ISO/TS 21633:2021

TECHNICAL ISO/TS
SPECIFICATION 21633
First edition
2021-08
Label-free impedance technology to
assess the toxicity of nanomaterials in
vitro
Technologie de l'impédance électrique sans marqueur pour évaluer la
toxicité des nanomatériaux in vitro
Reference number
©
ISO 2021
© ISO 2021
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 2021 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviations. 2
5 Background . 4
5.1 General . 4
5.2 Electrochemical impedance technique . 4
6 Basic principles, instruments . 6
6.1 Basics of electrochemical impedance technique . 6
6.2 Types of instrument . 6
6.2.1 Electrochemical impedance-based instruments for in vitro analysis of
toxicity on cell monolayers . 6
6.2.2 Impedance-based flow cytometry . 6
6.2.3 Electrochemical impedance-based spectroscopy . 7
6.2.4 Electrical impedance tomography . 7
7 Application for in vitro toxicity assessment . 7
7.1 General . 7
7.2 Normalized cell index.10
8 Technical limitations .11
Annex A (informative) Basic procedures using the xCELLigence system .12
Annex B (informative) Case studies using standard operating procedure for setting up an
xCELLigence experiment with various cellular models .17
Bibliography .21
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.
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 2021 – All rights reserved

TECHNICAL SPECIFICATION ISO/TS 21633:2021(E)
Label-free impedance technology to assess the toxicity of
nanomaterials in vitro
1 Scope
This document describes a methodology of a label free and real-time detection for non-invasive
monitoring of cell-based assays to assess toxicity of nanomaterials to eukaryotic and prokaryotic cells.
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 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 10993-1, Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk
management process
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from larger sizes are predominantly exhibited in this
length range.
3.2
nanomaterial
NM
material with any external dimension in the nanoscale (3.1), or having internal structure or surface
structure in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object (3.3) [and nanostructured material (3.4)].
3.3
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale (3.1)
3.4
nanostructured material
material having internal nanostructure or surface nanostructure
3.5
nanoparticle
NP
nano-object (3.3) with all external dimensions in the nanoscale (3.1) where the lengths of the longest
and the shortest axes of the nano-object do not differ significantly
[SOURCE: ISO/TS 80004-2:2015, 4.4, modified — Note 1 to entry has been deleted.]
3.6
test sample
material, device, device portion, component, extract or portion thereof that is subjected to biological or
chemical testing or evaluation
3.7
cell index
CI
dimensionless parameter obtained from the electrochemical impedance measurement
3.8
electrochemical impedance
effective resistance of an electric circuit or component to alternating current, arising from the combined
effects of ohmic resistance and reactance.
3.9
impedance-based flow cytometry
IFC
technique used to detect and measure physical and chemical characteristics of a population of cells or
particles
Note 1 to entry: A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer
instrument.
3.10
electrochemical impedance spectroscopy
EIS
method that measures the impedance of a system in dependence of the AC potentials frequency and
therefore that determines both the resistive and capacitive (dielectric) properties of materials
3.11
electrical impedance tomography
EIT
technique in which electrical measurements between many pairs of appropriately positioned surface
electrodes are used to produce images of underlying body structures
4 Abbreviations
AC Alternating current
AgNPs Silver nanoparticles
AuNPs Gold nanoparticles
BSA Bovine serum albumin
CB Carbon black
CI Cell index
CeO Cerium oxide
2 © ISO 2021 – All rights reserved

CuO Copper oxide
DIC Differential interference contrast
DMEM Dulbecco’s modified Eagle’s medium
DMSO Dimethylsulfoxide
ECIS Electric cell-substrate impedance sensing
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid
EIS Electrochemical impedance spectroscopy
EIT Electrical impedance tomography
EMEM Eagle's minimum essential medium
Fe O Ferric oxide
2 3
FBS Fetal bovine serum
HTS High throughput system
IC half-maximal inhibition concentration
IFC Impedance-based flow cytometry
IMEs Interdigitated microelectrodes
Mn O Manganese oxide
2 3
NCI Normalized CI
Ni Nickel
PBS Phosphate buffered saline
QD Quantum dot
RTCA Real-time cell analyzer
RPMI Roswell park memorial institute medium
SiO Silicon dioxide
SPR Surface plasmon resonance
TiO Titanium dioxide
ZrO Zirconium oxide
ZnO Zinc oxide
5 Background
5.1 General
Several in vitro assay systems that have been developed for the assessment of the toxicity of
different chemical compounds have also been implemented to assess the toxicity of NMs. Due to their
physicochemical properties, NMs may behave differently than the chemical compounds for which
these assay systems were developed and therefore, when they were used with NMs, discrepancies in
[1]
results among assays were often observed . As a result, investigators were prompted to consider the
interaction of NMs with the assay systems as a possible source for the observed discrepancies.
The detection systems of these toxicity assays are mostly optical in nature and rely on absorbance,
luminescence or fluorescence to quantify the products of the assay systems (e.g. tetrazolium salts).
NMs may therefore interfere directly with the assay readout by altering the absorbance, luminescence
[2]
or fluorescence of the products of these assay systems . Depending on their material, shape and size,
certain NMs may absorb, scatter and emit light at the assay detection wavelength. Carbon-based NPs, for
example, CB are known to absorb light in the visible spectrum whereas metal oxides, metal hydroxides,
[3]
and metal carbonate NPs are known to scatter light . AuNPs with a strong SPR absorb more light
[1]
than iron oxide NPs and larger NPs absorb more light than smaller NPs . Similar to AuNPs, AgNPs
[4]
also have strong plasmon resonances . Such absorptive abilities of these NPs may therefore interfere
with the absorptive properties of products obtained from different assay systems. NMs may also
interfere directly with the assay by interacting with the chemical reaction product. Due to their large
surface area per unit mass and surface reactivity, compared to large particles NMs may also display
an increased adsorption capacity thereby increasing the possible interaction between nanoparticles
[3][5]
and assay components . Finally, NMs may also catalyse the conversion of substrate to product. The
large surface area per unit mass and surface reactivity may lead to an excess in surface energy with
[6]
subsequent enhancement in the catalytic activity of NMs .
This document is therefore based on current information about electrochemical impedance technique
that does not rely on optical measurements to determine the degree of cell viability or cytotoxicity and
also provide ki
...