 10 sites are to be
injected with the sample extract liquid and 10 sites with the blank extract liquid.(i.e., 5 sites with saline and 5 sites with vegetable
oil extracts) and 10 sites (i.e., 5 each) with the corresponding blank extract liquids.
9. Procedure
9.1 Preparation of Rabbits—On the day (no more than 24 h) before the test, closely clip the fur on the animal’s back on both
sides of the spinal column over a sufficiently large test area. Avoid mechanical irritation and trauma. Remove loose hair by means
of a vacuum. The use of a depilatory agent that does not cause skin irritation in place of or in addition to clipping may be desirable.
Swab the skin slightlygently with diluted alcohol, and dry the skin prior to injection.
9.2 Agitate each extract liquid vigorously prior to withdrawal of each injection dose to ensure even distribution of the extracted
matter. If the extract liquid appears to contain particulates, record this and consider it when reporting the results.
9.3 Inject intracutaneously 0.2 mL of the sample extract liquid per site at five sites on one side of the dorsal midline of each
of twothree rabbits. Similarly, at five
...