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

General Information

Status

Published

Publication Date

11-May-2023

ICS

07.120 - Nanotechnologies

Technical Committee

ISO/TC 229 - Nanotechnologies

Drafting Committee

ISO/TC 229/WG 3 - Health, Safety and Environmental Aspects of Nanotechnologies

Current Stage

6060 - International Standard published

Start Date

12-May-2023

Due Date

21-Jan-2023

Completion Date

12-May-2023

Ref Project

Relations

Revises

ISO/TS 19337:2016 - Nanotechnologies — Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicity

Effective Date

23-Apr-2020

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ISO 19337:2023 - Nanotechnologies — Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicity
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ISO/PRF 19337 - Nanotechnologies — Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicity
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ISO/PRF 19337 - Nanotechnologies — Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicity Released:7/14/2022

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Standards Content (Sample)

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

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
© 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

iii
© ISO 2023 – All rights reserved
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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.
© ISO 2023 – All rights reserved
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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.
© ISO 2023 – All rights reserved
---------------------- Page: 5 ----------------------
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

© ISO 2023 – All rights reserved
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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;
© ISO 2023 – All rights reserved
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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.
© ISO 2023 – All rights reserved
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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
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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
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Contents Page