1、Designation: F 2103 01 (Reapproved 2007)e2Standard Guide forCharacterization and Testing of Chitosan Salts as StartingMaterials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications1This standard is issued under the fixed designation F 2103; the number immediately followi
2、ng the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.e1NOTEFormatting and grammar w
3、ere corrected editorially throughout in April 2007.e2NOTEMercury warning was editorially added in April 2008.INTRODUCTIONBiopolymers from marine sources have been studied and used in commercial applications andproduct development for a number of years. Chitosan, a linear polysaccharide consisting of
4、glucosamine and N-acetyl glucosamine derived mainly from crustacean shells, has been used in manytechnical applications such as water purification (as a flocculant), in cosmetics, and recently as aproposed fat-binding weight control product. In solution, the cationic nature of chitosan gives thispol
5、ymer a mucoadhesive property. Chitosan salts can be used as a matrix or scaffold material as wellas in non-parenteral delivery systems for challenging drugs. Chitosan salts have been shown toincrease the transport of polar drugs across the nasal epithelial surface. The purpose of this guide isto ide
6、ntify key parameters relevant for the functionality and characterization of chitosan salts for thedevelopment of new commercial applications of chitosan salts for the biomedical and pharmaceuticalindustries.1. Scope1.1 This guide covers the evaluation of chitosan saltssuitable for use in biomedical
7、or pharmaceutical applications,or both, including, but not limited to, tissue-engineered medi-cal products (TEMPS).1.2 This guide addresses key parameters relevant for thefunctionality, characterization, and purity of chitosan salts.1.3 As with any material, some characteristics of chitosanmay be al
8、tered 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 polymershould be evaluated using test methods that are appropriate toensure safety an
9、d efficacy.1.4 WarningMercury has been designated by EPA andmany state agencies as a hazardous material that can causecentral nervous system, kidney, and liver damage. Mercury, orits vapor, may be hazardous to health and corrosive tomaterials. Caution should be taken when handling mercury andmercury
10、-containing products. See the applicable product Ma-terial Safety Data Sheet (MSDS) for details and EPAs website(http:/www.epa.gov/mercury/faq.htm) for additional informa-tion. Users should be aware that selling mercury or mercury-containing products, or both, in your state may be prohibited bystate
11、 law.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referen
12、ced Documents2.1 ASTM Standards:2D 2196 Test Methods for Rheological Properties of Non-Newtonian Materials by Rotational (Brookfield type) Vis-cometerF 619 Practice for Extraction of Medical PlasticsF 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and Devices1This guide is
13、 under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Feb. 1, 2007. Published February 2007. Originallyapproved in 2001. Last previous editi
14、on approved in 2001 as F 2103 01.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM Internationa
15、l, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.F 749 Practice for Evaluating Material Extracts by Intracu-taneous Injection in the RabbitF 756 Practice for Assessment of Hemolytic Properties ofMaterialsF 763 Practice for Short-Term Screening of Implant Mate-ri
16、alsF 813 Practice for Direct Contact Cell Culture Evaluation ofMaterials for Medical DevicesF 895 Test Method for Agar Diffusion Cell Culture Screen-ing for CytotoxicityF 981 Practice for Assessment of Compatibility of Bioma-terials for Surgical Implants with Respect to Effect ofMaterials on Muscle
17、and BoneF 1251 Terminology Relating to Polymeric Biomaterials inMedical and Surgical DevicesF 1439 Guide for Performance of Lifetime Bioassay for theTumorigenic Potential of Implant MaterialsF 1903 Practice for Testing For Biological Responses toParticles in vitroF 1904 Practice for Testing the Biol
18、ogical Responses toParticles in vivoF 1905 Practice For Selecting Tests for Determining thePropensity of Materials to Cause ImmunotoxicityF 1906 Practice for Evaluation of Immune Responses InBiocompatibility Testing Using ELISA Tests, LymphocyteProliferation, and Cell Migration2.2 Ph. Eur. Document:
19、Ph. Eur. Monograph Chitosan Chloride, Nov. 200032.3 ISO Documents:ISO 10993 Biological Evaluation of Medical Devices4ISO 10993-1 Biological Evaluation of Medical DevicesPart 1: Evaluation and Testing4ISO 10993-3Part 3: Tests for Genotoxicity, Carcinogenic-ity and Reproductive Toxicity4ISO 10993-9Par
20、t 9: Framework for Identification andQuantification of Potential Degradation Products4ISO 10993-17Part 17: Methods for Establishment ofAllowable Limits for Leachable Substances Using Health-Based Risk Assessment4ISO 13408-1: 1998: Aseptic Processing of Health CareProductsPart 1: General Requirements
21、42.4 ICH Documents:International Conference on Harmonization (1997) Guid-ance for Industry M3 Nonclinical Safety Studies for theConduct of Human Clinical Trials for Pharmaceuticals 62FR 629225International Conference on Harmonization (1996) Guide-line for Industry S2A Specific Aspects of RegulatoryG
22、enotoxicity Tests for Pharmaceuticals 61 FR 181995International Conference on Harmonization (1997) Guid-ance for Industry S2B Genotoxicity: A Standard Batteryfor Genotoxicity Testing of Pharmaceuticals 62 FR624725International Conference on Harmonization (1994) Guide-line for Industry S5A Detection
23、of Toxicity to Reproduc-tion for Medicinal Products 59 FR 487465International Conference on Harmonization (1996) Guid-ance for Industry S5B Detection of Toxicity to Reproduc-tion for Medicinal Products: Addendum on Toxicity toMale Fertility 61 FR 153605International Conference on Harmonization (1996
24、) Guide-line for Industry S1A The Need for Long-term RodentCarcinogenicity Studies of Pharmaceuticals 61 FR 81535International Conference on Harmonization (1998) Guid-ance for Industry S1B Testing for Carcinogenicity ofPharmaceuticals 63 FR 89835International Conference on Harmonization (1995) Guide
25、-line for Industry S1C Dose Selection for CarcinogenicityStudies of Pharmaceuticals 60 FR 112785International Conference on Harmonization (1997) S1CRGuidance for Industry Addendum to Dose Selection forCarcinogenicity Studies of Pharmaceuticals: Addition of aLimit Dose and Related Notes 62 FR 642595I
26、nternational Conference on Harmonization (ICH) Q1A ICHHarmonized Tripartite Guidance for Stability Testing ofNew Drug Substances and Products (September 23,1994)52.5 FDA Documents:FDA Guideline on Validation of the Limulus AmebocyteTest as an End-Product Endotoxin Test for Human andAnimal Parenteral
27、 Drugs, Biological Products and Health-care Products DHHS, December 19876FDA Interim Guidance for Human and Veterinary DrugProducts and Biologicals. Kinetic LAL Techniques-DHHS, July 15, 199162.6 ANSI Documents:ANSI/AAMI/ISO 11737-1: 1995 Sterilization of MedicalDevicesMicrobiological MethodsPart 1:
28、 Estimationof Bioburden on Product4ANSI/AAMI/ISO 11737-2: 1998 Sterilization of MedicalDevicesMicrobiological MethodsPart 2: Tests of Ste-rility Performed in the Validation of a Sterilization Pro-cess42.7 AAMI Documents:AAMI TIR No. 191998: Guidance for ANSI/AAMI/ISO109937: 1995, Biological Evaluati
29、on of MedicalDevicesPart 7: Ethylene Oxide Sterilization Residuals7AAMI/ISO 141601998: Sterilization of Single-UseMedical Devices Incorporating Materials of AnimalOriginValidation and Routine Control of Sterilizationby Liquid Chemical Sterilants7AAMI ST67/CDV-2: 1999: Sterilization of MedicalDevices
30、Requirements for Products Labeled “Sterile”73Available from EDQM, Publications and Services European Pharmacopoeia,BP 907 226, avenue de Colmar, F-67029 Strasbourg Cedex 1, France.4Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.an
31、si.org.5Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, PO Box 758,1211 Geneva 13, Switzerland.6Available from Food and Drug Administration (FDA), 5600 Fishers Ln.,Rockville, MD 20857, http:/www.fda.gov.7Association for the Advancement of Medical Instrumentation, 111 N. GlebeRd., Suite
32、 220, Arlington, VA 222014795.F 2103 01 (2007)e222.8 EN Documents:EN 12442-1 Animal Tissues and Their Derivative Utilizedin the Manufacture of Medical DevicesPart 1: Analysisand Management of Risk8EN 12442-Part 3: Validation of the Elimination and/orInactivation of Virus and Transmissible Agents83.
33、Terminology3.1 Definitions:3.1.1 chitosan, na linear polysaccharide consisting ofb(14) linked 2-acetamido-2-deoxy-D-glucopyranose(GlcNAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN).3.1.1.1 DiscussionChitosan is a polysaccharide derivedby N-deacetylation of chitin.3.1.2 decomposition, nstructural cha
34、nges of chitosans as aresult of exposure to environmental, chemical, or thermalfactors, such as temperatures greater than 200C.3.1.2.1 DiscussionDecomposition can result in deleteri-ous changes to the chitosan.3.1.3 degradation, nchange in the chemical structure,physical properties, or appearance of
35、 a material.3.1.3.1 DiscussionDegradation of polysaccharides occursby means of cleavage of the glycosidic bonds, usually by acidcatalyzed hydrolysis. Degradation can also occur thermally.Note that degradation is not synonymous with decomposition.Degradation is often used as a synonym for depolymeriz
36、ationwhen referring to polymers.3.1.4 degree of deacetylation, nthe fraction or percentageof glucosamine units (deacetylated monomers) in a chitosanpolymer molecule.3.1.5 depolymerization, nreduction in length of a polymerchain to form shorter polymeric units.3.1.5.1 DiscussionDepolymerization may r
37、educe thepolymer chain to oligomeric or monomeric units, or both. Inchitosan, hydrolysis of the glycosidic bonds is the primarymechanism.3.1.6 endotoxin, na high-molecular-weight lipopolysac-charide (LPS) complex associated with the cell wall ofgram-negative bacteria that is pyrogenic in humans.3.1.
38、6.1 DiscussionThough endotoxins are pyrogens, notall pyrogens are endotoxins.3.1.7 molecular mass average (molecular weight average),nthe given molecular weight (Mw) of a chitosan will alwaysrepresent an average of all of the molecules in the population.The most common ways to express the Mw are as
39、the numberaverage (Mn) and the weight average (Mw). The two averagesare defined by the following equations:Mn5(iNiMi(iNiandMw5(iWiMi(iWi5(iNiMi2(iNiMiwhere:Ni= number of molecules having a specific molecularweight Miandwi= weight of molecules having a specific molecularweight Mi. In a polydisperse m
40、olecular population therelation Mw Mnis always valid. The coefficient Mw/Mnis referred to as the polydispersity index, and willtypically be in the range 1.5 to 3.0 for commercialchitosans.3.1.8 pyrogen, nany substance that produces fever whenadministered parenterally.4. Significance and Use4.1 This
41、guide contains a listing of those characterizationparameters that are directly related to the functionality ofchitosan. This guide can be used as an aid in the selection andcharacterization of the appropriate chitosan or chitosan salt fora particular application. This standard is intended to givegui
42、dance in the methods and types of testing necessary toproperly characterize, assess, and ensure consistency in theperformance of a particular chitosan. It may have use in theregulation of devices containing chitosan by appropriate au-thorities.4.2 The chitosan salts covered by this guide may be gell
43、ed,extruded, or otherwise formulated into biomedical devices foruse as tissue-engineered medical products or drug deliverydevices for implantation as determined to be appropriate, basedon supporting biocompatibility and physical test data. Recom-mendations in this guide should not be interpreted as
44、aguarantee of clinical success in any tissue-engineered medicalproduct or drug delivery application.4.3 To ensure that the material supplied satisfies require-ments for use in TEMPs, several general areas of characteriza-tion should be considered. These include identity of chitosan,physical and chem
45、ical characterization and testing, impuritiesprofile, and performance-related tests.5. Chemical and Physical Test Methods5.1 Identity of ChitosanThe identity of chitosan andchitosan salts can be established by several methods including,but not limited to the following:5.1.1 Chitosan chloride monogra
46、ph Ph. Eur.5.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)Almost all organic chemical compounds absorb infrared radia-tion at frequencies characteristic for the functional groups inthe compound. A FT-IR spectrum will show absorption bandsrelating to bond stretching and bending and can therefor
47、e serveas a unique fingerprint of a specific compound. Cast a chitosanfilm from a 0.25 % (w/v) solution of chitosan (in 1 % aceticacid) or chitosan salt (dissolved in water) by drying approxi-mately 500 L of the sample onto a disposable IR card9for 3to4hat60C. Record a background spectrum between 40
48、00and 400 cm-1 using 128 scans at a resolution of 4 cm-1. Recordthe IR spectrum of a dried blank IR card, then record the IR8Available from European Committee for Standardization, CEN ManagementCentre, 36 rue de Stassart, B-1050 Brussels, Belgium.9No suitable commercially available IR cards are avai
49、lable for the IR analysis ofchitosan glutamate salt. Alternative methods are under investigation.F 2103 01 (2007)e23spectrum of the sample using 128 scans at a resolution of 4cm-1, percent transmission mode. Label the peaks. Typicalfrequencies (cm-1) for chitosan are as follows:Chitosan Base(as Acetate)Chitosan Chloride Chitosan Glutamate3362b 3344b 1555b1556 1605 13961406 1513 11541153 1379 1085s1083s 11541086sThe peak designators are: sh: sharp; s: strong; m: medium;w: weak; and b: broad.5.2 Physical and Chemical Characterizat
