EN ISO 29701:2010

SLOVENSKI STANDARD
01-maj-2011
Nanotehnologija - Endotoksinski preskus vzorcev iz nanomaterialov za sisteme in
vitro - Preskus Limulus amebocyte lysate (LAL) (ISO 29701:2010)
Nanotechnologies - Endotoxin test on nanomaterial samples for in vitro systems -
Limulus amebocyte lysate (LAL) test (ISO 29701:2010)
Nanotechnologien - Endotoxinprüfung an Proben aus nanomaterial für In-vitro-Systeme -
Limulus-Amoebozyten-Lysat-Prüfung (LAL-Prüfung) (ISO 29701:2010)
Nanotechnologies - Essai de détection d'endotoxines sur des échantillons de
nanomatériaux pour des systèmes in vitro - Essai au lysat d'amébocyte de Limule (LAL)
(ISO 29701:2010)
Ta slovenski standard je istoveten z: EN ISO 29701:2010
ICS:
07.120 Nanotehnologije Nanotechnologies
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 29701
NORME EUROPÉENNE
EUROPÄISCHE NORM
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)
Nanotechnologies - Essai de détection d'endotoxines sur Nanotechnologien - Endotoxinprüfung an Proben aus
des échantillons de nanomatériaux pour des systèmes in nanomaterial für In-vitro-Systeme - Limulus-Amoebozyten-
vitro - Essai au lysat d'amébocyte de Limule (LAL) (ISO Lysat-Prüfung (LAL-Prüfung) (ISO 29701:2010)
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 national 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 in 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, 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
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 29701:2010: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
The text of 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 Standard 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 this 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 Standard: 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 United Kingdom.
Endorsement notice
The text of ISO 29701:2010 has been approved by CEN as a EN ISO 29701:2010 without any modification.

INTERNATIONAL ISO
STANDARD 29701
First edition
2010-09-15
Nanotechnologies — Endotoxin test on
nanomaterial samples for in vitro
systems — Limulus amebocyte lysate
(LAL) test
Nanotechnologies — Essai de détection d'endotoxines sur des
échantillons de nanomatériaux pour des systèmes in vitro — Essai au
lysat d'amébocyte de Limule (LAL)

Reference number
ISO 29701:2010(E)
©
ISO 2010
ISO 29701:2010(E)
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ii © ISO 2010 – All rights reserved

ISO 29701:2010(E)
Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Terms and definitions .1
3 Abbreviated terms .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 sample.3
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 test sample.4
6.5 Laboratory environment .4
7 Test methods .5
7.1 Principle.5
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 report.7
Annex A (informative) Examples 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 method.16
Bibliography.19

ISO 29701:2010(E)
Foreword
ISO (the International Organization 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 has 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.
International 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. Publication 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 rights.
ISO 29701 was prepared by Technical Committee ISO/TC 229, Nanotechnologies.

iv © ISO 2010 – All rights reserved

ISO 29701:2010(E)
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, 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 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 nanomaterials 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 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 ce
...