ISO/TR 16196:2016
(Main)Nanotechnologies — Compilation and description of sample preparation and dosing methods for engineered and manufactured nanomaterials
Nanotechnologies — Compilation and description of sample preparation and dosing methods for engineered and manufactured nanomaterials
ISO/TR 16196:2016 provides guidance regarding the preparation of nanomaterials for eco- and bio- toxicological testing. It provides guidance regarding factors pertaining to sample preparation and dose determination that might be useful in toxicological, including ecotoxicological, testing of engineered and manufactured nanoscale materials. The descriptions of sample preparation method factors for both in vitro and in vivo toxicological testing of engineered and manufactured nanoscale materials include considerations about physico-chemical properties, media, methods for transformation and accumulation studies, health effects and dosimetry. The document is not intended to be a literature review nor a thorough assessment of the quality of the methods or data generated. The document is intended to complement other international efforts. The focus of this document is on factors that might lead to results that are not relevant to safety evaluations. When featured, referenced methods are considered for their general interest and potential applicability. It is likely that most of the described methods are not generally applicable to all nanomaterials but they do demonstrate important factors and limitations that are common for a variety of nanomaterials.
Nanotechnologies — Compilation et description de la préparation des échantillons et des méthodes de dosage pour les nanomatériaux d'ingénierie et manufacturés
General Information
Buy Standard
Standards Content (Sample)
TECHNICAL ISO/TR
REPORT 16196
First edition
2016-10-01
Nanotechnologies — Compilation and
description of sample preparation and
dosing methods for engineered and
manufactured nanomaterials
Nanotechnologies — Compilation et description de la préparation
des échantillons et des méthodes de dosage pour les nanomatériaux
d’ingénierie et manufacturés
Reference number
ISO/TR 16196:2016(E)
©
ISO 2016
---------------------- Page: 1 ----------------------
ISO/TR 16196:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TR 16196:2016(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 3
5 Background . 3
5.1 Discussion of the importance of sample preparation and dosing . 3
5.2 Discussion of specific issues when testing the toxicology of manufactured nanomaterials 4
5.2.1 Physical properties . 4
5.2.2 Sample preparation . 4
5.2.3 Administration of doses. 5
5.2.4 Discussion of relationship between this document and ISO/TR 13014 . 5
5.3 Discussion of relevant dosing for toxicological screening . 5
5.4 Discussion of the relationship between this document and ISO/TR 16197 . 5
5.5 Review of other relevant international activities and documents . 5
6 Physico-chemical properties . 6
6.1 Particle size, shape, size distribution and degree of agglomeration . . 6
6.2 Chemical description — Composition and identification . 6
6.3 Specific surface area . 6
6.4 Surface chemistry . 6
6.5 Isoelectric point, zeta potential and Hamaker constant . 7
6.6 Influence of water chemistry on nanomaterial properties and dispersion behavior . 8
6.7 Preparation of liquid dispersions . 8
6.8 Crystal structure . 8
6.9 Surface energy or interfacial tension . 8
6.10 Solubility . 8
7 Considerations for preparing samples of nanomaterials in exposure media in
ecotoxicity studies . 9
7.1 General . 9
7.1.1 Introduction to existing knowledge . 9
7.1.2 Environmental behaviour . 9
7.1.3 Degradation and transformation . 9
7.1.4 Bioaccumulation . 9
7.2 Test method applicability and dosimetry .10
7.2.1 Environmental distribution .10
7.2.2 Degradation and transformation .10
7.2.3 Bioaccumulation .11
8 Methods for preparing nanomaterials for toxicological studies in mammals that
ensure correct dosing .11
8.1 Issues to consider for stock dispersion preparation .11
8.2 Importance of monitoring stability of test dispersions during experiments .11
8.3 Special considerations for physiological salines used in mammalian studies .12
8.4 Routes of delivery and behaviour of nanomaterial dispersions in mammalian studies .12
8.4.1 Respiratory tract exposures .12
8.4.2 Oral exposure .12
8.4.3 Dermal exposures .13
8.4.4 Injection routes .13
9 Methods for preparing samples of nanomaterials for use in cell cultures .13
9.1 Cell cultures and dispersion of nanomaterials in culture media .13
9.1.1 General.13
© ISO 2016 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/TR 16196:2016(E)
9.1.2 Considerations for nanomaterial dispersion in the dosing solution .13
9.1.3 Consideration of relevant exposure scenarios .14
9.2 Cell cultures .14
Bibliography .16
iv © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TR 16196:2016(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 229, Nanotechnologies.
© ISO 2016 – All rights reserved v
---------------------- Page: 5 ----------------------
ISO/TR 16196:2016(E)
Introduction
This document provides guidance regarding the preparation of nanomaterials for toxicological,
including eco-toxicological, testing. The goal of this document is to assist health and environmental
scientists and scientists and experts from other disciplines to understand, plan, choose and address
issues relevant to nanomaterials before and during conducting toxicological tests. These issues include
the effects of the properties of the material on preparation methods and of the media into which the
samples of nanomaterials will be added. Failure to consider these effects might lead to erroneous
conclusions regarding the relationship between the nature of the nanomaterial and observed
toxicological responses. In particular, the composition and other characteristics of test media can affect
the dose to which an organism that is the subject of a test will be exposed. Information on preparation
of the test material is necessary prior to any biological or ecological evaluation. Information such as
[1]
this is consistent with other ISO documents. For example, ISO 10993-18 specifically addresses the
[2]
evaluation of the chemical characterization of materials used in medical devices, ISO 14971 specifies
that a toxicological risk analysis should take into account the chemical nature of the materials,
[3] [55]
ISO/TR 13014 addresses issues pertaining to the materials themselves and ISO/TS 19337 points
out the need to clarify whether observed toxic effects come from tested nano-objects themselves or
from other uncontrolled sources. Some examples are provided of methods that establish test conditions
that are relatable to environmentally relevant conditions.
This document uses a number of technical terms which have been defined earlier in other documents.
Some of these terms have been defined in multiple documents, in different areas of science and
technology, providing potentially or seemingly conflicting definitions. This document does not provide
new, authoritative definitions for the terms used herein. Instead, this clause provides short descriptions
for the terms used. Where possible, reference is made to existing documents.
vi © ISO 2016 – All rights reserved
---------------------- Page: 6 ----------------------
TECHNICAL REPORT ISO/TR 16196:2016(E)
Nanotechnologies — Compilation and description of
sample preparation and dosing methods for engineered
and manufactured nanomaterials
1 Scope
This document provides guidance regarding the preparation of nanomaterials for eco- and bio-
toxicological testing. It provides guidance regarding factors pertaining to sample preparation and dose
determination that might be useful in toxicological, including ecotoxicological, testing of engineered
and manufactured nanoscale materials.
The descriptions of sample preparation method factors for both in vitro and in vivo toxicological testing
of engineered and manufactured nanoscale materials include considerations about physico-chemical
properties, media, methods for transformation and accumulation studies, health effects and dosimetry.
The document is not intended to be a literature review nor a thorough assessment of the quality of the
methods or data generated. The document is intended to complement other international efforts.
The focus of this document is on factors that might lead to results that are not relevant to safety
evaluations. When featured, referenced methods are considered for their general interest and
potential applicability. It is likely that most of the described methods are not generally applicable to
all nanomaterials but they do demonstrate important factors and limitations that are common for a
variety of nanomaterials.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
particle
minute piece of matter with defined physical boundaries
Note 1 to entry: A physical boundary can also be described as an interface.
Note 2 to entry: A particle can move as a unit.
Note 3 to entry: This general particle definition applies to nano-objects.
[SOURCE: ISO/TS 80004-2:2015, 3.1]
3.2
structure
arrangement defined by four different aspects (crystallinity, crystal structure, molecular structure and
microstructure)
© ISO 2016 – All rights reserved 1
---------------------- Page: 7 ----------------------
ISO/TR 16196:2016(E)
3.2.1
crystallinity
presence or absence of crystalline structure in the arrangement of the atoms of which a material
consists
3.2.2
crystal structure
lattice structure in which atoms of an individual crystal are arranged, using lattice parameters and
lattice type, such as face-centred cubic, hexagonal close-packed, body-centred, cubic, etc.
3.2.3
molecular structure
arrangement of atoms of an individual molecule
3.2.4
microstructure
arrangement of individual crystals or amorphous phases in a polycrystalline or multiphase material
3.3
measurand
quantity intended to be measured or a quantity that is being determined by measurement
Note 1 to entry: The specification of a measurand requires knowledge of the kind of quantity, description of the
state of the phenomenon, body, or substance carrying the quantity, including any relevant component, and the
chemical entities involved. The measurement, including the measuring system and the conditions under which
the measurement is carried out, might change the phenomenon, body, or substance so that the quantity being
measured may differ from the measurand as defined.
[SOURCE: ISO/IEC Guide 99, 2007, 2.3 — modified]
3.4
nanomaterial
NM
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
Note 2 to entry: See also engineered nanomaterial, manufactured nanomaterial, incidental nanomaterial.
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.5
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-2:2015, 2.2]
3.6
nanoparticle
NP
nano-object with all external dimensions in the nanoscale where the lengths of the longest and the
shortest axes of the nano-object do not differ significantly
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms, such as
nanofibre or nanoplate, may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
2 © ISO 2016 – All rights reserved
---------------------- Page: 8 ----------------------
ISO/TR 16196:2016(E)
3.7
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from a larger size are predominantly exhibited in this
length range.
[SOURCE: ISO/TS 80004-2:2015, 2.1]
4 Abbreviated terms
BET Brunauer–Emmett–Teller isotherm
CNT carbon nanotube
DLS dynamic light scattering
ICP-MS inductively coupled plasma mass spectrometry
NOAA nano-objects, and their aggregates and agglomerates greater than 100 nm
NOM natural organic material
TEM transmission electron microscopy
5 Background
5.1 Discussion of the importance of sample preparation and dosing
Nanomaterials are diverse, being based on endless combinations of composition, particle size and
distribution, surface chemistry and many other key properties. With this diversity, nanomaterials
cannot be treated as a single class of substances. Just as in other areas of toxicology, the assessment
of biological effects should consider how samples and doses are prepared and dosimetry is assured so
that the observed effects are meaningful and test results can be used in a realistic way such as in safety
assessments.
Screening tests are used for rapid evaluations and are typically conducted using cell culture or other
in vitro techniques due to fast response time, cost, infrastructure and time constraints, factors that
limit most whole animal studies. The purpose of a screening test is to provide an indicator of potential
adverse outcomes and effects on human health or the environment. Although there are many definitions
available for the term screening test, for the purposes of this document, a screening test can be
generally defined as a relatively simple, inexpensive test that can be administered easily and provides
rapid results. The screening tests should reflect the compromise between simplicity, rapidity and low-
cost while still providing results that have meaning to safety-relevant situations, the way that samples
are prepared and the doses administered will ideally be relatable to realistic situations. Therefore, the
points considered in this document also apply to screening tests and should also be taken into account
in tiered testing to ensure consistent conditions with each step of the tier.
© ISO 2016 – All rights reserved 3
---------------------- Page: 9 ----------------------
ISO/TR 16196:2016(E)
5.2 Discussion of specific issues when testing the toxicology of manufactured
nanomaterials
5.2.1 Physical properties
5.2.1.1 General
The subject that is sometimes referred to as “nanotoxicology” considers the same issues as the broader
subject of toxicology with additional scrutiny paid to elements based on a very small size. These include,
but are not limited to, physical properties that are described in detail below in 5.2.1.2 to 5.2.1.5.
5.2.1.2 Size
Smaller particles can have the ability to reach target sites that larger forms cannot. In addition, smaller
particles present larger surface areas possibly increasing their chemical reactivity. An example might
be inhaled particles (<100 nm) that can reach the alveolar region of the lungs.
5.2.1.3 Size distribution
Most nanoparticles are not of a single size, therefore there is a distribution of sizes. For some materials,
there might be nano-sized fractions and aggregated larger particles present of the same composition.
The toxicological contributions of each should be distinguished where possible.
5.2.1.4 Dissolution
Nanomaterials have a greater surface to mass ratio so sparingly soluble nanomaterials more readily
dissolve in solutions than the same material in bulk form. Examples include amorphous silicon dioxide,
zinc oxide and silver. When a material has any appreciable solubility, the relative contributions of
toxicological effects due to particulates versus dissolved species should be considered. In some cases
where dissolution is complete, observed effects might be due to the ionized/dissolved fraction rather
than the nanoparticles, even though the initial test substance was a nanoparticle.
5.2.1.5 Transformation
Because nanomaterials are not a single class of materials there are an enormous number of uses for
them. Some of these uses might involve intentional and unintentional transformations. An example of
an intentional transformation is the use of aluminium nanomaterials in energetic applications where at
[4]
least some of the aluminium is converted to alumina . An example of an unintentional transformation
is the release of CNTs from matrices into environmental settings in which they would be subject to
photochemical processes, oxidation, biotransformation, etc. CNTs might be subject to combustion
processes if they are part of matrices that are burned. With each transformation it is important to
consider if changes have occurred that require recharacterization of the test nanomaterial.
5.2.2 Sample preparation
Toxicological studies are generally performed to evaluate the potential hazards of materials. For sample
preparation, care should be taken to ensure that the material is prepared in a way that is appropriate
for the toxicological evaluation that will be applied. The OECD “Guidance on Sample Preparation and
[5]
Dosimetry for the Safety Testing of Manufactured Nanomaterials” provides information regarding
the critical aspects that should be considered for preparation of nanomaterial samples for testing and
the use of relevant dose and dose metrics. It is important to prepare and administer nanomaterials in a
way that is representative of a potential exposure, i.e. which can be linked to an exposure scenario. As
[6][7][8][9]
with assays in general, potential interferences should be considered .
4 © ISO 2016 – All rights reserved
---------------------- Page: 10 ----------------------
ISO/TR 16196:2016(E)
5.2.3 Administration of doses
After sample preparation it is important to quantify and characterize the administered dose and the
received dose.
5.2.4 Discussion of relationship between this document and ISO/TR 13014
ISO/TR 13014 provides essential guidance to all researchers who attempt to assess relationships
between nanomaterial exposures and linked biological responses. However, as of the time of writing,
the practices described in ISO/TR 13014 are not widely practiced. It is not possible to find many
references in literature where materials are characterized as described in ISO/TR 13014. ISO/TR 13014
was used as a benchmark in considering whether the studies cited in the references contained in this
document should be considered.
5.3 Discussion of relevant dosing for toxicological screening
Humans and the environment may be exposed to nanomaterials via a number of routes, e.g. inhalation,
ingestion, dermal for humans, or environment via water. The exposure concentrations for some of these
scenarios can be determined, e.g. airborne nanomaterials in the workplace or particles per mass unit
of oil-in-water emulsions applied to the skin. Currently, the exposure concentrations of engineered and
manufactured nanomaterials for the environment is unknown. While certainty of these concentrations
is required for quantitative risk assessment, it is not required for the hazard characterization typically
associated with screening level assays.
However, care should be employed by the investigator conducting screening assays and by the
assessors evaluating the data so that the relationship between effect and dose is not over-interpreted.
Whenever possible, investigators should use dose levels that approximate the estimated concentrations
to realistic exposure dose; therefore, in vitro studies of cell cultures from the respiratory tract should
use concentrations that relate to lung burden observed following inhalation, or in vitro studies of
keratinocytes should use concentration levels that are consistent with dose applied to the skin.
Other dose levels might and should be used to demonstrate dose-related responses. These additional
dose levels are frequently exaggerated levels of real exposures, but they serve to stress the system
investigated. The use of single dose level studies is discouraged.
5.4 Discussion of the relationship between this document and ISO/TR 16197
This document was developed in concert with a sister document ISO/TR 16197. ISO/TR 16197 discusses
methods used to prepare samples in various relevant media for toxicological studies, and also discusses
issues of relevant dose metrics for toxicological testing considering the various routes of administration.
ISO/TR 16197 complements this document by moving from sample preparation and dosimetry into a
more detailed discussion of the various methods used to perform toxicological screening. When using
ISO/TR 16197, it is important to consider this document since difficulties associated with sample
preparation and dosimetry are often the bottleneck in performing a good toxicological assessment of
nanomaterials.
5.5 Review of other relevant international activities and documents
Readers/users of this document should also take note of an OECD document, “Guidance on Sample
[5]
Preparation and Dosimetry for the Safety Testing of Manufactured Nanomaterials” . This document
provides a perspective based on a detailed review of the literature at the time of the document’s
publication including potential issues to be addressed.
© ISO 2016 – All rights reserved 5
---------------------- Page: 11 ----------------------
ISO/TR 16196:2016(E)
6 Physico-chemical properties
6.1 Particle size, shape, size distribution and degree of agglomeration
The size of a particle can change once it is put into a new medium. For example, some particles might
form homo- or hetero-agglomerates and others might de-agglomerate. A study was performed using
micro- and nano-sized carboxylated polystyrene beads to assess the effect on size using daphnia as the
[10]
target organism . It was observed that the larger particles were taken up more readily but were also
more readily cleared.
6.2 Chemical description — Composition and identification
The possibility that the chemical composition of a nanomaterial, where the composition considers the
chemical constituents primarily on the surface, might change due to reaction with the
...
TECHNICAL ISO/TR
REPORT 16196
First edition
Nanotechnologies — Compilation and
description of sample preparation and
dosing methods for engineered and
manufactured nanomaterials
Nanotechnologies — Compilation et description de la préparation
des échantillons et des méthodes de dosage pour les nanomatériaux
d’ingénierie et manufacturés
PROOF/ÉPREUVE
Reference number
ISO/TR 16196:2016(E)
©
ISO 2016
---------------------- Page: 1 ----------------------
ISO/TR 16196:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TR 16196:2016(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 3
5 Background . 3
5.1 Discussion of the importance of sample preparation and dosing . 3
5.2 Discussion of specific issues when testing the toxicology of manufactured nanomaterials 4
5.2.1 Physical properties . 4
5.2.2 Sample preparation . 4
5.2.3 Administration of doses. 5
5.2.4 Discussion of relationship between this document and ISO/TR 13014 . 5
5.3 Discussion of relevant dosing for toxicological screening . 5
5.4 Discussion of the relationship between this document and ISO/TR 16197 . 5
5.5 Review of other relevant international activities and documents . 5
6 Physico-chemical properties . 6
6.1 Particle size, shape, size distribution and degree of agglomeration . . 6
6.2 Chemical description — Composition and identification . 6
6.3 Specific surface area . 6
6.4 Surface chemistry . 6
6.5 Isoelectric point, zeta potential and Hamaker constant . 7
6.6 Influence of water chemistry on nanomaterial properties and dispersion behavior . 8
6.7 Preparation of liquid dispersions . 8
6.8 Crystal structure . 8
6.9 Surface energy or interfacial tension . 8
6.10 Solubility . 8
7 Considerations for preparing samples of nanomaterials in exposure media in
ecotoxicity studies . 9
7.1 General . 9
7.1.1 Introduction to existing knowledge . 9
7.1.2 Environmental behaviour . 9
7.1.3 Degredation and transformation . 9
7.1.4 Bioaccumulation . 9
7.2 Test method applicability and dosimetry .10
7.2.1 Environmental distribution .10
7.2.2 Degredation and transformation .10
7.2.3 Bioaccumulation .11
8 Methods for preparing nanomaterials for toxicological studies in mammals that
ensure correct dosing .11
8.1 Issues to consider for stock dispersion preparation .11
8.2 Importance of monitoring stability of test dispersions during experiments .11
8.3 Special considerations for physiological salines used in mammalian studies .12
8.4 Routes of delivery and behaviour of nanomaterial dispersions in mammalian studies .12
8.4.1 Respiratory tract exposures .12
8.4.2 Oral exposure .12
8.4.3 Dermal exposures .13
8.4.4 Injection routes .13
9 Methods for preparing samples of nanomaterials for use in cell cultures .13
9.1 Cell cultures and dispersion of nanomaterials in culture media .13
9.1.1 General.13
© ISO 2016 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO/TR 16196:2016(E)
9.1.2 Considerations for nanomaterial dispersion in the dosing solution .13
9.1.3 Consideration of relevant exposure scenarios .14
9.2 Cell cultures .14
Bibliography .16
iv PROOF/ÉPREUVE © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TR 16196:2016(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 229, Nanotechnologies.
© ISO 2016 – All rights reserved PROOF/ÉPREUVE v
---------------------- Page: 5 ----------------------
ISO/TR 16196:2016(E)
Introduction
This document provides guidance regarding the preparation of nanomaterials for toxicological,
including eco-toxicological, testing. The goal of this document is to assist health and environmental
scientists and scientists and experts from other disciplines to understand, plan, choose and address
issues relevant to nanomaterials before and during conducting toxicological tests. These issues include
the effects of the properties of the material on preparation methods and of the media into which the
samples of nanomaterials will be added. Failure to consider these effects might lead to erroneous
conclusions regarding the relationship between the nature of the nanomaterial and observed
toxicological responses. In particular, the composition and other characteristics of test media can affect
the dose to which an organism that is the subject of a test will be exposed. Information on preparation
of the test material is necessary prior to any biological or ecological evaluation. Information such as
[1]
this is consistent with other ISO documents. For example, ISO 10993-18 specifically addresses the
[2]
evaluation of the chemical characterization of materials used in medical devices, ISO 14971 specifies
that a toxicological risk analysis should take into account the chemical nature of the materials,
[3] [55]
ISO/TR 13014 addresses issues pertaining to the materials themselves and ISO/TS 19337 points
out to help clarify whether observed toxic effects come from tested nano-objects themselves or from
other uncontrolled sources. Some examples are provided of methods that establish test conditions that
are relatable to environmentally relevant conditions.
This document uses a number of technical terms which have been defined earlier in other documents.
Some of these terms have been defined in multiple documents, in different areas of science and
technology, providing potentially or seemingly conflicting definitions. This document does not provide
new, authoritative definitions for the terms used herein. Instead, this clause provides short descriptions
for the terms used. Where possible, reference is made to existing documents.
vi PROOF/ÉPREUVE © ISO 2016 – All rights reserved
---------------------- Page: 6 ----------------------
TECHNICAL REPORT ISO/TR 16196:2016(E)
Nanotechnologies — Compilation and description of
sample preparation and dosing methods for engineered
and manufactured nanomaterials
1 Scope
This document provides guidance regarding the preparation of nanomaterials for eco- and bio-
toxicological testing. It provides guidance regarding factors pertaining to sample preparation and dose
determination that might be useful in toxicological, including ecotoxicological, testing of engineered
and manufactured nanoscale materials.
The descriptions of sample preparation method factors for both in vitro and in vivo toxicological testing
of engineered and manufactured nanoscale materials include considerations about physico-chemical
properties, media, methods for transformation and accumulation studies, health effects and dosimetry.
The document is not intended to be a literature review nor a thorough assessment of the quality of the
methods or data generated. The document is intended to complement other international efforts.
The focus of this document is on factors that might lead to results that are not relevant to safety
evaluations. When featured, referenced methods are considered for their general interest and
potential applicability. It is likely that most of the described methods are not generally applicable to
all nanomaterials but they do demonstrate important factors and limitations that are common for a
variety of nanomaterials.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
particle
minute piece of matter with defined physical boundaries
Note 1 to entry: A physical boundary can also be described as an interface.
Note 2 to entry: A particle can move as a unit.
Note 3 to entry: This general particle definition applies to nano-objects.
[SOURCE: ISO/TS 80004-2:2015, 3.1]
3.2
structure
defined by four different aspects — crystallinity, crystal structure, and molecular structure
© ISO 2016 – All rights reserved PROOF/ÉPREUVE 1
---------------------- Page: 7 ----------------------
ISO/TR 16196:2016(E)
3.2.1
crystallinity
presence or absence of crystalline structure in the arrangement of the atoms of which a material
consists
3.2.2
crystal structure
lattice structure in which atoms of an individual crystal are arranged, using lattice parameters and
lattice type, such as face-centered cubic, hexagonal close-packed, body-centred, cubic, etc.
3.2.3
molecular structure
arrangement of atoms of an individual molecule
3.2.4
microstructure
arrangement of individual crystals or amorphous phases in a polycrystalline or multiphase material
3.3
measurand
quantity intended to be measured or a quantity that is being determined by measurement
Note 1 to entry: The specification of a measurand requires knowledge of the kind of quantity, description of the
state of the phenomenon, body, or substance carrying the quantity, including any relevant component, and the
chemical entities involved. The measurement, including the measuring system and the conditions under which
the measurement is carried out, might change the phenomenon, body, or substance so that the quantity being
measured may differ from the measurand as defined.
[SOURCE: ISO/IEC Guide 99, 2007, 2.3 — modified]
3.4
nanomaterial
NM
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
Note 2 to entry: See also engineered nanomaterial, manufactured nanomaterial, incidental nanomaterial.
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.5
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-2:2015, 2.2]
3.6
nanoparticle
NP
nano-object with all external dimensions in the nanoscale where the lengths of the longest and the
shortest axes of the nano-object do not differ significantly
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms, such as
nanofibre or nanoplate, may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
2 PROOF/ÉPREUVE © ISO 2016 – All rights reserved
---------------------- Page: 8 ----------------------
ISO/TR 16196:2016(E)
3.7
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from a larger size are predominantly exhibited in this
length range.
[SOURCE: ISO/TS 80004-2:2015, 2.1]
4 Abbreviated terms
BET Brunauer–Emmett–Teller isotherm
CNT carbon nanotube
DLS dynamic light scattering
ICP-MS inductively coupled plasma mass spectrometry
NOAA nano-objects, and their aggregates and agglomerates greater than 100 nm
NOM natural organic material
TEM transmission electron microscopy
5 Background
5.1 Discussion of the importance of sample preparation and dosing
Nanomaterials are diverse, being based on endless combinations of composition, particle size and
distribution, surface chemistry and many other key properties. With this diversity, nanomaterials
cannot be treated as a single class of substances. Just as in other areas of toxicology, the assessment
of biological effects should consider how samples and doses are prepared and dosimetry is assured so
that the observed effects are meaningful and test results can be used in a realistic way such as in safety
assessments.
Screening tests are used for rapid evaluations and are typically conducted using cell culture or other
in vitro techniques due to fast response time, cost, infrastructure and time constraints, factors that
limit most whole animal studies. The purpose of a screening test is to provide an indicator of potential
adverse outcomes and effects on human health or the environment. Although there are many definitions
available for the term screening test, for the purposes of this document, a screening test can be
generally defined as a relatively simple, inexpensive test that can be administered easily and provides
rapid results. The screening tests should reflect the compromise between simplicity, rapidity and low-
cost while still providing results that have meaning to safety-relevant situations, the way that samples
are prepared and the doses administered will ideally be relatable to realistic situations. Therefore, the
points considered in this document also apply to screening tests and should also be taken into account
in tiered testing to ensure consistent conditions with each step of the tier.
© ISO 2016 – All rights reserved PROOF/ÉPREUVE 3
---------------------- Page: 9 ----------------------
ISO/TR 16196:2016(E)
5.2 Discussion of specific issues when testing the toxicology of manufactured
nanomaterials
5.2.1 Physical properties
5.2.1.1 General
The subject that is sometimes referred to as “nanotoxicology” considers the same issues as the broader
subject of toxicology with additional scrutiny paid to elements based on a very small size. These include,
but are not limited to, physical properties that are described in detail below in 5.2.1.2 to 5.2.1.5.
5.2.1.2 Size
Smaller particles can have the ability to reach target sites that larger forms cannot. In addition, smaller
particles present larger surface areas possibly increasing their chemical reactivity. An example might
be inhaled particles (<100 nm) that can reach the alveolar region of the lungs.
5.2.1.3 Size distribution
Most nanoparticles are not of a single size, therefore there is a distribution of sizes. For some materials,
there might be nano-sized fractions and aggregated larger particles present of the same composition.
The toxicological contributions of each should be distinguished where possible.
5.2.1.4 Dissolution
Nanomaterials have a greater surface to mass ratio so sparingly soluble nanomaterials more readily
dissolve in solutions than the same material in bulk form. Examples include amorphous silicon dioxide,
zinc oxide and silver. When a material has any appreciable solubility, the relative contributions of
toxicological effects due to particulates versus dissolved species should be considered. In some cases
where dissolution is complete, observed effects might be due to the ionized/dissolved fraction rather
than the nanoparticles, even though the initial test substance was a nanoparticle.
5.2.1.5 Transformation
Because nanomaterials are not a single class of materials there are an enormous number of uses for
them. Some of these uses might involve intentional and unintentional transformations. An example of
an intentional transformation is the use of aluminium nanomaterials in energetic applications where at
[4]
least some of the aluminium is converted to alumina . An example of an unintentional transformation
is the release of CNTs from matrices into environmental settings in which they would be subject to
photochemical processes, oxidation, biotransformation, etc. CNTs might be subject to combustion
processes if they are part of matrices that are burned. With each transformation it is important to
consider if changes have occurred that require recharacterization of the test nanomaterial.
5.2.2 Sample preparation
Toxicological studies are generally performed to evaluate the potential hazards of materials. For sample
preparation, care should be taken to ensure that the material is prepared in a way that is appropriate
for the toxicological evaluation that will be applied. The OECD “Guidance on Sample Preparation and
[5]
Dosimetry for the Safety Testing of Manufactured Nanomaterials” provides information regarding
the critical aspects that should be considered for preparation of nanomaterial samples for testing and
the use of relevant dose and dose metrics. It is important to prepare and administer nanomaterials in a
way that is representative of a potential exposure, i.e. which can be linked to an exposure scenario. As
[6][7][8][9]
with assays in general, potential interferences should be considered .
4 PROOF/ÉPREUVE © ISO 2016 – All rights reserved
---------------------- Page: 10 ----------------------
ISO/TR 16196:2016(E)
5.2.3 Administration of doses
After sample preparation it is important to quantify and characterize the administered dose and the
received dose.
5.2.4 Discussion of relationship between this document and ISO/TR 13014
ISO/TR 13014 provides essential guidance to all researchers who attempt to assess relationships
between nanomaterial exposures and linked biological responses. However, as of the time of writing,
the practices described in ISO/TR 13014 are not widely practiced. It is not possible to find many
references in literature where materials are characterized as described in ISO/TR 13014. ISO/TR 13014
was used as a benchmark in considering whether the studies cited in the references contained in this
document should be considered.
5.3 Discussion of relevant dosing for toxicological screening
Humans and the environment may be exposed to nanomaterials via a number of routes, e.g. inhalation,
ingestion, dermal for humans, or environment via water. The exposure concentrations for some of these
scenarios can be determined, e.g. airborne nanomaterials in the workplace or particles per mass unit
of oil-in-water emulsions applied to the skin. Currently, the exposure concentrations of engineered and
manufactured nanomaterials for the environment is unknown. While certainty of these concentrations
is required for quantitative risk assessment, it is not required for the hazard characterization typically
associated with screening level assays.
However, care should be employed by the investigator conducting screening assays and by the
assessors evaluating the data so that the relationship between effect and dose is not over-interpreted.
Whenever possible, investigators should use dose levels that approximate the estimated concentrations
to realistic exposure dose; therefore, in vitro studies of cell cultures from the respiratory tract should
use concentrations that relate to lung burden observed following inhalation, or in vitro studies of
keratinocytes should use concentration levels that are consistent with dose applied to the skin.
Other dose levels might and should be used to demonstrate dose-related responses. These additional
dose levels are frequently exaggerated levels of real exposures, but they serve to stress the system
investigated. The use of single dose level studies is discouraged.
5.4 Discussion of the relationship between this document and ISO/TR 16197
This document was developed in concert with a sister document ISO/TR 16197. ISO/TR 16197 discusses
methods used to prepare samples in various relevant media for toxicological studies, and also discusses
issues of relevant dose metrics for toxicological testing considering the various routes of administration.
ISO/TR 16197 complements this document by moving from sample preparation and dosimetry into a
more detailed discussion of the various methods used to perform toxicological screening. When using
ISO/TR 16197, it is important to consider this document since difficulties associated with sample
preparation and dosimetry are often the bottleneck in performing a good toxicological assessment of
nanomaterials.
5.5 Review of other relevant international activities and documents
Readers/users of this document should also take note of an OECD document, “Guidance on Sample
[5]
Preparation and Dosimetry for the Safety Testing of Manufactured Nanomaterials” . This document
provides a perspective based on a detailed review of the literature at the time of the document’s
publication including potential issues to be addressed.
© ISO 2016 – All rights reserved PROOF/ÉPREUVE 5
---------------------- Page: 11 ----------------------
ISO/TR 16196:2016(E)
6 Physico-chemical properties
6.1 Particle size, shape, size distribution and degree of agglomeration
The size of a particle can change once it is put into a new medium. For example, some particles might
form homo- or hetero-agglomerates and others might de-agglomerate. A study was performed using
micro- and nano-sized carboxylated polystyrene beads to assess the effect on size using daphnia as the
[10]
target organism . It was observed that the larger particles were taken up more readily but were also
more readily cleared.
6.2 Chemical description — Composition and identification
The possibility that the chemical composition of a nanomaterial, where the composition considers th
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.