1、January 2011 Translation by DIN-Sprachendienst.English price group 13No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).IC
2、S 07.030!$m16“1741419www.din.deDDIN EN ISO 29701www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.26Nanotechnologies Endotoxin test on nanomaterial samples for in vitro systems Limulus amebocyte lysate (LAL) test (ISO 29701:2010) Eng
3、lish translation of DIN EN ISO 29701:2011-01 Nanotechnologien Endotoxinprfung an Proben aus anomaterial fr In-vitro-Systeme Limulus-Amoebozyten-Lysat-Prfung (LAL-Prfung) (ISO 29701:2010) Englische bersetzung von DIN EN ISO 29701:2011-01 NNanotechnologies Essai de dtection dendotoxines sur des chanti
4、llons de nanomatriaux pour des systmes in vitro Essai au lysat dambocyte de Limule (LAL) (ISO 29701:2010) Traduction anglaise de DIN EN ISO 29701:2011-01 0 .11 2DIN EN ISO 29701:2011-01 2 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee
5、ISO/TC 229 “Nanotechnologies” in collaboration with Technical Committee CEN/TC 352 “Nanotechnologies”. BSI, United Kingdom, holds the secretariats of both Technical Committees. The responsible German body involved in its preparation was the Normenausschuss Materialprfung (Materials Testing Standards
6、 Committee), Working Committees NA 062-08-17 AA Nanotechnologien and NA 062-08-17-03 UA Gesundheits- und Umweltaspekte. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 10993-12 DIN EN ISO 10993-12 ISO 14644-1 DIN EN ISO 14644-1 ISO 1464
7、4-2 DIN EN ISO 14644-2 ISO 14644-7 DIN EN ISO 14644-7 National Annex NA (informative) Bibliography DIN EN ISO 10993-12, Biological evaluation of medical devices Part 12: Sample preparation and reference materials DIN EN ISO 14644-1, Cleanrooms and associated controlled environments Part 1: Classific
8、ation of air cleanliness DIN EN ISO 14644-2, Cleanrooms and associated controlled environments Part 2: Specifications for testing and monitoring to prove continued compliance with ISO 14644-1 DIN EN ISO 14644-7, Cleanrooms and associated controlled environments Part 7: Separative devices (clean air
9、hoods, gloveboxes, isolators and mini-environments) EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 29701 September 2010 ICS 11.100.10; 07.030 English Version Nanotechnologies Endotoxin test on nanomaterial samples for in vitro systems Limulus amebocyte lysate (LAL) test (ISO 29701:2010) Na
10、notechnologies Essai de dtection dendotoxines sur des chantillons de nanomatriaux pour des systmes in vitro Essai au lysat dambocyte de Limule (LAL) (ISO 29701:2010) Nanotechnologien Endotoxinprfung an Proben aus anomaterial fr In-vitro-Systeme Limulus-Amoebozyten-Lysat-Prfung (LAL-Prfung) (ISO 2970
11、1:2010) This European Standard was approved by CEN on 22 August 2010. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographi
12、cal references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CE
13、N member into its own language and notified to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, I
14、reland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marni
15、x 17, B-1000 Brussels 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 29701:2010: ENContents DIN EN ISO 29701:2011-01 EN ISO 29701:2010 (E) 2 PageForeword3 Introduction .4 1 Scope 5 2 Terms and definitions .5 3 Abbreviated
16、 terms .6 4 Pre-test considerations.7 4.1 Storage of nanomaterials7 4.2 Storage containers 7 4.3 Handling of nanomaterials7 5 Test sample 7 5.1 Aqueous dispersion 7 5.2 Aqueous extract.7 6 Preparation of test sample7 6.1 Dispersion method 7 6.2 Extraction method .8 6.3 Concentration.8 6.4 Storage of
17、 test sample 8 6.5 Laboratory environment .8 7 Test methods9 7.1 Principle9 7.2 Alternative test methods.9 7.3 Selection and validation of the test method .10 7.4 Test procedures.10 8 Assessment of results 10 8.1 General10 8.2 Guidance on application of test .11 9 Test report 11 Annex A (informative
18、) Examples of potential interferences to LAL test .12 Annex B (informative) Gel-clot method.13 Annex C (informative) Endpoint photometric method.17 Annex D (informative) Kinetic method 20 Bibliography 23 Foreword The text of ISO 29701:2010 has been prepared by Technical Committee ISO/TC 229 “Nanotec
19、hnologies” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 29701:2010 by Technical Committee CEN/TC 352 “Nanotechnologies” the secretariat of which is held by BSI. This European Standard shall be given the status of a national standard, either by publica
20、tion of an identical text or by endorsement, at the latest by March 2011, and conflicting national standards shall be withdrawn at the latest by March 2011. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall
21、not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmar
22、k, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 29701:2010 has been approved
23、 by CEN as a EN ISO 29701:2010 without any modification. DIN EN ISO 29701:2011-01 EN ISO 29701:2010 (E) 3 Introduction Endotoxins (lipopolysaccharides LPS) are part of the outer membrane of the cell wall of Gram-negative bacteria such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemoph
24、ilus. Endotoxins can cause a variety of systemic reactions in mammals, including humans, such as fever, disseminated intravascular coagulation, hypotension, shock and death: the responses are mediated by production of various kinds of cytokines, activation of the complement cascade, activation of th
25、e coagulation cascade, etc. Endotoxins are present in the ordinary environment. Since most test samples of nanomaterials intended for in vitro and in vivo test systems require various preparation procedures, endotoxins might contaminate the test nanomaterials if the samples are prepared without spec
26、ial care. For the purpose of toxicity screening or biocompatibility testing of nanomaterials, or mechanism studies on the possible toxicity induced by nanomaterials, various cell-based in vitro test systems and in vivo animal models are being developed and employed. In in vitro test systems, macroph
27、ages and other relevant mammalian cells are frequently used as the test cells especially for nanomaterials because they are primarily the responsible surveillance cells in the body. However, these cells are highly reactive to endotoxins; therefore it is difficult to distinguish the response to endot
28、oxins from that to nanomaterials. Consequently, contamination by endotoxins would confound the result of tests in vitro. Contamination by endotoxins of test samples may be reduced if appropriate precautions are followed in preparation of the test sample. Therefore the preliminary detection of endoto
29、xins is required to minimize the contamination by endotoxins or confirm the insignificant levels of endotoxins in the test sample. It is also important to quantify endotoxin levels for the adequate interpretation of data obtained by in vitro biological test systems. Since endotoxins may contaminate
30、medical devices and medicines for parenteral use, quantitative and semi-quantitative assay methods to test for endotoxins both in vivo and in vitro have been developed and used for regulatory purposes as well as laboratory standard operational procedures for nanomaterials (see Reference 6). The bact
31、erial endotoxin test using Limulus amebocyte lysate (LAL) reagent has been developed as an in vitro assay method to test for the presence of endotoxin contamination as an alternative to the pyrogenicity test using rabbits, and methods are described in the pharmacopoeia of many countries. This Intern
32、ational Standard provides considerations for the application of the LAL test to nanomaterial samples intended for in vitro biological tests. DIN EN ISO 29701:2011-01 EN ISO 29701:2010 (E) 4 1 Scope This International Standard describes the application of a test using Limulus amebocyte lysate (LAL) r
33、eagent for the evaluation of nanomaterials intended for cell-based in vitro biological test systems. The test is suitable for use with nanomaterial samples dispersed in aqueous media, e.g. water, serum or reaction medium, and to such media incubated with nanomaterials for an appropriate duration at
34、37 C. This International Standard is restricted to test samples for in vitro systems, but the methods can also be adapted to nanomaterials to be administered to animals by parenteral routes. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 coa
35、gulogen clottable protein in LAL which is known to play a central role in gel-clot formation by endotoxins NOTE Coagulogen derived from Japanese horseshoe crab (Tachypleus tridentatus) consists of a total 175 amino acids with the molecular weight of 19,723 (see Reference 7). 2.2 coagulin resulting f
36、ragments of coagulogen after limited proteolysis of clotting enzyme in LAL NOTE A coagulin derived from Japanese horseshoe crab (Tachypleus tridentatus) consists of the N-terminal fragment peptides (Ala1 Arg18) and the C-terminal fragment peptides (Gly47 Phe175) (see Reference 7). 2.3 endotoxin part
37、 of the outer membrane of the cell envelope of Gram-negative bacteria NOTE The main active ingredient is lipopolysaccharides (LPS). 2.4 endotoxin unit EU standard unit of endotoxin activity NOTE 1 The endotoxin unit was defined by the World Health Organization (WHO) Expert Committee on Biological St
38、andardization (ECBS) in 1996, relative to the activity of 0,1 ng of WHO reference standard endotoxin (RSE) from Escherichia coli 0113:HK10:K(-) or 10 EU/ng (see Reference 8). NOTE 2 EU is equal to international unit (IU) of endotoxin. DIN EN ISO 29701:2011-01 EN ISO 29701:2010 (E) 5 2.5 lambda label
39、led sensitivity of LAL for gel-clot method or the lowest endotoxin concentration on the standard curve for chromogenic or turbidimetric methods, expressed in EU/mL 2.6 Limulus amebocyte lysate LAL aqueous extract of the blood corpuscle of horseshoe crabs, Limulus polyphemus or Tachypleus tridentatus
40、 2.7 Limulus amebocyte lysate test LAL test test for measuring bacterial endotoxins using Limulus amebocyte lysate reagent NOTE The LAL test is called “bacterial endotoxin test (BET)” in pharmacopoeia. 2.8 optical density OD optical absorbance of an optical element for a given wavelength per unit di
41、stance 2.9 test sample aqueous dispersion or aqueous extract of nanomaterials under investigation 3 Abbreviated terms BET bacterial endotoxin test CSE control standard endotoxin ECBS expert committee on biological standardization EF endotoxin-free EU endotoxin unit I/EC inhibition/enhancement contro
42、l LAL Limulus amebocyte lysate LPS lipopolysaccharide OD optical density RSE reference standard endotoxin WHO World Health Organization DIN EN ISO 29701:2011-01 EN ISO 29701:2010 (E) 6 4 Pre-test considerations 4.1 Storage of nanomaterials Nanomaterials because of their high surface area can collect
43、 many of the contaminants including endotoxins from the environment. For this reason, nanomaterials shall be collected and stored in endotoxin-free, sealable containers (e.g. glassware) upon arrival until use. Suitable blanks such as endotoxin-free metal oxide powders like titanium dioxide, silicon
44、dioxide, etc. shall be used to verify the absence of endotoxin contamination. NOTE 1 It is advisable that plastics like polypropylene be avoided for the storage of nanomaterials, due to the possible interference with the LAL test as shown in Annex A. NOTE 2 Endotoxin-free metal oxide powders can be
45、obtained by heat-treatment (see 4.2). 4.2 Storage containers Glassware and other heat stable storage containers for storage of nanomaterials and test samples should be treated by heating to a temperature of greater than 250 C for at least 30 min or other validated combinations of temperature and tim
46、e (e.g. 180 C for at least 3 h, or 650 C for 1 min) to eliminate endotoxins. Commercially available sterile endotoxin-free polystyrene containers can be used. 4.3 Handling of nanomaterials Dust found in the indoor environment usually contains significant amounts of endotoxins. Special attention shal
47、l be paid to avoid contact between dust and nanomaterials during sampling and handling. A clean air laboratory condition is required (recommended in 6.5). 5 Test sample 5.1 Aqueous dispersion Nanomaterials which are dispersed in aqueous liquid may be subjected to the LAL test directly or after dilut
48、ion with endotoxin-free water. 5.2 Aqueous extract Endotoxin-free reaction medium, physiological saline solution or other extraction vehicles incubated with nanomaterials may be used as test sample for the LAL test. 6 Preparation of test sample 6.1 Dispersion method Test dispersions might be prepare
49、d by one or more of the following: hand grinding; mechanical milling; ultrasonication. The dispersion medium will depend on the purpose and the particular in vitro test. NOTE Nanomaterials can have high surface area, porosity, hydrophobicity and other properties that can make this step difficult. Therefore, methods development might be required. DIN EN ISO 29701:2011-01 EN ISO 29701:2010 (E) 7 6.2 Extraction method The extraction conditions, such as extraction medium, incubation time, incubation te
