EN ISO 29701:2010

Nanotechnologies - Endotoxin test on nanomaterial samples for in vitro systems - Limulus amebocyte lysate (LAL) test (ISO 29701:2010)

ISO 29701:2010 describes the application of a test using Limulus amebocyte lysate (LAL) reagent 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 37 °C.
ISO 29701:2010 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.

Nanotechnologien - Endotoxinprüfung an Proben aus Nanomaterial für In-vitro-Systeme - Limulus-Amoebozyten-Lysat-Prüfung (LAL-Prüfung) (ISO 29701:2010)

Die vorliegende Internationale Norm beschreibt die Anwendung einer Prüfung mit einem Limulus-Amoebozyten-Lysat-Reagenz (LAL) für die Evaluierung von Nanomaterialien, die für biologische In vitro-Prüfsysteme auf Zellbasis bestimmt sind. Die Prüfung ist für die Anwendung mit in wässrigen Medien, z. B. Wasser, Serum oder Reaktionsmedium, dispergierten Nanomaterialproben geeignet und für derartige Medien, die für eine angemessene Dauer bei 37 °C mit Nanomaterialien inkubiert wurden.
Diese Internationale Norm ist auf Prüfobjekte für In vitro-Systeme beschränkt, jedoch können die Verfahren auch an Nanomaterialien angepasst werden, die Tieren auf parenteralen Wegen zugeführt werden.

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)

L'ISO 29701:2010 décrit l'application d'un essai utilisant le réactif LAL (lysat d'amébocytes de limule) en vue d'évaluer des nanomatériaux destinés à des systèmes d'essai biologique sur cultures cellulaires in vitro. L'essai est approprié à une utilisation avec des échantillons de nanomatériaux dispersés dans un milieu aqueux, par exemple de l'eau, du sérum physiologique ou un milieu de réaction, ainsi qu'à des milieux incubés avec des nanomatériaux pendant un temps donné à 37 °C.
L'ISO 29701:2010 est conçue pour des échantillons destinés à des systèmes in vitro, toutefois, les méthodes peuvent également être adaptées à des nanomatériaux étant administrés à des animaux par voies parentérales.

Nanotehnologija - Endotoksinski preskus vzorcev iz nanomaterialov za sisteme in vitro - Preskus Limulus amebocyte lysate (LAL) (ISO 29701:2010)

Ta mednarodni standard določa preskus za zaznavanje in kvantifikacijo endotoksinov, ki lahko kontaminirajo vzorce v vodnih nanomaterialih za biološke preskusne sisteme in vitro z uporabo reagenta Limulus Amebocyte Lysate (LAL).

General Information

• Status: Published
• Publication Date: 14-Sep-2010
• Withdrawal Date: 30-Mar-2011
• ICS: 07.030 - Physics. Chemistry
• ICS: 07.120 - Nanotechnologies
• ICS: 11.100.10 - In vitro diagnostic test systems
• ICS: 71.100.01 - Products of the chemical industry in general
• Technical Committee: CEN/TC 352 - Nanotechnologies
• Drafting Committee: CEN/TC 352 - Nanotechnologies
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 15-Sep-2010
• Completion Date: 15-Sep-2010
• Ref Project: SIST EN ISO 29701:2011 - Nanotechnologies - Endotoxin test on nanomaterial samples for in vitro systems - Limulus amebocyte lysate (LAL) test (ISO 29701:2010)

Overview

EN ISO 29701:2010 (ISO 29701:2010) - "Nanotechnologies - Endotoxin test on nanomaterial samples for in vitro systems - Limulus amebocyte lysate (LAL) test" - defines how to apply the Limulus amebocyte lysate (LAL) test to nanomaterial samples. The standard covers LAL testing of nanomaterials dispersed in aqueous media (e.g., water, serum, reaction medium) and media incubated with nanomaterials (typically at 37 °C). It is focused on samples intended for cell‑based in vitro biological test systems, and notes that methods can be adapted for parenteral animal administration.

Key topics and requirements

• Scope and definitions: clear terminology for endotoxin (LPS), endotoxin units (EU), LAL, coagulogen/coagulin and test sample types.
• Pre-test considerations: storage and handling of nanomaterials and containers to minimise contamination; laboratory environment controls.
• Test sample preparation: guidance on preparing aqueous dispersions and aqueous extracts, dispersion and extraction methods, concentration and storage of test samples.
• Test methods and validation: principles and selection of LAL test formats (gel‑clot, chromogenic/endpoint photometric, turbidimetric/kinetic approaches are included in informative annexes), plus validation requirements and use of inhibition/enhancement controls.
• Interference assessment: identification of potential nanomaterial interferences with LAL assays and controls to detect inhibition or enhancement.
• Results and reporting: assessment of endotoxin levels (in EU/mL) and recommended test report contents to support interpretation for in vitro systems.

Practical applications

• Toxicity screening and biocompatibility: ensure endotoxin contamination does not confound cell‑based assays (macrophage and other cytokine‑responsive cells are highly sensitive to LPS).
• Quality control for nanomaterials: routine endotoxin testing of aqueous dispersions used in research, safety testing or preclinical studies.
• Regulatory and safety assessment: supports reproducible endotoxin measurement for laboratories performing in vitro biological tests and for adapting methods for parenteral product testing.

Who should use this standard

• Nanomaterials researchers and laboratories performing in vitro toxicology
• Biocompatibility and preclinical testing labs
• Medical device and nanotechnology product manufacturers managing endotoxin risk
• Quality assurance and regulatory scientists validating LAL assays for nanomaterial samples

Related standards and context

• LAL-based bacterial endotoxin test (BET) methods are described in pharmacopoeias and regulatory guidance; ISO 29701 applies those principles specifically to nanotechnologies (prepared by ISO/TC 229). Informative annexes in the standard provide practical methods (gel‑clot, photometric, kinetic) and examples of interferences to aid implementation.

Keywords: EN ISO 29701:2010, ISO 29701, LAL test, endotoxin testing, nanomaterials, in vitro, Limulus amebocyte lysate, LPS, endotoxin unit.

Standards Content (Sample)

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 cells in the body. However, these cells are highly reactive to endotoxins; therefore it is difficult 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. Therefore 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 test
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 for 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 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.

INTERNATIONAL STANDARD ISO 29701:2010(E)

Nanotechnologies — 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 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 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
coagulogen
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 fragments 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 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
Standardization (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.
ISO 29701:2010(E)
2.5
lambda
λ
labelled 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
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 distance
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 control
LAL Limulus amebocyte lysate
LPS lipopolysaccharide
OD optical density
RSE reference standard endotoxin
WHO World Health Organization
2 © ISO 2010 – All rights reserved

ISO 29701:2010(E)
4 Pre-test considerations
4.1 Storage of nanomaterials
Nanomaterials because of their high surface area can collect 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 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 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 time (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
shall 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 dilution
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 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 that can make this
step difficult. Therefore, methods development might be required.
ISO 29701:2010(E)
6.2 Extraction method
The extraction conditions, such as extraction medium, incubation time, incubation temperature and the
concentration of the test sample may simulate the incubation condition of the in vitro test concerned. Reaction
medium without pH indicator (e.g. phenol red) or buffered saline are preferable for the extraction medium to
avoid the interference with colour. The extraction medium should be certified endotoxin-free or reconstituted
from endotoxin-free reagents. Addition of antibiotics and antimycotics to the extraction medium might be
effective to prevent respectively bacterial and fungi growth. Interference effects of the added anti-biotic agents
to the LAL test should be validated (see 7.3.3). After extraction, the extraction mixture shall be centrifuged to
remove the particulates, and the supernatant, which shall serve as a test sample for the LAL test, should be
collected to endotoxin-free tubes or containers with endotoxin-free pipette tips. The extraction conditions
including centrifugation shall be justified and recorded. In particular, centrifugation condition shall be
determined according to the nanomaterials concerned.
NOTE 1 0,05 % polysorbate 20 is proposed as an extraction vehicle for airborne endotoxin from glass fibre filters (see
Reference [9]).
NOTE 2 0,1 % vitamin E surfactant (vitamin E d-α-tocopheryl polyethylene glycol-1000 succinate) was found to
improve the extraction of endotoxin from carbon nano-objects (see Reference [10]).
NOTE 3 For more information on the extraction methods, see ISO 10993-12:2007.
6.3 Concentration
Test sample shall be reconstituted in endotoxin-free water to the highest concentration in the cell-based in
vitro test concerned, if necessary.
6.4 Storage of test sample
The test sample shall, if possible, be tested as soon as possible after preparation because degradation of
endotoxins in the test sample or bacterial growth during storage can occur. The test sample shall be stored in
an endotoxin-free, sealable container (see 4.2) at a temperature of between 2 °C and 8 °C. If the test sample
is stored longer than 24 h, the stability and homogeneity of the test sample under the storage conditions shall
be verified.
6.5 Laboratory environment
6.5.1 Tap water and air cleanliness
Tap water and dust found in the indoor environment usually contain significant amounts of endotoxins.
Nanomaterials shall be processed with endotoxin-free medium and endotoxin-free laboratory-ware to ensure
aseptic sample preparation. A clean room, a clean air hood, or an equivalent clean air device with an air
cleanliness of ISO Class 5 (see ISO 14644-1) shall be used in laboratory circumstances where airborne
endotoxin contamination is a demonstrated problem, unless otherwise justified. For guidance on air
cleanliness, see ISO 14644-1, ISO 14644-2 and ISO 14644-7.
6.5.2 Equipment and laboratory-ware
Equipment and laboratory glassware used for the preparation of the test sample 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, and 650 °C for 1 min) to eliminate endotoxins. Heat-labile or other materials
which are not suitable for heat-treatment shall be treated with measures other than heat treatment to reduce
endotoxins. Rinsing with endotoxin-free water after soaking the materials in strong alkali or oxidizing solution
is a reliable method to remove endotoxins. If strong alkali or an oxidizing solution is used, the method needs
to be validated to ensure that the method reduces the presence of endotoxins and that no residuals remain
after treatment that interfere with the test. With respect to heat-labile laboratory-ware such as containers,
tubes, tips for micropipettes, endotoxin-free plastic products are commercially available.
NOTE It is advisable to use polystyrene products when plastic products are used.
4 © ISO 2010 – All rights reserved

ISO 29701:2010(E)
6.5.3 Rinse water
Water is one of the sources of endotoxins detected in equipment and laboratory-ware. Distilled water may be
used for rinsing the equipment and laboratory-ware after endotoxin reduction treatments. However, distilled
water prepared in-house might be contaminated with endotoxins due to inadequate equipment or
inappropriate handling, although distillation has been shown to be effective in removing endotoxins from
contaminated water (see Reference [11]). The endotoxin level in the distilled water prepared in-house shall be
measured periodically to validate that it contains insignificant levels of endotoxins. If endotoxin contamination
in distilled water is unavoidable, commercially available endotoxin-free water should be used.
7 Test methods
7.1 Principle
Endotoxins activate a factor in the LAL and trigger a proteolytic cascade (see Reference [12]). The clotting
enzyme, which is released from the proclotting enzyme by one of the activated factors, catalyses a proteolysis
of coagulogen in the LAL and the resulting fragments, coagulins, spontaneously bind to each other through
disulfide linkage to develop the turbidity of the LAL and finally form a gel-clot. The gel-clot formation is
principally determined by visual inspection after inverting test tubes. This method requires no optical reader
and the procedures are easy to perform. The most sensitive gel-clot method using commercially available
reagents measures 0,015 EU/mL.
NOTE One of the practical procedures for the gel-clot method is described in Annex B.
7.2 Alternative test methods
7.2.1 Endpoint photometric methods
The optical density (OD) of the reaction mixture is measured after a certain period of reaction time. With
regard to endpoint photometric methods, there are two techniques; the turbidimetric technique measuring the
turbidity of the reaction mixture and the chromogenic technique measuring p-nitroaniline (p-NA) liberated from
a synthetic substrate, such as Boc-Leu-Gly-Arg-p-NA or Boc-Thr-Gly-Arg-p-NA for the clotting enzyme. Due to
the low sensitivity and a technical difficulty stopping the progress of turbidity generation at a designated time
point, the simple turbidimetric method is replaced with the kinetic turbidimetric method described in 7.2.2.
There are at least two procedures for measuring p-NA in the reaction mixture:
⎯ one measures the OD of p-NA directly at a wavelength of 405 nm, and
⎯ the other measures the diazotized magenta derivative of p-NA photometrically at a wavelength of
between 540 nm and 550 nm.
The sensitivity of endpoint photometric method using commercially available reagents by measuring the OD at
a wavelength of 405 nm is 0,01 EU/mL while that of the diazo-coupling method is 0,001 EU/mL.
NOTE One of the practical procedures for the endpoint photometric method is described in Annex C.
7.2.2 Kinetic methods
The time required to reach the predetermined OD of the reaction mixture or the rate of colour or turbidity
development is determined by an optical reader. With regard to kinetic procedures, the OD of p-NA liberated
from the synthetic peptide stated above or turbidity of the reaction mixture is read at multiple time points as
the reaction proceeds, and thus several types of automated instruments have been developed. To detect
endotoxins more precisely and accurately with kinetic methods, sophisticated automated instruments are
necessary. The best sensitivity of the kinetic method using a commercially available automated instrument is
0,001 EU/mL.
NOTE One of the practical procedures for the kinetic method is described in Annex D.
ISO 29701:2010(E)
7.3 Selection and validation of the test method
7.3.1 Considerations of minimum required sensitivity
The critical concentration for endotoxins in in vitro test systems to produce biological reactions varies
according to the in vitro test systems employed; 0,01 ng of endotoxin/mL induced IL-1β and TNF-α in human
macrophages (see Reference [13]) whereas 0,048 ng of endotoxin induced insignificant biological responses
in human dendritic cells or peripheral blood mononuclear cells (see Reference [13]). It is highly desirable to
know the allowable contamination level of endotoxins in in vitro test systems to select the appropriate LAL test.
The sensitivity of the LAL reagent should be better than or at least equivalent to the allowable contamination
level in the in vitro test concerned.
7.3.2 Inhibition/enhancement potential to test by test sample
7.3.2.1 It is reported that several materials interfere with the LAL test. Some nanomaterials may have
been recently synthesized and have unknown inhibition/enhancement potential on the LAL test due to their
properties.
NOTE Examples of potential interferences are available in Annex A.
7.3.2.2 Interference by colour or turbidity of the test sample is a matter of concern when endpoint
photometric methods
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