ISO 19337:2023
(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
This document describes the characteristics of working suspensions of nano-objects to be considered when conducting in vitro assays to evaluate inherent nano-object toxicity. In addition, the document identifies applicable measurement methods for these characteristics. This document is applicable to nano-objects, and their aggregates and agglomerates greater than 100 nm. This document intends to help clarify whether observed toxic effects come from tested nano-objects themselves or from uncontrolled sources.
Nanotechnologies — Caractéristiques des suspensions de nano-objets utilisées pour les tests in vitro évaluant la toxicité inhérente aux nano-objets
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INTERNATIONAL ISO
STANDARD 19337
Second edition
2023-05
Nanotechnologies — Characteristics of
working suspensions of nano-objects
for in vitro assays to evaluate inherent
nano-object toxicity
Nanotechnologies — Caractéristiques des suspensions de nano-objets
utilisées pour les tests in vitro évaluant la toxicité inhérente aux nano-
objets
Reference number
ISO 19337:2023(E)
© ISO 2023
---------------------- Page: 1 ----------------------
ISO 19337:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2023 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 19337:2023(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 Stability of working suspensions . 3
5.2.1 General . 3
5.2.2 Representative size change of secondary particles of nano-objects . 3
5.2.3 Concentration change of nano-objects . 3
5.3 Concentration of metal ions . 3
5.4 Concentration of culture medium components . 4
5.4.1 General . 4
5.4.2 Proteins . 4
5.4.3 Calcium. 4
5.5 Contamination . 4
6 Reporting . 5
6.1 General . 5
6.2 Name of nano-objects and manufacturing information . 5
6.3 Composition and metallic elements included in the nano-object sample . 5
6.4 Culture medium and serum . 5
6.5 Measurement results . 5
6.6 Optional methods . 6
Annex A (normative) Flow of measurements .7
Annex B (informative) Measurement and evaluation of stability . 8
Annex C (informative) Measurement of metal ions . 9
Annex D (informative) Measurement of culture medium components .11
Annex E (informative) Contamination .12
Bibliography .13
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ISO 19337:2023(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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use
of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed
patent rights in respect thereof. As of the date of publication of this document, ISO had not received
notice of (a) patent(s) which may be required to implement this document. However, implementers are
cautioned that this may not represent the latest information, which may be obtained from the patent
database available at www.iso.org/patents. ISO shall not be held responsible for identifying any or all
such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, 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
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
This second edition cancels and replaces the first edition (ISO/TS 19337:2016) which has been
technically revised.
The main changes are as follows:
— “the flow of measurements” has been improved as shown in Figure A.1;
— the status of Annex A has been changed from informative to normative;
— “5.2 Endotoxin” has been replaced by “5.5 Contamination”.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
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ISO 19337:2023(E)
Introduction
Before nano-objects enter onto the market, their possible impact on human health and the environment
should be carefully evaluated.
In vitro toxicity assays using cultured cells are frequently used as a tool in screening materials for
possible hazardous properties. The 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 of their unique behaviour in cell
culture medium. For example, immediately after the introduction of nano-object samples into the
culture medium, the nano-objects can undergo changes in
a) ionic dissolution,
b) corona formation, or
c) aggregation/agglomeration
leading to alteration in particles size and sedimentation. Therefore, it is critical to consider the above-
mentioned phenomena in clarifying if the observed effects are related to the tested nano-object itself
or from uncontrolled sources and to avoid false interpretation of assay results. For example, the corona
formation, metal ion release from the nano-objects and impurities (residual from the nano-object
[1]
synthesis process) can interfere with some in vitro assays , producing inaccurate results. Additionally,
the formation of agglomerates and aggregates can alter the toxicity of a suspension. It is important to
carefully assess and describe the characteristics of the suspension of nano-objects being tested.
Therefore, the rigorous characterization of the working suspension prior and during in vitro toxicity
assays on these characteristics is essential to exclude the in vitro experimental artefacts. In this
document, the essential characteristics related to these three phenomena and applicable measurement
methods were summarized. On the other hands, this document does not include a strategy to select the
appropriate techniques from the applicable measurement methods because the working suspensions
that contain nano-object samples for in vitro toxicity assays has the different materials components,
concentration and sizes; thus, the appropriate selection is depending on the investigators. While
the related informative annexes and the list of references in the Bibliography are included in this
document to assist with appropriate method selection by investigators to make allowance for the
characterization method selection, optional methods are also given in this document. In Clause 6, the
details of the characterization methods/procedures are described; therefore, the appropriateness of
the characterization can be judged.
The intention of this document is for reliable test results on nano-object toxicity to be shared and
communicated among stakeholders of nano-objects, such as regulators, general public, manufacturers
and end users.
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INTERNATIONAL STANDARD ISO 19337:2023(E)
Nanotechnologies — Characteristics of working
suspensions of nano-objects for in vitro assays to evaluate
inherent nano-object toxicity
1 Scope
This document describes the characteristics of working suspensions of nano-objects to be considered
when conducting in vitro assays to evaluate inherent nano-object toxicity. In addition, the document
identifies applicable measurement methods for these characteristics.
This document is applicable to nano-objects, and their aggregates and agglomerates greater than
100 nm.
This document intends to help clarify whether observed toxic effects come from tested nano-objects
themselves or from uncontrolled sources.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/TS 80004-2 and the following
apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
culture medium
aqueous solution of nutrients required for cell growth
3.2
secondary particle
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 (3.1) and nano-object sample
1
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ISO 19337:2023(E)
3.5
contamination
trace amounts of extrinsic substances present in the nano-object samples that affect cellular growth
4 Abbreviated terms
AAS atomic absorption spectrometry
BCA bicinchoninate acid
BSA Bovine serum albumin
C-U/F ultrafiltration assisted by centrifugation
DLS dynamic light scattering
AF4 asymmetrical-flow field-flow fractionation
ICP-AES inductively coupled plasma-atomic emission spectrometry
ICP-MS inductively coupled plasma mass spectrometry
LAL limulus amebocyte lysate
LD laser diffraction
LPS liposaccharides
MAT monocyte activation test
PCR polymerase chain reaction
ppt parts per trillion
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 identify certain
characteristics that can impact the biological system tested. Working suspensions for individual dose
shall be divided into two samples, one for toxicity assay and another for characteristics measurements.
Clause 5 specifies essential characteristics of the working suspension, listed below, and measurement
methods that are applicable to them:
— stability of working suspensions;
— concentration of metal ions;
— concentration of culture medium components;
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ISO 19337:2023(E)
— contamination.
Measurements of those characteristics shall be made for each dose of working suspensions. The flow of
measurements shall be in accordance with Annex A and Figure A.1.
5.2 Stability of working suspensions
5.2.1 General
Stability of working suspension is a key characteristic as it directly impacts the in vitro assay
[2],[3],[4]
conditions 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 can affect the stability of the suspended
nano-objects. The suspension stability shall be evaluated for the two characteristics, i.e. the relative
change of representative size of secondary particles of nano-objects and the relative change of the
concentration of nano-objects in the working suspension. The change in size of secondary particles of
nano-objects can result from agglomeration of smaller particles in culture media. The relative change
of nano-object concentration can result 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 an in vitro toxicity assay.
[5]
ISO/TR 13097 is recommended as comprehensive guidance for stability of working suspension.
5.2.2 Representative size change of secondary particles of nano-objects
An appropriate method shall be selected to directly measure the representative size change of
[3],[6] [7] [8]
secondary particles of nano-objects from among DLS , LD and SLS . Other methods not listed in
this document can be used and reported according to the optional methods in 6.6.
See Annex B for measurements.
5.2.3 Concentration change of nano-objects
An appropriate method shall be selected to measure the concentration change of nano-objects
[3],[6],[8] [9],[10],[11]
suspended in the biological media from among the static light scattering , ICP-MS , UV-
[12] [13]
Vis absorption, X-ray transmission and the total organic carbon analysis . Other methods not
listed in this document can be used and reported according to the optional methods in 6.6.
See Annex B for measurements.
5.3 Concentration of metal ions
Metal ions, produced as a result of nano-object test sample dissolution, can contribute to observed 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 U/F, C-U/F TFF or
centrifugation. 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 ICP-AES, ICP-MS, AAS and the colorimetric method. It shall be noted that many constituents in
culture media such as Na and Cl can interfere with metals analysis for some spectrometry techniques,
[14]-[16]
especially ICP-MS . Other methods not listed in this document can be used and reported according
to the optional methods in 6.6. Measurement results of concentrations shall be expressed in the unit
of molarity, mass/mass or mass/volume. The measurements can be omitted when a toxic effect is not
observed to the cells in the working suspensions. See Annex A for an example of flow of measurements.
See Annex C for measurements.
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ISO 19337:2023(E)
5.4 Concentration of culture medium components
5.4.1 General
A nano-object sample added to a culture medium to generate a working suspension can adsorb
[1]
components of the culture medium . This adsorption can result in starvation stress to the test cells.
The concentration of protein components and calcium, as surrogates for the nutritional components
in the solvent shall be measured after allowing enough time after the addition of nano-object sample
to the culture medium. If culture medium components other than protein and calcium that can
significantly affect the stability of the working suspension for in vitro toxicity assays are known, the
concentration of those components shall be measured as well. The measurements can be omitted when
a toxic effect is not observed to the cells in the working suspensions. See Annex A for an example of flow
of measurements.
Nano-object sample in culture medium shall be incubated with the same conditions of in vitro test.
Nano-objects can affect pH, osmolality and other essential characteristics in the culture medium.
5.4.2 Proteins
An appropriate method shall be chosen for the protein concentration measurement from among BCA,
[17],[18]
Bradford, Lowry, and ultraviolet, refractive index and SLS methods coupled with the AF4 . When
[19] [20] [21]
BCA , Bradford or Lowry method are chosen, the protein concentration in the solvent shall
be measured after separation of particulate matter from the working suspension. Results of protein
concentration measurement shall be expressed in the unit of mass/volume.
See Annex D for measurements.
5.4.3 Calcium
An appropriate me
...
DRAFT INTERNATIONAL STANDARD
ISO/DIS 19337
ISO/TC 229 Secretariat: BSI
Voting begins on: Voting terminates on:
2022-09-08 2022-12-01
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
ICS: 07.120
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
This document is circulated as received from the committee secretariat.
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 19337:2022(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022
---------------------- Page: 1 ----------------------
ISO/DIS 19337:2022(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 19337
ISO/TC 229 Secretariat: BSI
Voting begins on: Voting terminates on:
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
ICS: 07.120
COPYRIGHT PROTECTED DOCUMENT
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
© ISO 2022
THEREFORE SUBJECT TO CHANGE AND MAY
This document is circulated as received from the committee secretariat.
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
NOT BE REFERRED TO AS AN INTERNATIONAL
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on STANDARD UNTIL PUBLISHED AS SUCH.
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
IN ADDITION TO THEIR EVALUATION AS
or ISO’s member body in the country of the requester. BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
ISO copyright office
USER PURPOSES, DRAFT INTERNATIONAL
CP 401 • Ch. de Blandonnet 8
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
CH-1214 Vernier, Geneva
POTENTIAL TO BECOME STANDARDS TO
Phone: +41 22 749 01 11
WHICH REFERENCE MAY BE MADE IN
Reference number
Email: copyright@iso.org
NATIONAL REGULATIONS.
Website: www.iso.org ISO/DIS 19337:2022(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
Published in Switzerland
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
ii
© ISO 2022 – All rights reserved
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022
---------------------- Page: 2 ----------------------
ISO/DIS 19337:2022(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 Stability of working suspensions . 3
5.2.1 General . 3
5.2.2 Representative size change of secondary particles of nano-objects . 3
5.2.3 Concentration change of nano-objects . 3
5.3 Concentration of metal ions . 3
5.4 Concentration of culture medium components . 4
5.4.1 General . 4
5.4.2 Proteins . 4
5.4.3 Calcium. 4
5.5 Contamination . 4
6 Reporting . 5
6.1 General . 5
6.2 Name of nano-objects and manufacturing information . 5
6.3 Composition and metallic elements included in the nano-object sample . 5
6.4 Culture medium and serum . 5
6.5 Measurement results . 5
6.6 Optional methods . 6
Annex A (normative) Flow of measurements .7
Annex B (informative) Measurement and evaluation of stability . 9
Annex C (informative) Measurement of metal ions .10
Annex D (informative) Measurement of culture medium components .12
Annex E (informative) Contamination .13
Bibliography .14
iii
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---------------------- Page: 3 ----------------------
ISO/DIS 19337:2022(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 of the voluntary nature of standards, 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 www.iso.org/
iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
This second edition cancels and replaces the first edition (ISO/TS 19337:2016) which has been
technically revised.
The main changes are as follows:
— [to be completed]
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
© ISO 2022 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/DIS 19337:2022(E)
Introduction
Before nano-objects enter onto the market, their possible impact on human health and the environment
should be carefully evaluated.
In vitro toxicity assays using cultured cells are frequently used as a tool in screening materials for
possible hazardous properties. The 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 of their unique behaviour in cell
culture medium. For example, immediately after the introduction of nano-object samples into the
culture medium, the nano-objects can undergo changes in (1) ionic dissolution, (2) corona formation
or (3) aggregation/agglomeration leading to alteration in particles size and sedimentation. Therefore,
it is critical to consider the above mentioned phenomena in clarifying if the observed effects are related
to the tested nano-object itself or from 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.
ISO 19337 describes the essential characteristics and applicable measurement methods of working
suspensions that contain nano-object samples for in vitro toxicity assays. The 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 ISO 19337 does not
describe a procedure for validation of working suspension.
v
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DRAFT INTERNATIONAL STANDARD ISO/DIS 19337:2022(E)
Nanotechnologies — Characteristics of working
suspensions of nano-objects for in vitro assays to evaluate
inherent nano-object toxicity
1 Scope
This ISO 19337 describes characteristics of working suspensions of nano-objects to be considered
when conducting in vitro assays to evaluate inherent nano-object toxicity. In addition, the document
identifies applicable measurement methods for these characteristics.
This document is applicable to nano-objects, and their aggregates and agglomerates greater than 100
nm.
NOTE This ISO19337 intends to help clarify whether observed toxic effects come from tested nano-objects
themselves or from uncontrolled sources.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
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 terms and definitions contained in ISO/TS 80004-2 and the
following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
culture medium
aqueous solution of nutrients required for cell growth
3.2
secondary particle
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
1
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---------------------- Page: 6 ----------------------
ISO/DIS 19337:2022(E)
3.5
contamination
trace amounts of extrinsic substances present in the nano-object samples that affect cellular growth
4 Abbreviated terms
AAS atomic absorption spectrometry
BCA bicinchoninate acid
BSA Bovine serum albumin
C-U/F ultrafiltration assisted by centrifugation
DLS dynamic light scattering
AF4 asymmetrical-flow field-flow fractionation
ICP-AES inductively coupled plasma-atomic emission spectrometry
ICP-MS inductively coupled plasma mass spectrometry
LAL limulus amebocyte lysate
LD laser diffraction
MAT monocyte activation test
ppt parts per trillion
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 identify certain
characteristics that might impact the biological system tested. Working suspensions for individual dose
shall be divided into two samples, one for toxicity assay and another for characteristics measurements.
This clause specifies essential characteristics of the working suspension, listed below, and measurement
methods that are applicable to them.
— Stability of working suspensions
— Concentration of metal ions
— Concentration of culture medium components
— Contamination
Measurements of those characteristics shall be made for each dose of working suspensions. See Annex A
for the normative flow of measurements.
2
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ISO/DIS 19337:2022(E)
5.2 Stability of working suspensions
5.2.1 General
Stability of working suspension is a key characteristic as it directly impacts the in vitro assay
[2],[3],[4]
conditions 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 suspension stability shall be evaluated for the two characteristics, i.e. the relative
change of representative size of secondary particles of nano-objects and the relative change of the
concentration of nano-objects in the working suspension. The change in size of secondary particles of
nano-objects can result from agglomeration of smaller particles in culture media. The relative change
of nano-object concentration can result 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.
[5]
NOTE ISO/TR 13097 is recommended as a comprehensive guidance for stability of working suspension.
5.2.2 Representative size change of secondary particles of nano-objects
An appropriate method shall be selected to directly measure the representative size change of
[3],[6]
secondary particles of nano-objects from among dynamic light scattering (DLS), laser diffraction
[7] [8]
(LD) and static light scattering (SLS). Other methods not listed in this document can be used and
reported in accordance with 'Optional methods' in 6.6.
See Annex B (informative) for measurements.
5.2.3 Concentration change of nano-objects
An appropriate method shall be selected to measure the concentration change of nano-objects suspended
[3],[6],[8]
in the biological media from among the static light scattering, inductively coupled plasma mass
[9],[10],[11] [12]
spectrometry (ICP-MS), ultraviolet-visible (UV-Vis) absorption, X-ray transmission and the
[13]
total organic carbon analysis. Other methods not listed in this document can be used and reported
in accordance with 'Optional methods' in 6.6.
See Annex B (informative) for measurements.
5.3 Concentration of metal ions
Metal ions, produced as a result of nano-object test sample dissolution, can contribute to observed 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) tangential flow filtration (TFF) or centrifugation. 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) and the colorimetric method. It shall be noted that many constituents in culture media such as
[14-
Na and Cl can interfere with metals analysis for some spectrometry techniques, especially ICP-MS.
16]
Other methods not listed in this document can be used and reported in accordance with ‘Optional
methods' in 6.6. Measurement results of concentrations shall be expressed in the unit of molarity,
mass/mass or mass/volume. The measurements can be omitted when a toxic effect is not observed to
the cells in the working suspensions. See Annex A for an example of flow of measurements.
See Annex C (informative) for measurements.
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© ISO 2022 – All rights reserved
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ISO/DIS 19337:2022(E)
5.4 Concentration of culture medium components
5.4.1 General
A nano-object sample added to a culture medium to generate a working suspension may adsorb
[1]
components of the culture medium. This adsorption can result in starvation stress to the test cells.
The concentration of protein components and calcium, as surrogates for the nutritional components
in the solvent shall be measured after allowing enough time after the addition of nano-object sample
to the culture medium. If culture medium components other than protein and calcium that may
significantly affect the stability of working suspension for in vitro toxicity assays are known, the
concentration of those components shall be measured as well. The measurements can be omitted when
a toxic effect is not observed to the cells in the working suspensions. See Annex A for an example of flow
of measurements.
NOTE Nano-object sample in culture medium shall be incubated with the same c
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