ASTM F2103-01(2007)
(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.
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 (Reapproved 2007)
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 2. Referenced Documents
1.1 This guide covers the evaluation of chitosan salts 2.1 ASTM Standards:
suitable for use in biomedical or pharmaceutical applications, D 2196 Test Methods for Rheological Properties of Non-
or both, including, but not limited to, tissue-engineered medi- Newtonian Materials by Rotational (Brookfield type) Vis-
cal products (TEMPS). cometer
1.2 This guide addresses key parameters relevant for the F 619 Practice for Extraction of Medical Plastics
functionality, characterization, and purity of chitosan salts. F 748 Practice for Selecting Generic Biological Test Meth-
1.3 As with any material, some characteristics of chitosan ods for Materials and Devices
may be altered by processing techniques (such as molding, F 749 Practice for Evaluating Material Extracts by Intracu-
extrusion, machining, assembly, sterilization, and so forth) taneous Injection in the Rabbit
required for the production of a specific part or device. F 756 Practice for Assessment of Hemolytic Properties of
Therefore, properties of fabricated forms of this polymer Materials
should be evaluated using test methods that are appropriate to F 763 Practice for Short-Term Screening of Implant Mate-
ensure safety and efficacy. rials
1.4 This standard does not purport to address all of the F 813 PracticeforDirectContactCellCultureEvaluationof
safety concerns, if any, associated with its use. It is the Materials for Medical Devices
responsibility of the user of this standard to establish appro- F 895 Test Method forAgar Diffusion Cell Culture Screen-
priate safety and health practices and determine the applica- ing for Cytotoxicity
bility of regulatory limitations prior to use. F 981 Practice for Assessment of Compatibility of Bioma-
terials for Surgical Implants with Respect to Effect of
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee For referenced ASTM standards, visit the ASTM website, www.astm.org, or
F04.42 on Biomaterials and Biomolecules for TEMPs. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved Feb. 1, 2007. Published February 2007. Originally Standards volume information, refer to the standard’s Document Summary page on
approved in 2001. Last previous edition approved in 2001 as F 2103 – 01. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2103–01 (2007)
Materials on Muscle and Bone ance for Industry S1B Testing for Carcinogenicity of
F 1251 Terminology Relating to Polymeric Biomaterials in Pharmaceuticals 63 FR 8983
Medical and Surgical Devices
International Conference on Harmonization (1995) Guide-
F 1439 Guide for Performance of Lifetime Bioassay for the
line for Industry S1C Dose Selection for Carcinogenicity
Tumorigenic Potential of Implant Materials
Studies of Pharmaceuticals 60 FR 11278
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
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:
AnimalParenteralDrugs,BiologicalProductsandHealth-
ISO 10993 Biological Evaluation of Medical Devices 6
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- 6
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-
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
5 Origin—Validation and Routine Control of Sterilization
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
5 2.8 EN Documents:
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-
tion for Medicinal Products: Addendum on Toxicity to
3. Terminology
Male Fertility 61 FR 15360
International Conference on Harmonization (1996) Guide-
3.1 Definitions:
line for Industry S1A The Need for Long-term Rodent
3.1.1 chitosan, n—a linear polysaccharide consisting of
Carcinogenicity Studies of Pharmaceuticals 61 FR 8153
b(1→4) linked 2-acetamido-2-deoxy-D-glucopyranose
International Conference on Harmonization (1998) Guid-
3 6
Available from EDQM, Publications and Services European Pharmacopoeia, Available from Food and Drug Administration (FDA), 5600 Fishers Ln.,
BP 907 226, avenue de Colmar, F-67029 Strasbourg Cedex 1, France. Rockville, MD 20857, http://www.fda.gov.
4 7
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St., Association for the Advancement of Medical Instrumentation, 111 N. Glebe
4th Floor, New York, NY 10036, http://www.ansi.org. Rd., Suite 220, Arlington, VA 22201–4795.
5 8
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.
F2103–01 (2007)
(GlcNAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN). properly characterize, assess, and ensure consistency in the
Chitosan is a polysaccharide derived by N-deacetylation of performance of a particular chitosan. It may have use in the
chitin. regulation of devices containing chitosan by appropriate au-
3.1.2 decomposition,n—structuralchangesofchitosansasa thorities.
result of exposure to environmental, chemical, or thermal
4.2 The chitosan salts covered by this guide may be gelled,
factors, such as temperatures greater than 200°C. Decomposi-
extruded, or otherwise formulated into biomedical devices for
tion can result in deleterious changes to the chitosan.
use as tissue-engineered medical products or drug delivery
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
chain to form shorter polymeric units. Depolymerization may
5. Chemical and Physical Test Methods
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
– –
average (M ) and the weight average (M ). The two averages
n w
acid) or chitosan salt (dissolved in water) by drying approxi-
are defined by the following equations:
mately 500 µL of the sample onto a disposable IR card for 3
NM to4hat 60°C. Record a background spectrum between 4000
(i i i
–
M 5 -1
n
N and400cm-1using128scansataresolutionof4cm .Record
(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
WM NM
( (
– i i i i i i cm , percent transmission mode. Label the peaks. Typical
M 5 5
w
-1
W NM
( (
i i i i i frequencies (cm ) for chitosan are as follows:
Chitosan Base Chitosan Chloride Chitosan Glutamate
where:
(as Acetate)
N = number of molecules having a specific molecular
i
3362b 3344b 1555b
weight M and
i
1556 1605 1396
w = weight of molecules having a specific molecular
i
1406 1513 1154
weight M. In a polydisperse molecular population the
1153 1379 1085s
i
– – –
1083s 1154
relation M > M is always valid. The coefficient M /
w n w
1086s
–
M is referred to as the polydispersity index, and will
n
The peak designators are: sh: sharp; s: strong; m: medium;
typically be in the range 1.5 to 3.0 for commercial
w: weak; and b: broad.
chitosans.
5.2 Physical and Chemical Characterization of Chitosan:
3.1.8 pyrogen, n—any substance that produces fever when
5.2.1 The composition and sequential structure of chitosan
administered parenterally.
can be a key functional attribute of any chitosan or chitosan
salt. Variations in the composition or the sequential structure,
4. Significance and Use
or both, may, but not necessarily, cause differences in perfor-
4.1 This guide contains a listing of those characterization
mance of a chitosan in a particular end use. This information
parameters that are directly related to the functionality of
chitosan. This guide can be used as an aid in the selection and
characterization of the appropriate chitosan or chitosan salt for
a particular application. This standard is intended to give
NosuitablecommerciallyavailableIRcardsareavailablefortheIRanalysisof
guidance in the methods and types of testing necessary to chitosan glutamate salt. Alternati
...
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