1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 29701:2010Nanotechnologies Endotoxintest on nanomaterial samplesfor in vitro systems Limulusamebocyte lysate (LAL) testBS EN ISO 29701:2010 BRITISH STANDARDNational for
2、ewordThis British Standard is the UK implementation of EN ISO29701:2010.The UK participation in its preparation was entrusted to TechnicalCommittee NTI/1, Nanotechnologies.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purpo
3、rt to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 61399 9ICS 07.030; 11.100.10Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of the
4、Standards Policy and Strategy Committee on 31 October 2010.Amendments issued since publicationDate Text affectedEUROPEAN 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 s
5、ystems - Limulus amebocyte lysate (LAL) test (ISO 29701:2010) Nanotechnologies - 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 nanomaterial fr I
6、n-vitro-Systeme - Limulus-Amoebozyten-Lysat-Prfung (LAL-Prfung) (ISO 29701:2010) 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 na
7、tional standard without any alteration. Up-to-date lists and bibliographical 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 i
8、n any other language made by translation under the responsibility of a CEN 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
9、, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, 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 NOR
10、MALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 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: EBS EN ISO 29701:2010 EN ISO 29701:2010 (E) 3 Foreword The text of
11、ISO 29701:2010 has been prepared by Technical Committee ISO/TC 229 “Nanotechnologies” 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 S
12、tandard shall be given the status of a national standard, either by publication 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 t
13、his document may be the subject of patent rights. CEN and/or CENELEC shall 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 St
14、andard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, 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 Unit
15、ed Kingdom. Endorsement notice The text of ISO 29701:2010 has been approved by CEN as a EN ISO 29701:2010 without any modification. BS EN ISO 29701:2010ISO 29701:2010(E) ISO 2010 All rights reserved iiiContents Page Foreword iv Introduction.v 1 Scope1 2 Terms and definitions .1 3 Abbreviated terms .
16、2 4 Pre-test considerations.3 4.1 Storage of nanomaterials .3 4.2 Storage containers 3 4.3 Handling of nanomaterials .3 5 Test sample3 5.1 Aqueous dispersion 3 5.2 Aqueous extract 3 6 Preparation of test sample .3 6.1 Dispersion method 3 6.2 Extraction method .4 6.3 Concentration 4 6.4 Storage of te
17、st sample4 6.5 Laboratory environment .4 7 Test methods .5 7.1 Principle5 7.2 Alternative test methods.5 7.3 Selection and validation of the test method.6 7.4 Test procedures.6 8 Assessment of results 6 8.1 General .6 8.2 Guidance on application of test.7 9 Test report7 Annex A (informative) Example
18、s of potential interferences to LAL test.8 Annex B (informative) Gel-clot method 9 Annex C (informative) Endpoint photometric method 13 Annex D (informative) Kinetic method16 Bibliography19 BS EN ISO 29701:2010ISO 29701:2010(E) iv ISO 2010 All rights reservedForeword ISO (the International Organizat
19、ion for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established h
20、as the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Int
21、ernational Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Public
22、ation as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent ri
23、ghts. ISO 29701 was prepared by Technical Committee ISO/TC 229, Nanotechnologies. BS EN ISO 29701:2010ISO 29701:2010(E) ISO 2010 All rights reserved vIntroduction Endotoxins (lipopolysaccharides LPS) are part of the outer membrane of the cell wall of Gram-negative bacteria such as E. coli, Salmonell
24、a, Shigella, Pseudomonas, Neisseria, Haemophilus. 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
25、 of the complement cascade, activation of the 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 nanomateri
26、als if the samples are prepared without special 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
27、 employed. In in vitro test systems, macrophages 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 di
28、fficult to distinguish the response to endotoxins 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. T
29、herefore the preliminary detection of endotoxins 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 tes
30、t systems. Since endotoxins may contaminate 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 f
31、or nanomaterials (see Reference 6). The bacterial 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
32、pharmacopoeia of many countries. This International Standard provides considerations for the application of the LAL test to nanomaterial samples intended for in vitro biological tests. BS EN ISO 29701:2010BS EN ISO 29701:2010INTERNATIONAL STANDARD ISO 29701:2010(E) ISO 2010 All rights reserved 1Nano
33、technologies Endotoxin test on nanomaterial samples for in vitro systems Limulus amebocyte lysate (LAL) test 1 Scope This International Standard describes the application of a test using Limulus amebocyte lysate (LAL) reagent for the evaluation of nanomaterials intended for cell-based in vitro biolo
34、gical 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 37 C. This International Standard is restricted to test samples for in vitro syst
35、ems, 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 coagulogen clottable protein in LAL which is known to play a central role in gel-clo
36、t 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 fragments of coagulogen after limited proteolysis of clotting enzyme in LAL NOTE A
37、 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 of the outer membrane of the cell envelope of Gram-negative bacteria NOTE The ma
38、in 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 Standardization (ECBS) in 1996, relative to the activity of 0,1 ng of WHO reference
39、 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. BS EN ISO 29701:2010ISO 29701:2010(E) 2 ISO 2010 All rights reserved2.5 lambda labelled sensitivity of LAL for gel-clot method or the lowest endot
40、oxin 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 2.7 Limulus amebocyte lysate test LAL test test for measuring
41、 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 distance 2.9 test sample aqueous dispersion or aqueous extract o
42、f 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 control LAL Limulus amebocyte lysate LPS lipopolysaccharide OD optic
43、al density RSE reference standard endotoxin WHO World Health Organization BS EN ISO 29701:2010ISO 29701:2010(E) ISO 2010 All rights reserved 34 Pre-test considerations 4.1 Storage of nanomaterials Nanomaterials because of their high surface area can collect many of the contaminants including endotox
44、ins 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 dioxide, etc. shall be used to verify the a
45、bsence 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 obtained by heat-treatment (see 4.2). 4.2 S
46、torage 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 time (e.g. 180 C for at least 3 h, or 650 C fo
47、r 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 shall be paid to avoid contact between dust and
48、 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 dilution with endotoxin-free water. 5.2 Aqueous
49、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 prepared 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 th
