ASTM F2103-01
(Guide)Standard Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications
Standard Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications
SCOPE
1.1 This guide covers the evaluation of chitosan salts suitable for use in biomedical or pharmaceutical applications, or both, including, but not limited to, tissue-engineered medical products (TEMPS).
1.2 This guide addresses key parameters relevant for the functionality, characterization, and purity of chitosan salts.
1.3 As with any material, some characteristics of chitosan may be altered by processing techniques (such as molding, extrusion, machining, assembly, sterilization, and so forth) required for the production of a specific part or device. Therefore, properties of fabricated forms of this polymer should be evaluated using test methods that are appropriate to ensure safety and efficacy.
1.4 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 and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:F2103–01
Standard Guide for
Characterization and Testing of Chitosan Salts as Starting
Materials Intended for Use in Biomedical and Tissue-
Engineered Medical Product Applications
This standard is issued under the fixed designation F 2103; 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.
INTRODUCTION
Biopolymers from marine sources have been studied and used in commercial applications and
product development for a number of years. Chitosan, a linear polysaccharide consisting of
glucosamine and N-acetyl glucosamine derived mainly from crustacean shells, has been used in many
technical applications such as water purification (as a flocculant), in cosmetics, and recently as a
proposed fat-binding weight control product. In solution, the cationic nature of chitosan gives this
polymer a mucoadhesive property. Chitosan salts can be used as a matrix or scaffold material as well
asinnonparenteraldeliverysystemsforchallengingdrugs.Chitosansaltshavebeenshowntoincrease
the transport of polar drugs across the nasal epithelial surface. The purpose of this guide is to identify
keyparametersrelevantforthefunctionalityandcharacterizationofchitosansaltsforthedevelopment
of new commercial applications of chitosan salts for the biomedical and pharmaceutical industries.
1. Scope D 2196 Test Methods for Rheological Properties of Non-
Newtonian Materials by Rotational (Brookfield) Viscom-
1.1 This guide covers the evaluation of chitosan salts
eter
suitable for use in biomedical or pharmaceutical applications,
F 619 Practice for Extraction of Medical Plastics
or both, including, but not limited to, tissue-engineered medi-
F 748 Practice for Selecting Generic Biological Test Meth-
cal products (TEMPS).
ods for Materials and Devices
1.2 This guide addresses key parameters relevant for the
F 749 Practice for Evaluating Material Extracts by Intracu-
functionality, characterization, and purity of chitosan salts.
taneous Injection in the Rabbit
1.3 As with any material, some characteristics of chitosan
F 756 Practice for Assessment of Hemolytic Properties of
may be altered by processing techniques (such as molding,
Materials
extrusion, machining, assembly, sterilization, and so forth)
F 763 Practice for Short-Term Screening of Implant Mate-
required for the production of a specific part or device.
rials
Therefore, properties of fabricated forms of this polymer
F 813 PracticeforDirectContactCellCultureEvaluationof
should be evaluated using test methods that are appropriate to
Materials for Medical Devices
ensure safety and efficacy.
F 895 Test Method forAgar Diffusion Cell Culture Screen-
1.4 This standard does not purport to address all of the
ing for Cytotoxicity
safety concerns, if any, associated with its use. It is the
F 981 Practice for Assessment of Compatibility of Bioma-
responsibility of the user of this standard to establish appro-
terials for Surgical Implants with Respect to Effect of
priate safety and health practices and determine the applica-
Materials on Muscle and Bone
bility of regulatory limitations prior to use.
F 1251 Terminology Relating to Polymeric Biomaterials in
2. Referenced Documents
Medical and Surgical Devices
F 1439 Guide for Performance of Lifetime Bioassay for the
2.1 ASTM Standards:
Tumorigenic Potential of Implant Materials
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.42 on Tissue Characterization. Annual Book of ASTM Standards, Vol 06.01.
Current edition approved May 10, 2001. Published June 2001. Annual Book of ASTM Standards, Vol 13.01.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2103
F 1903 Practice for Testing for Biological Responses to International Conference on Harmonization (1997) S1C[R]
Particles In Vitro Guidance for Industry Addendum to Dose Selection for
F 1904 Practice for Testing the Biological Responses to Carcinogenicity Studies of Pharmaceuticals:Addition of a
3 6
Particles In Vivo Limit Dose and Related Notes 62 FR 64259
F 1905 Practice for Selecting Tests for Determining the International Conference on Harmonization (ICH) Q1AICH
Propensity of Materials to Cause Immunotoxicity Harmonized Tripartite Guidance for Stability Testing of
F 1906 Practice for Evaluation of Immune Responses in New Drug Substances and Products (September 23,
Biocompatibility Testing Using ELISATests, Lymphocyte 1994)
Proliferation, and Cell Migration 2.5 FDA Documents:
2.2 Ph. Eur. Document: FDA Guideline on Validation of the Limulus Amebocyte
Ph. Eur. Monograph Chitosan Chloride, Nov. 2000 Test as an End-Product Endotoxin Test for Human and
2.3 ISO Documents: Animal Parenteral Drugs, Biological Products and Health-
5 7
ISO 10993 Biological Evaluation of Medical Devices care Products DHHS, December 1987
ISO 10993-1 Biological Evaluation of Medical Devices— FDA Interim Guidance for Human and Veterinary Drug
Part 1: Evaluation and Testing Products and Biologicals. Kinetic LAL Techniques-
ISO10993-3—Part3: TestsforGenotoxicity,Carcinogenic- DHHS, July 15, 1991
ity and Reproductive Toxicity 2.6 ANSI Documents:
ISO 10993-9—Part 9: Framework for Identification and ANSI/AAMI/ISO 11737-1: 1995 Sterilization of Medical
Quantification of Potential Degradation Products Devices—Microbiological Methods—Part 1: Estimation
ISO 10993-17—Part 17: Methods for Establishment of of Bioburden on Product
AllowableLimitsforLeachableSubstancesUsingHealth- ANSI/AAMI/ISO 11737-2: 1998 Sterilization of Medical
Based Risk Assessment Devices—Microbiological Methods—Part 2:Tests of Ste-
ISO 13408-1: 1998: Aseptic Processing of Health Care rility Performed in the Validation of a Sterilization Pro-
5 5
Products—Part 1: General Requirements cess
2.4 ICH Documents: 2.7 AAMI Documents:
International Conference on Harmonization (1997) Guid- AAMI TIR No. 19—1998: Guidance for ANSI/AAMI/ISO
ance for Industry M3 Nonclinical Safety Studies for the 10993–7: 1995, Biological Evaluation of Medical
Conduct of Human ClinicalTrials for Pharmaceuticals 62 Devices—Part 7: Ethylene Oxide Sterilization Residuals
FR 62922 AAMI/ISO 14160—1998: Sterilization of Single-Use
International Conference on Harmonization (1996) Guide- Medical Devices Incorporating Materials of Animal
line for Industry S2A Specific Aspects of Regulatory Origin—Validation and Routine Control of Sterilization
6 8
Genotoxicity Tests for Pharmaceuticals 61 FR 18199 by Liquid Chemical Sterilants
International Conference on Harmonization (1997) Guid- AAMI ST67/CDV-2: 1999: Sterilization of Medical
ance for Industry S2B Genotoxicity: A Standard Battery Devices—Requirements for Products Labeled “Sterile”
for Genotoxicity Testing of Pharmaceuticals 62 FR 2.8 EN Documents:
62472 EN 12442-1 Animal Tissues and Their Derivative Utilized
International Conference on Harmonization (1994) Guide- in the Manufacture of Medical Devices—Part 1:Analysis
line for Industry S5A Detection of Toxicity to Reproduc- and Management of Risk
tion for Medicinal Products 59 FR 48746 EN 12442-Part 3: Validation of the Elimination and/or
International Conference on Harmonization (1996) Guid- Inactivation of Virus and Transmissible Agents
ance for Industry S5B Detection of Toxicity to Reproduc-
3. Terminology
tion for Medicinal Products: Addendum on Toxicity to
Male Fertility 61 FR 15360
3.1 Definitions:
International Conference on Harmonization (1996) Guide- 3.1.1 chitosan, n—a linear polysaccharide consisting of
line for Industry S1A The Need for Long-term Rodent
b(1→4) linked 2-acetamido-2-deoxy-D-glucopyranose
Carcinogenicity Studies of Pharmaceuticals 61 FR 8153 (GlcNAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN).
International Conference on Harmonization (1998) Guid- Chitosan is a polysaccharide derived by N-deacetylation of
ance for Industry S1B Testing for Carcinogenicity of chitin.
Pharmaceuticals 63 FR 8983 3.1.2 decomposition,n—structuralchangesofchitosansasa
International Conference on Harmonization (1995) Guide- result of exposure to environmental, chemical, or thermal
line for Industry S1C Dose Selection for Carcinogenicity factors, such as temperatures greater than 200°C. Decomposi-
Studies of Pharmaceuticals 60 FR 11278 tion can result in deleterious changes to the chitosan.
4 7
Available from EDQM, Publications and Services European Pharmacopoeia, Available from U.S. Food and DrugAdministration, 5600 Fishers Lane,
BP 907 226, avenue de Colmar, F-67029 Strasbourg Cedex 1, France. Rockville, MD 20857–0001.
5 8
Available from American National Standards Institute, 25 W. 43rd St., 4th Association for the Advancement of Medical Instrumentation, 111 N. Glebe
Floor, New York, NY 10036. Rd., Suite 220, Arlington, VA 22201–4795.
6 9
Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, PO Box 758, Available from European Committee for Standardization, CEN Management
1211 Geneva 13, Switzerland. Centre, 36 rue de Stassart, B-1050 Brussels, Belgium.
F2103
3.1.3 degradation, n—change in the chemical structure, devicesforimplantationasdeterminedtobeappropriate,based
physical properties, or appearance of a material. Degradation on supporting biocompatibility and physical test data. Recom-
of polysaccharides occurs by means of cleavage of the glyco- mendations in this guide should not be interpreted as a
sidic bonds, usually by acid catalyzed hydrolysis. Degradation guarantee of clinical success in any tissue-engineered medical
can also occur thermally. Note that degradation is not synony- product or drug delivery application.
mous with decomposition. Degradation is often used as a 4.3 To ensure that the material supplied satisfies require-
synonym for depolymerization when referring to polymers. ments for use in TEMPS, several general areas of character-
3.1.4 degree of deacetylation, n—the fraction or percentage ization should be considered. These are: identity of chitosan,
of glucosamine units (deacetylated monomers) in a chitosan physical and chemical characterization and testing, impurities
polymer molecule. profile, and performance-related tests.
3.1.5 depolymerization, n—reductioninlengthofapolymer
5. Chemical and Physical Test Methods
chain to form shorter polymeric units. Depolymerization may
reduce the polymer chain to oligomeric or monomeric units, or
5.1 Identity of Chitosan—The identity of chitosan and
both. In chitosan, hydrolysis of the glycosidic bonds is the
chitosan salts can be established by several methods including,
primary mechanism.
but not limited to the following:
3.1.6 endotoxin, n—a high molecular weight lipopolysac-
5.1.1 Chitosan chloride monograph Ph. Eur.
charide (LPS) complex associated with the cell wall of
5.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)—
gram-negative bacteria that is pyrogenic in humans. Though
Almost all organic chemical compounds absorb infrared radia-
endotoxins are pyrogens, not all pyrogens are endotoxins.
tion at frequencies characteristic for the functional groups in
3.1.7 molecular mass average (molecular weight average),
the compound. A FT-IR spectrum will show absorption bands
n—the given molecular weight (Mw) of a chitosan will always
relating to bond stretching and bending and can therefore serve
represent an average of all of the molecules in the population.
as a unique fingerprint of a specific compound. Cast a chitosan
The most common ways to express the Mw are as the number
film from a 0.25 % (w/v) solution of chitosan (in 1 % acetic
– –
acid) or chitosan salt (dissolved in water) by drying approxi-
average (M ) and the weight average (M ). The two averages
n w
mately 500 µL of the sample onto a disposable IR card for 3
are defined by the following equations:
to4hat 60°C. Record a background spectrum between 4000
NM
(
– i i i -1
and 400 cm-1 using 128 scans at a resolution of 4 cm . Record
M 5
n
N
(i i
the IR spectrum of a dried blank IR card, then record the IR
and
spectrum of the sample using 128 scans at a resolution of 4
-1
2 cm , percent transmission mode. Label the peaks. Typical
WM NM
(i i i (i i i
–
-1
M 5 5
w frequencies (cm ) for chitosan are as follows:
W NM
( (
i i i i i
Chitosan Base Chitosan Chloride Chitosan Glutamate
(as Acetate)
where:
N = number of molecules having a specific molecular
i
3362b 3344b 1555b
weight M and
1556 1605 1396
i
1406 1513 1154
w = weight of molecules having a specific molecular
i
1153 1379 1085s
weight M. In a polydisperse molecular population the
i
1083s 1154
– – –
1086s
relation M > M is always valid. The coefficient M /
w n w
–
The peak designators are: sh: sharp; s: strong; m: medium;
M is referred to as the polydispersity index, and will
n
typically be in the range 1.5 to 3.0 for commercial w: weak; and b: broad.
5.2 Physical and Chemical Characterization of Chitosan:
chitosans.
5.2.1 The composition and sequential structure of chitosan
3.1.8 pyrogen, n—any substance that produces fever when
can be a key functional attribute of any chitosan or chitosan
administered parenterally.
salt. Variations in the composition or the sequential structure,
4. Significance and Use or both, may, but not necessarily, cause differences in perfor-
mance of a chitosan in a particular end use. This information
4.1 This guide contains a listing of those characterization
may be determined by the following method: High-
parameters that are directly related to the functionality of
1 13
resolution H- and C-nuclear magnetic resonance spectros-
chitosan. This guide can be used as an aid in the selection and
copy (NMR).
characterization of the appropriate chitosan or chitosan salt for
5.2.2 The degree of deacetylation of chitosan can be estab-
a particular application. This standard is intended to give
lished using a number of techniques including, but not limited
guidance in the methods and types of testing necessary to
to, the following:
properly characterize, assess, and ensure consistency in the
1 13
5.2.2.1 High-resolution H- and C-Nuclear Magnetic
performance of a particular chitosan. It may have use in the
Resonance Spectroscopy (NMR)—Chitosan salts should be
regulation of devices containing chitosan by appropriate au-
thorities.
4.2 The chitosan salts covered by this guide may be gelled,
extruded, or otherwise formulated into biomedical devices for
No suitable commercially available IR cards are available for the IR analysis
use as tiss
...
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