ISO/TS 19337:2016
(Main)Nanotechnologies — Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicity
Nanotechnologies — Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicity
ISO/TS 19337:2016 describes characteristics of working suspensions of nano-objects to be considered when conducting in vitro assays to evaluate inherent nano-object toxicity. In addition, this Technical Specification identifies applicable measurement methods for these characteristics. This Technical Specification is applicable to nano-objects, and their aggregates and agglomerates greater than 100 nm. NOTE This Technical Specification intends to help clarify whether observed toxic effects come from tested nano-objects themselves or from other uncontrolled sources.
Nanotechnologies — Caracteristiques des suspensions de nano-objets utilisées pour les tests in vitro évaluant la toxicité inherente aux nano-objets
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Standards Content (Sample)
TECHNICAL ISO/TS
SPECIFICATION 19337
First edition
2016-03-15
Nanotechnologies — Characteristics of
working suspensions of nano-objects
for in vitro assays to evaluate inherent
nano-object toxicity
Nanotechnologies — Caracteristiques des suspensions de nano-objets
utilisées pour les tests in vitro évaluant la toxicité inherente aux
nano-objets
Reference number
ISO/TS 19337:2016(E)
©
ISO 2016
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ISO/TS 19337:2016(E)
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ISO/TS 19337:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Characteristics and measurement methods . 2
5.1 General . 2
5.2 Endotoxin . 2
5.3 Stability of working suspensions . 2
5.3.1 General. 2
5.3.2 Representative size change of secondary particles of nano-objects . 3
5.3.3 Concentration change of nano-objects . 3
5.4 Concentration of metal ions. 3
5.5 Concentration of culture medium components . 3
5.5.1 General. 3
5.5.2 Proteins . 4
5.5.3 Calcium . 4
6 Reporting . 4
6.1 General . 4
6.2 Name of nano-objects and manufacturer . 4
6.3 Metallic elements included in the nano-object sample . 4
6.4 Culture medium and serum . 4
6.5 Measurement results . 4
6.6 Deviation . 5
Annex A (informative) Flow of measurements. 6
Annex B (informative) Measurement and evaluation of stability . 7
Annex C (informative) Measurement of metal ions. 8
Annex D (informative) Measurement of culture medium components. 9
Bibliography .10
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ISO/TS 19337:2016(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 WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 229, Nanotechnologies.
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ISO/TS 19337:2016(E)
Introduction
Before nano-objects enter into the market, their possible impact on human health and the environment
needs to be carefully evaluated.
In vitro toxicity assays using cultured cells are frequently used as a tool in screening hazardous
materials. This testing provides essential information for understanding the mechanisms of biological
effects induced by the materials. However, nano-objects require specific considerations with respect
to the in vitro toxicity assays, because their behaviour is distinct from water soluble chemicals. For
example, immediately after the introduction of nano-object samples into the culture medium, the nano-
objects undergo changes, such as (a) dissolution, which is the dissolving of nano-objects into their ionic
counterparts, (b) corona formation, which is the adsorption of the components of culture medium onto
the nano-object surface, or (c) changes in aggregation/agglomeration state, leading to alteration in
particles size and sedimentation. Therefore, it is critical to consider the aforementioned phenomena in
clarifying if the observed effects are related to the tested nano-object itself or from other uncontrolled
sources and to avoid false interpretation of assay results.
The rigorous characterization of the working suspension prior and during in vitro toxicity assays
is essential to exclude the in vitro experimental artefacts. For example, the corona formation, metal
ion release from the nano-objects and impurities (residual from the nano-object synthesis process)
[1]
can interfere with some in vitro assays, producing inaccurate results. Additionally, the formation
of agglomerates and aggregates can alter the toxicity of a suspension. Therefore, it is important to
carefully assess and describe the characteristics of the suspension of nano-objects being tested.
This Technical Specification describes the essential characteristics and applicable measurement
methods of working suspension containing nano-object samples for in vitro toxicity assays. Intention is
that reliable test results on nano-object toxicity could be shared and communicated among stakeholders
of nano-objects, such as regulators, general public, manufacturers and end users. This Technical
Specification does not describe a procedure for validation of working suspension.
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TECHNICAL SPECIFICATION ISO/TS 19337:2016(E)
Nanotechnologies — Characteristics of working
suspensions of nano-objects for in vitro assays to evaluate
inherent nano-object toxicity
1 Scope
This Technical Specification describes characteristics of working suspensions of nano-objects to be
considered when conducting in vitro assays to evaluate inherent nano-object toxicity. In addition, this
Technical Specification identifies applicable measurement methods for these characteristics.
This Technical Specification is applicable to nano-objects, and their aggregates and agglomerates
greater than 100 nm.
NOTE This Technical Specification intends to help clarify whether observed toxic effects come from tested
nano-objects themselves or from other uncontrolled sources.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 29701, Nanotechnologies — Endotoxin test on nanomaterial samples for in vitro systems — Limulus
amebocyte lysate (LAL) test
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
culture medium
aqueous solution of nutrients required for cell growth
3.2
secondary particle
complex agglomerate/aggregate of primary particle(s), proteins and other medium components
3.3
stability
properties to remain unchanged over a given time under stated or reasonably expected conditions of
storage and use for an in vitro toxicity assay
3.4
working suspension
suspension prepared for an in vitro assay that includes culture medium and nano-object sample
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ISO/TS 19337:2016(E)
4 Abbreviated terms
For the purposes of this Technical Specification, the following abbreviated terms apply.
AAS atomic absorption spectrometry
BCA bicinchoninic acid
C-U/F ultrafiltration assisted by centrifugation
DLS dynamic light scattering
FFFF flow field-flow fractionation
ICP-AES inductively coupled plasma-atomic emission spectrometry
ICP-MS inductively coupled plasma mass spectrometry
LD laser diffraction
SLS static light scattering
TFF tangential flow filtration
TOC total organic carbon
U/F ultrafiltration
UV-Vis ultraviolet-visible
5 Characteristics and measurement methods
5.1 General
To characterize the working suspension for in vitro toxicity assays, it is necessary to determine
certain characteristics that might impact the biological system tested. This Clause specifies essential
characteristics of the working suspension, listed below, and measurement methods that are applicable
to them.
— Presence of endotoxins,
— stability of working suspensions,
— concentration of metal ions, and
— concentration of culture medium components.
Measurements of those characteristics shall be made for each dose of working suspensions. The
measurement of endotoxin can be made alternatively for the stock nano-object suspension to be dosed.
See Annex A for an example of flow of measurements.
5.2 Endotoxin
Contamination with endotoxins, part of the outer membrane of Gram-negative bacteria may significantly
alter the results of the in vitro toxicity test. Therefore, it is critical to quantify the concentrations of
endotoxins in the working suspension. The concentration of endotoxins in the working suspension shall
be measured by Limulus amebocyte lysate (LAL) test in accordance with ISO 29701 and the monocyte
[2][3]
activation test (MAT).
5.3 Stability of working suspensions
5.3.1 General
Stability of working suspension is a key characteristic as it directly impacts the in vitro assay conditions
[4][5]
in terms of the dose of the nano-objects to the cells. Aggregation/agglomeration and gravitational
settling of the nano-objects are major issues that may affect the stability of the suspended nano-objects.
The stability shall be evaluated for the two characteristics, i.e. the relative change of representative
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ISO/TS 19337:2016(E)
size of secondary particles of nano-objects and the relative change of the concentration of nano-objects
in the working suspension, resulting from gravitational settling during an in vitro toxicity assay, by
considering experimental duration required for the in vitro toxicity assay. Evaluation results of the
stability shall be expressed in the unit of per cent (%) over the timescale for in vitro toxicity assay.
[6]
NOTE ISO/TR 13097 is recommended as a comprehensive guidance for stability of working suspension.
5.3.2 Representative size change of secondary particles of nano-objects
An appropriate method shall be selected to directly measure the representative size change of
[4][7]
secondary particles of nano-objects from among dynamic light scattering (DLS), laser diffraction
[8] [9]
(LD) and static light scattering (SLS). Other methods deviating from this Technical Specification
can be used and reported in accordance with 6.6.
See Annex B for measurements.
5.3.3 Concentration change of nano-objects
An appropriate method shall be selected to measure the concentration change of nano-objects
[4][7][10]
suspended in the biological media from among the light scattering, inductively coupled plasma
[11][12][13] [14]
mass spectrometry (ICP-MS), ultraviolet-visible (UV-Vis) absorption, X-ray transmission
[15]
and the total organic carbon analysis. Other methods deviating from this Technical Specification
can be used and reported in accordance with 6.6.
See Annex B for measurements.
5.4 Concentration of metal ions
Metal ions, produced as a result of nano-object test sample dissolution, can contribute to test cell
toxicity. The concentration of metal ions in the working suspension shall be measured after separation
of particulate matter. Particulate matter can be separated from the ionic fraction by ultra-filtration
(U/F), ultra filtration assisted by centrifugation (C-U/F) or tangential flow filtration (TFF). The
measurement shall be made for all metallic elements that are included in the nano-object sample. An
appropriate method shall be selected to measure the metal ion concentrations from among inductively
coupled plasma-atomic emission spectrometry (ICP-AES), ICP-MS, atomic absorption spectrometry
(AAS)
...
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