SIST EN ISO 10808:2011

SLOVENSKI STANDARD
SIST EN ISO 10808:2011
01-november-2011
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SUHVNXãDQMHWRNVLþQRVWLSULYGLKRYDQMX,62
Nanotechnologies - Characterization of nanoparticles in inhalation exposure chambers
for inhalation toxicity testing (ISO 10808:2010)
Nanotechnologien - Charakterisierung von Nanopartikeln in Inhalationskammern zur
Prüfung auf Toxizität nach Inhalation (ISO 10808:2010)
Nanotechnologies - Caractérisation des nanoparticules dans les chambres d'inhalation
par exposition pour les essais de toxicité par inhalation (ISO 10808:2010)
Ta slovenski standard je istoveten z: EN ISO 10808:2010
ICS:
07.120 Nanotehnologije Nanotechnologies
SIST EN ISO 10808:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 10808:2011

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SIST EN ISO 10808:2011


EUROPEAN STANDARD
EN ISO 10808

NORME EUROPÉENNE

EUROPÄISCHE NORM
December 2010
ICS 07.030
English Version
Nanotechnologies - Characterization of nanoparticles in
inhalation exposure chambers for inhalation toxicity testing (ISO
10808:2010)
Nanotechnologies - Caractérisation des nanoparticules Nanotechnologien - Charakterisierung von Nanopartikeln in
dans les chambres d'inhalation par exposition pour les Inhalationskammern zur Prüfung auf Toxizität nach
essais de toxicité par inhalation (ISO 10808:2010) Inhalation (ISO 10808:2010)
This European Standard was approved by CEN on 10 December 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-CENELEC 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-CENELEC 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 10808:2010: E
worldwide for CEN national Members.

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SIST EN ISO 10808:2011
EN ISO 10808:2010 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 10808:2011
EN ISO 10808:2010 (E)
Foreword
This document (EN ISO 10808:2010) has been prepared by Technical Committee ISO/TC 229
“Nanotechnologies” in collaboration with 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 June 2011, and conflicting national standards shall be withdrawn at
the latest by June 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 10808:2010 has been approved by CEN as a EN ISO 10808:2010 without any modification.

3

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SIST EN ISO 10808:2011

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SIST EN ISO 10808:2011

INTERNATIONAL ISO
STANDARD 10808
First edition
2010-12-15

Nanotechnologies — Characterization of
nanoparticles in inhalation exposure
chambers for inhalation toxicity testing
Nanotechnologies — Caractérisation des nanoparticules dans les
chambres d'inhalation par exposition pour les essais de toxicité par
inhalation




Reference number
ISO 10808:2010(E)
©
ISO 2010

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SIST EN ISO 10808:2011
ISO 10808:2010(E)
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All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
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Published in Switzerland

ii © ISO 2010 – All rights reserved

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SIST EN ISO 10808:2011
ISO 10808:2010(E)
Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Terms and definitions .1
3.1 Particle measuring systems.2
4 Test substance monitoring method .4
4.1 Principle .4
4.1.1 Exposure .4
4.1.2 Particle properties.4
4.2 Preparation of system.4
4.3 Study.5
5 Specific monitoring method.5
5.1 Requirements for number-based particle size distribution and mass concentration .5
5.2 Measurement of number-based particle size distribution .5
5.3 Mass concentration measurement .6
5.4 Inhalation exposure chamber .6
6 Assessment of results .7
7 Test report.7
Annex A (informative) Example of nanoparticle characterization for inhalation toxicity testing.9
Bibliography.17

© ISO 2010 – All rights reserved iii

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SIST EN ISO 10808:2011
ISO 10808:2010(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 10808 was prepared by Technical Committee ISO/TC 229, Nanotechnologies.

iv © ISO 2010 – All rights reserved

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SIST EN ISO 10808:2011
ISO 10808:2010(E)
Introduction
The number of nanotechnology-based consumer products containing silver, gold, carbon, zinc oxide, titanium
dioxide and silica nanoparticles is growing very rapidly. The population at risk of exposure to nanoparticles
continues to increase as the applications expand. In particular, workers in nanotechnology-based industries
are at risk of being exposed to nanoparticles. If nanoparticles are liberated from products, the public could be
exposed as well. Although toxicity screening using instillation of nanomaterials provides important information,
it does not reflect the actual scenario of inhalation exposure and does not provide the data required for
inhalation exposure risk assessment. In addition, while inhalation toxicology using rats is the norm at this time,
[10]
it is desirable to replace this antiquated method with a human-relevant assay .
The inhalation toxicity of nanoparticles is of particular concern in ensuring the health of workers and
consumers. In order to conduct inhalation toxicity studies of nano-sized particles, the monitoring of
concentration, size and distribution of nano-sized particles in the inhalation chamber is necessary. The
conventional methods of fine or coarse particle monitoring, such as weight-based mass dose monitoring, are
considered insufficient for nanoparticles, since nano-specific parameters (particle surface area, particle
number, etc.) might be critical determinants, and if so, should also be monitored.
This International Standard proposes a battery of inhalation toxicity testing chamber monitoring, including a
differential mobility analyzing system (DMAS), for measuring particle number, size, distribution, surface area
and estimated mass dose, as well as morphological examination using transmission electron microscopy
(TEM) or scanning electron microscopy (SEM) equipped with an energy dispersive X-ray analyzer
(TEM-EDXA) for chemical composition.
This International Standard also includes conventional mass dose monitoring and other physicochemical
monitoring, for use when deemed a necessary parameter for toxicity determination. This method evaluates
nano-sized particle surface area, mass dose, particle distribution, composition and dispersion to support
[13][17][18]
effective analysis of inhalation toxicity testing results .

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SIST EN ISO 10808:2011

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SIST EN ISO 10808:2011
INTERNATIONAL STANDARD ISO 10808:2010(E)

Nanotechnologies — Characterization of nanoparticles in
inhalation exposure chambers for inhalation toxicity testing
1 Scope
This International Standard specifies requirements for, and gives guidance on, the characterization of airborne
nanoparticles in inhalation exposure chambers for the purpose of inhalation toxicity studies in terms of particle
mass, size distribution, number concentration and composition.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 10312, Ambient air —Determination of asbestos fibres — Direct transfer transmission electron
microscopy method
ISO 15900, Determination of particle size distribution — Differential electrical mobility analysis for aerosol
particles
ISO/TS 27687, Nanotechnologies — Terminology and definitions for nano-objects — Nanoparticle, nanofibre
and nanoplate
1)
OECD Test Guideline 403 (TG 403), Acute Inhalation Toxicity
1)
OECD Test Guideline 412 (TG 412), Subacute Inhalation Toxicity: 28-Day Study
1)
OECD Test Guideline 413 (TG 413), Subchronic Inhalation Toxicity: 90-Day Study
1)
OECD Guidance Document 39 (GD 39), Acute Inhalation Toxicity Testing
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 15900 and ISO/TS 27687 and the
following apply.

1) Organization for Economic Cooperation and Development (OECD) publication.
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SIST EN ISO 10808:2011
ISO 10808:2010(E)
3.1 Particle measuring systems
3.1.1
differential electrical mobility classifier
DEMC
differential electrical mobility spectrometer
DEMS
classifier that is able to select aerosol particle sizes from a distribution that enters it and pass only selected
sizes to the exit
NOTE 1 A DEMC classifies aerosol particle sizes by balancing the electrical force on each particle in an electrical field
with its aerodynamic drag force. Classified particles have different sizes due to their number of electrical charges and a
narrow range of electrical mobility determined by the operating conditions and physical dimensions of the DEMC.
NOTE 2 Adapted from ISO 15900:2009, definition 2.7.
3.1.2
differential mobility analyzing system
DMAS
system used to measure the size distribution of submicrometre aerosol particles consisting of a DEMC,
a particle charge conditioner, flow meters, a particle detector, interconnecting plumbing, a computer and
suitable software
NOTE Adapted from ISO 15900:2009, definition 2.8.
3.1.3
condensation particle counter
CPC
instrument that detects particles and that can be used to calculate particle number concentration given the
known flow rates into the detector
NOTE 1 The range of particles detected are usually smaller than several hundred nanometers and larger than a few
nanometers. A CPC is one possible detector for use with a DEMC.
NOTE 2 In some cases, a condensation particle counter may be called a condensation nucleus counter (CNC).
NOTE 3 Adapted from ISO 15900:2009, definition 2.5.
3.2
inhalation exposure chamber
inhalation chamber
exposure chamber
system prepared to expose experimental animals to an inhaled test substance of predetermined duration and
dose by either the nose-only or whole-body method
NOTE 1 The term “nose-only” is synonymous with “head-only” or “snout-only”.
NOTE 2 Adapted from OECD TG 403, 412, 413.
3.3
nanoparticle generation system
device used to make nanoparticle aerosol with controlled size distribution and concentration
3.4
breathing zone
location from which the experimental animal breathes
NOTE 1 For an unrestrained, non-caged animal, this will be the entire volume of the inhalation chamber. For a
restrained or caged animal, this will be the range of motion for the animal's nose. For a masked animal, this will be the
small volume in front of the nostrils.
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SIST EN ISO 10808:2011
ISO 10808:2010(E)
NOTE 2 The term “breathing zone” is used to ensure test atmosphere samples are obtained from the same location as
that in which the animal breathes. An undesirable sampling approach would be one where concentration measurements
are obtained at the top of the inhalation chamber while the animal is exposed at the bottom.
3.5
geometric mean diameter
GMD
measure of central tendency of particle size distribution using the logarithm of particle diameters, computed
for the DMAS by
n
ΔNdln
()
∑ ii
im=
ln(GMD)=
N
where
d is the midpoint diameter for the size channel, i;
i
N is the total concentration;
ΔN is the concentration within the size channel, i;
i
m is the first channel;
n is the last channel.
NOTE The GMD is normally computed from particle counts and when noted may be based on surface area or
particle volume with appropriate weighting.
3.6
geometric standard deviation
GSD
measure of width or spread of particle sizes, computed for the DMAS by
n 2
⎡⎤
Ndln − ln GMD
()
∑ ii
⎣⎦
im=
ln(GSD)=
N−1
3.7
count median diameter
CMD
diameter equal to GMD for particle counts assuming a logarithmic normal distribution
NOTE The general form of the relationship as described in ISO 9276-5 is
2
rp− s
()
CMD==xx e
50,rp50,
where
e is the base of natural logarithms, e = 2,718 28;
p is the dimensionality (type of quantity) of a distribution, where
p = 0 is the number,
p = 1 is the length,
p = 2 is the area, and
p = 3 is the volume or mass;
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SIST EN ISO 10808:2011
ISO 10808:2010(E)
r is the dimensionality (type of quantity) of a distribution, where
r = 0 is the number,
r = 1 is the length,
r = 2 is the area, and
r = 3 is the volume or mass;
s is the standard deviation of the density distribution;
x is the median particle size of a cumulative distribution of dimensionality, r.
50,r
4 Test substance monitoring method
4.1 Principle
4.1.1 Exposure
Precise characterization of the test substance exposure is essential for an inhalation toxicology study. The
objective in nanoparticle inhalation toxicology is to establish a quantitative relationship between the observed
toxicological outcome and the dose metrics used in terms of test substance physical and chemical properties.
4.1.2 Particle properties
The specific chemical and physical properties of the nanoparticle should be determined to the extent possible;
however, because these may not be known a priori, as many parameters as possible should be determined.
Nanoparticle composition, number and mass concentrations, median and mean size and size distribution,
surface area, electrical charge, surface character, hygroscopicity and shape might be important parameters
for dosimetry.
4.2 Preparation of system
4.2.1 During development of the nanoparticle generating system and prior to interfacing with the exposure
chamber(s), measurements should be performed to verify aerosol particle composition and purity and to
establish the stability. During exposure tests, analysis should be conducted continuously and/or intermittently
depending on the method of analysis to determine the consistency of particle size distribution without
disrupting the inhalation exposure.
[3]
NOTE A nanoparticle generating system for silver and other metals is described in ISO 10801 .
4.2.2 Inhalation chambers and supporting equipment shall be prepared in accordance with OECD TG 403,
OECD TG 412 and OECD TG 413.
4.2.3 Inhalation chambers and supporting equipment shall be prepared for nanoparticle exposure studies.
NOTE 1 Aerosolized nanoparticles can be deposited to walls by Brownian diffusion and particle size change due to
aggregation/agglomeration. This deposition process depends on the particle size, electrostatic charge, particle number
concentration and residence time. See standard texts on aerosol science, References [11], [19] and [20].
NOTE 2 Charge neutralization might be required, depending on the purpose of the study.
If charge distribution is considered a characterization requirement, this shall be specified and measured in the
study.
NOTE 3 To reduce deposition losses, conductive tubing of the minimum length practical to use with the tubing diameter
is selected to interface with instrumentation.
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SIST EN ISO 10808:2011
ISO 10808:2010(E)
4.2.4 An inhalation chamber or chambers and supporting equipment, such as sampling probes and
manifolds, shall be characterized to ensure compliance with OECD TG 403, OECD TG 412 and
OECD TG 413, for determining any sampling bias.
NOTE Sampling manifold consists of tubing, solenoid valves and/or other elements required for routing samples from
each chamber to online monitoring equipment.
4.2.5 Measurement instruments used in inhalation testing should be calibrated and/or tested in accordance
with ISO 15900.
The DMAS is usually calibrated at the factory and this should be documented in the report.
NOTE In addition, in the course of using the DMAS, it must be routinely calibrated as well.
4.3 Study
4.3.1 The study shall be conducted in accordance with OECD TG 403, OECD TG 412, OECD TG 413 and
OECD GD 39.
4.3.2 During the exposure period the concentrations of the test substance should be held as constant as
practicable and monitored continuously and/or intermittently depending on the method of analysis.
4.3.3 Breathing zone sampling shall be conducted to establish exposure.
4.3.4 The rate of air flow in the supply and chamber(s), should be monitored continuously in order to
document compliance with OECD TG 403, OECD TG 412, OECD TG 413 and OECD GD 39.
Airflow meters should be employed to establish that the parameter is within limits.
4.3.5 Temperature and humidity inside the inhalation chamber and as close to the breathing zone as
practical shall be monitored continuously.
Temperature and humidity sensors with transducers should be employed to establish that the parameter is
within limits.
4.3.6 Exhaust air from the chambers containing nanoparticles shall be treated by appropriate filtration, and,
if necessary or appropriate, chemical scrubbing, before being vented to the atmosphere.
5 Specific monitoring method
5.1 Requirements for number-based particle size distribution and mass concentration
Measurement of number-based particle size distribution and measurement of total particle mass concentration
are two essential measurements in the characterization of nanoparticles in inhalation toxicity testing. Particle
size distribution measurement is essential because the knowledge of particle size is crucial for the evaluation
of the result of toxicity testing. Mass concentration, on the other hand, has been used as the dosimetric
parameter in every inhalation toxicity test and is indispensable in nanoparticle toxicity testing. Therefore, these
two measurements shall always be made in nanoparticle inhalation toxicity testing and carried out using
appropriate methods.
5.2 Measurement of number-based particle size distribution
5.2.1 The method used shall be able to monitor particle size distribution in a continuous manner during
particle exposures with time resolution appropriate to checking the stability of particle size distribution and
concentration.
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SIST EN ISO 10808:2011
ISO 10808:2010(E)
5.2.2 The measurable range of particle sizes and concentrations in the animal's breathing zone shall cover
those of the nanoparticle aerosols exposed to the test system during the toxicity test.
5.2.3 Particle size and concentration measurements in the animal's breathing zone should be accurate for
nanoparticle toxicity testing, and can be validated by means such as calibration against appropriate reference
standards (see ISO/IEC 17025).
5.2.4 The resolution of particle sizing shall be accurate and the range of particle sizes measured shall be
sufficiently broad to permit conversion from number-based distribution to surface area-based or volume-based
distribution.
NOTE For particle size distribution, measurement with DMAS is the only currently available method that meets all the
above requirements in the size range below 100 nm (see ISO 15900).
Particles larger than 100 nm may be measured by other instruments using optical or electrical properties, time
[8]
of flight, or other aerodynamic properties .
5.3 Mass concentration measurement
The method selected shall be accurate and sensitive, and defined by the limit of quantification, for
nanoparticle aerosols exposed to test subject during the toxicity test.
NOTE 1 Beta attenuation monitor (BAM), tapered element oscillating microbalance (TEOM), piezoelectric
microbalance, filter gravimetric, and other methods based on chemical analysis of particles collected on filter media may
[4]
meet the requirements for nanoparticle mass concentration measurement .
NOTE 2 Mass concentration can be derived from number-based size distribution measurement data by making an
[14]
assumption regarding particle density, particularly for spherical particles which may match bulk material density .
However, significant errors in calculated mass concentration may result if particle density is inaccurate or unknown.
Derived mass concentration from number-based size distribution data should be accepted only when no other
accepted methods meet the measuring requirements.
5.4 Inhalation exposure chamber
−1 −1
5.4.1 Air flow shall be 10 h to 15 h air changes for whole body chamber. For nose-only exposure
chambers, the air flow shall be at least twice the respiratory minute volume of animals exposed (e.g. at least
0,5 l/min per exposure port for rats).
The chamber should be prepared such that distribution of nanoparticles inside it is uniform.
5.4.2 Temperature and humidity shall remain within study established limits.
NOTE OECD TG 413 prescribes that the temperature at which the test is performed be maintained at 22 °C (±3°).
Ideally, the relative humidity should be maintained between 30 % and 70 %, but in certain instances (e.g. tests
of aerosols) this may not be practicable.
5.4.3 Pressure inside the chamber shall remain slightly negative (u 5 mm water) to prevent leakage outside
the testing boundaries.
For nose-only exposure, pressure should be slightly positive to ensure animals will be properly exposed. Due
to potential leakage from this positive pressure, nose-only experiments should be conducted inside the
boundaries of an adequately designed fume hood (see OECD GD 39).
5.4.4 Supply air shall ensure an adequate oxygen content of at least 19 % as well as uniform conditions
throughout the exposure chamber.
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SIST EN ISO 10808:2011
ISO 10808:2010(E)
6 Assessment of results
6.1 The following nanoparticle data shall be obtained to assist with interpretation of the study results:
a) nanoparticle number-size (in nanometres) distribution, geometric mean diameter (GMD), and geometric
standard deviation (GSD) in each exposure chamber using DMAS, TEM, SEM, etc.;
b) particle morphology using TEM or SEM with an adaptation of ISO
...