ISO/TR 21624:2020

Nanotechnologies - Considerations for in vitro studies of airborne nano‐objects and their aggregates and agglomerates (NOAA)

This document collates information regarding the systems available for exposure and assessment of nano-objects and their aggregates and agglomerates (NOAA) for in vitro air exposure studies. It provides an overview of the various exposure systems and in vitro cell systems used to perform in vitro studies that simulate an inhalation toxicology study design.

Nanotechnologies — Considérations pour les études in vitro des nano-objets en suspension dans l’air et de leurs agrégats et agglomérats (NOAA)

General Information

• Status: Published
• Publication Date: 27-Apr-2020
• 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: 28-Apr-2020
• Completion Date: 28-Apr-2020

Relations

• Consolidated By: ISO 8985:2022 - Plastics - Ethylene/vinyl acetate copolymer (EVAC) thermoplastics - Determination of vinyl acetate content
• Effective Date: 06-Jun-2022

Overview

ISO/TR 21624:2020 - Nanotechnologies: Considerations for in vitro studies of airborne nano‑objects and their aggregates and agglomerates (NOAA) collates current knowledge on systems and biological models used to assess inhalation-relevant exposures of airborne nano‑objects in vitro. The technical report focuses on exposure and assessment systems that simulate inhalation toxicology study design, comparing air‑liquid interface (ALI) and submerged approaches, and summarizing considerations for particle generation, characterization, dosing and choice of cell systems.

Key topics and technical considerations

• Modes of exposure
  • Direct exposure (aerosol delivered to cells under submerged, intermittently submerged or ALI conditions) and indirect exposure (collection of aerosols and subsequent suspension).
  • ALI systems are preferred for inhalation-relevant testing because they better reproduce airway cell phenotype and particle deposition.
• Characterization of NOAA
  • Importance of monitoring particle size, aggregation/agglomeration state, life‑cycle transformations and how aerosol generation can alter particle properties.
• Choice of cell systems
  • Guidance on mono‑culture, co‑culture and three‑dimensional models that reflect epithelial differentiation, mucus production and barrier functions.
• Dose selection and metrics
  • Considerations for relevant concentrations, deposited dose assessment and use of in silico tools for dosimetry (e.g., to relate administered dose to deposition).
• Practical limitations
  • Effects of suspending media on agglomeration, sedimentation and dissolution under submerged conditions; potential for misleading toxicity outcomes if in vitro conditions diverge from inhalation realities.
• Adverse Outcome Pathways (AOPs)
  • Annex A discusses applying AOPs to design mechanistic, in vitro‑based approaches.

Practical applications

ISO/TR 21624:2020 provides a structured, pragmatic reference for:

• Designing inhalation‑relevant in vitro studies of nanoparticles and NOAA
• Selecting exposure chambers and ALI platforms for nanosafety testing
• Choosing biologically relevant cell models (mono‑ and co‑cultures, 3D) and dose metrics
• Interpreting differences between submerged and ALI exposures for regulatory or risk assessment contexts

Who should use this standard

• Nanotoxicology researchers and academic laboratories
• Industrial R&D and nanosafety teams developing inhalation exposure testing
• Respiratory toxicologists and in vitro assay developers
• Regulatory scientists and risk assessors seeking harmonized approaches for in vitro inhalation studies
• Instrument and exposure system manufacturers

Related standards

• ISO/TR 19601 (overview of aerosol generation and in vivo testing) - referenced in the report
• ISO 15900 and ISO/TS 80004 series - referenced for terminology and definitions

This technical report is a guidance/overview document (not a prescriptive protocol) aimed at harmonizing approaches for in vitro assessment of airborne nano‑objects and improving the physiological relevance of inhalation toxicity testing. Keywords: ISO/TR 21624:2020, NOAA, nanotechnologies, in vitro inhalation, air‑liquid interface, aerosol exposure, nanoparticle dosimetry, nanosafety.

Standards Content (Sample)

TECHNICAL ISO/TR
REPORT 21624
First edition
2020-04
Nanotechnologies — Considerations
for in vitro studies of airborne nano‐
objects and their aggregates and
agglomerates (NOAA)
Nanotechnologies — Considérations pour les études in vitro
des nano-objets en suspension dans l’air et de leurs agrégats et
agglomérats (NOAA)
Reference number
©
ISO 2020
© ISO 2020
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
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below or ISO’s member body in the country of the requester.
ISO copyright office
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Published in Switzerland
ii © ISO 2020 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Considerations for in vitro systems for assessing inhalation exposure to NOAA .3
5.1 Background . 3
5.2 Modes of exposure . 4
5.2.1 General. 4
5.2.2 Considerations for ALI exposure systems . 5
5.3 Considerations for characterizing NOAA tested in vitro studies of airborne
nanomaterials . 9
5.4 Choice of cell systems .10
5.4.1 General.10
5.4.2 Mono-culture systems .10
5.4.3 Co-cultures/three-dimensional systems.12
6 Choice of appropriate dose and dose metrics .15
6.1 General .15
6.2 In silico methods to assess dose/dose metrics and deposition .16
7 Summary .16
Annex A (informative) Application of adverse outcome pathways (AOPs) to design in vitro‐
based approaches .17
Bibliography .18
Foreword
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described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
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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
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iv © ISO 2020 – All rights reserved

Introduction
Inhalation is one of the prominent routes by which humans can come in contact with natural, unintended
and engineered nano-objects and their aggregates and agglomerates (NOAA). Due to the physiological,
biochemical and anatomical differences between humans and animals, as well as the considerable time,
cost and animal numbers required to conduct in vivo toxicity tests, there is much interest in developing
in vitro strategies for risk assessment that are based on human cells and mechanisms of toxicity. To
enable comparability of the results of in vitro assay and in vivo effects observed after inhalation of
NOAA, certain parameters should be considered, including:
a) the choice of cell types;
b) characterization of the NOAA throughout the assay, including life-cycle transformations;
c) the choice of nano-object concentration relevant to human exposures;
d) generation of NOAA form that mimics human exposures;
e) the use of relevant dispersants;
f) the use of appropriate mode of exposure (submerged or air liquid interface) and exposure
[1]
duration .
This document includes descriptions of the aforementioned parameters with regard to using in vitro-
based strategies for assessing specific aspects related to the inhalation toxicity of NOAA. For example,
for inhalation studies, it is critical to choose the proper equipment for generation, exposure to, and
characterization of the nano-objects. This document includes information about available in vitro
aerosol exposure chambers and biological models that have been used to assess the inhalation toxicity
of NOAA. This document does not include details regarding the techniques for aerosol generation or
characterization of specific nanomaterials (NMs), their life cycle transformations or in vivo testing. An
[2]
overview of the aerosol generation of NMs and in vivo testing is given in ISO/TR 19601 .
TECHNICAL REPORT ISO/TR 21624:2020(E)
Nanotechnologies — Considerations for in vitro studies
of airborne nano‐objects and their aggregates and
agglomerates (NOAA)
1 Scope
This document collates information regarding the systems available for exposure and assessment
of nano-objects and their aggregates and agglomerates (NOAA) for in vitro air exposure studies. It
provides an overview of the various exposure systems and in vitro cell systems used to perform in
vitro studies that simulate an inhalation toxicology study design.
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:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
aerosol
system of solid or liquid particles suspended in gas
[SOURCE: ISO 15900:2009, 2.1]
3.2
agglomerate
collection of weakly bound particles or aggregates (3.3) or mixtures of the two where the resulting
external surface area is similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces, or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-4:2011, 2.8]
3.3
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area may
be significantly smaller than the sum of calculated surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example covalent bonds, or those
resulting from sintering or complex physical entanglement.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-4:2011, 2.7]
3.4
engineered nanomaterial
nanomaterial designed for specific purpose or function
[SOURCE: ISO/TS 80004-1:2015, 2.8]
3.5
incidental nanomaterial
nanomaterial generated as an unintentional by-product of a process
Note 1 to entry: The process includes manufacturing, bio-technological or other processes.
Note 2 to entry: See “ultrafine particle” in ISO/TR 27628:2007, 2.21.
[SOURCE: ISO/TS 80004-1:2015, 2.10]
3.6
manufactured nanomaterial
nanomaterial intentionally produced to have specific properties or composition
[SOURCE: ISO/TS 80004-1:2015, 2.9, modified — “specific” has replaced “selected”.]
3.7
nanoparticle
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 3 times), terms such as nanofibre or
nanoplate may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
3.8
particle size distribution
distribution of particles as a function of particle size
Note 1 to entry: Particle size distribution may be expressed as cumulative distribution or a distribution density.
[SOURCE: ISO/TS 80004-6:2013, 3.1.2, modified — “(distribution of the fraction of material in a size
class, divided by the width of that class)” has been deleted from the Note 1 to entry.]
4 Abbreviated terms
Ag NPs silver nanoparticles
ALI air-liquid interface
AOP adverse outcome pathway
Au NPs gold nanoparticles
CCSP clara cell secretary protein
CD cluster of differentiation
CFTR cystic fibrosis transmembrane conductance regulator
CNT carbon nanotube
2 © ISO 2020 – All rights reserved

CO carbon dioxide
ENM engineered nanomaterial
IATA integrated approach to testing an assessment
ICAM-1 intercellular adhesion molecule 1
IL interleukin
ISDD in vitro sedimentation, diffusion and dosimetry
ISD3 in vitro sedimentation, diffusion, dissolution and dosimetry
KE key event
MIE molecular initiating event
MPPD multiple-path particle dosimetry
MT metallothionein
MUC 1 mucin 1
MWCNT multi-walled carbon nanotubes
NM nanomaterial
NOAA nano-objects and their aggregates and agglomerates
OECD Organisation for Economic Co-operation and Development
QCM quartz crystal microbalance
ROS reactive oxygen species
SiO silicon dioxide
SP-A surfactant protein A
SP-D surfactant protein D
TEER transepithelial electrical resistance
TiO titanium dioxide
VCAM-1 vascular cell adhesion molecule 1
5 Considerations for in vitro systems for assessing inhalation exposure to NOAA
5.1 Background
Nano-objects can be incidental or are manufactured from a variety of materials (e.g. metals, polymers,
metal oxides) and come in many different morphologies and combinations. Testing the toxicity of
inhaled NOAA using in vitro systems involves considerations of several parameters, including the
appropriate mode of exposure (see 5.2), characterization of test material (see 5.3) and choice of cell
types (see 5.4).
5.2 Modes of exposure
5.2.1 General
Both direct and indirect methods have been used to assess the aerosolized nano-objects using in vitro
systems. Direct methods involve exposing the test aerosol, generated using aerosol generators, to the
cells directly under submerged, intermittently submerged (e.g. on a rocking platform) or air-liquid
[3][4][5]
interface (ALI) conditions . Figure 1 presents the diagrammatic representation of exposure
to NOAA under submerged and ALI conditions. Indirect methods often involve collection of aerosols
(e.g. road-side ambient particles or nano-objects) using special apparatus (e.g. wetted rotating vane
impactors or liquid impingers) or on a filter-like substrate followed by suspension of the collected
[3][6][7][8][9]
sample in a culture medium before exposing the cells .
a)  Submerged culture b)  Air‐liquid interface culture
Figure 1 — Diagrammatic representation of submerged and air‐liquid interface (ALI) cultures
The indirect method allows for the identification of the potential toxicity of air borne particles. However,
the steps needed to prepare the suspensions of collected test material (when using indirect methods)
and the interaction of the test material with the medium in submerged systems (when using direct
methods) can cause changes to the properties of the test material leading to false assessments. This is
especially applicable to nano-objects due to the fact that both their agglomeration state and movements
are impacted by the use of a suspending medium, with their movement being controlled by Brownian
[10][11]
diffusion or sedimentation based on their physico-chemical parameters such as size and density .
As a result, the probability of agglomerates settling down onto the cell culture is higher than for single
particles. Additionally, dissolution kinetics of nano-objects under submerged conditions might differ
considerably from what the particles undergo in in vivo conditions. Since the size, form, and solubility
are the main parameters that determine nano-object toxicity, the toxicity outcomes observed in the
submerged systems might not capture what happens in the lung after nano-object exposure. Another
limitation of submerged systems is that the phenotype of the cells is usually different than that found
under in vivo conditions as seen by the lack of formation of tight junctions and mucus in epithelial cells,
[12][13]
an issue which is particularly important for inhalation models .
To overcome the limitations associated with the submerged systems, ALI systems are used and are
[14][15][16][18]
preferred for inhalation toxicity testing because of their physiological relevance . An ALI
system constitutes cells cultured on a semi-permeable membrane where their basal surface is exposed
to the culture medium and the apical surface is exposed to air. Such configuration is considered to be
physiologically relevant as it has been shown to drive differentiation of the cells that mimics their in
[12][13][19]
vivo phenotype . In addition to having the cells in an “in vivo-like” configuration/phenotype,
ALI systems allow a relatively direct deposition of NOAA on the cells that more closely mimics the
particle deposition that results following inhalation, which might be used to derive a concentration-
[20]
response relationship . It should be noted that, similar to liquid conditions, the generation of an
4 © ISO 2020 – All rights reserved

aerosol can also have an effect on particle characteristics. Thus, during and after aerosol generation,
proper characterization of the particles should be performed.
Figure 1 shows exposure of NOAA to cells under submerged conditions and at the ALI. Under submerged
conditions, the NOAA have to span the depth of the medium to reach the cells. At ALI conditions, the
NOAA settle directly on the cells due to minimal fluid layer over the cells. Several studies have compared
[15]
submerged and ALI systems exposed to NOAA and have reported differences in cellular responses .
For example, a higher expression of interleukin 6 (IL-6), IL-8, and hemeoxygenase-1 was observed
[20][21][22][23]
in cells exposed to NOAA at ALI as compared to submerged cultures . A few studies have
[24][25][26]
observed contradicting outcomes for the aforementioned biomarkers . Despite the differences
between the two modes of exposure, both the systems are still used to assess the toxicity of NOAA.
Submerged cell cultures can be used for identifying the toxic potency of air borne (e.g. environmental
or exhaust) particles. However, for evaluation of engineered and manufactured nano-objects, especially
when considering occupational exposure, the ALI culture system more appropriately represents factors
dealing with lung exposure.
5.2.2 Considerations for ALI exposure systems
5.2.2.1 General
Treating the cell systems at ALI requires exposure systems that can deliver the material to be tested
as an aerosol to the cell system in a form that is relevant to human exposure scenarios. The exposure
systems used to test NOAA could either be “closed-box” or “flow-through” type and typically involve
two basic components: an aerosol generator that generates the test atmosphere and the exposure
chamber that houses the cell system. Flow-through systems (as shown in Figure 2) also typically consist
of connectors and peripherals that transport, dilute, characterize and condition the aerosol before
delivering it to the cells inside the chamber, and an exhaust, which could be used for test atmosphere
sampling.
Figure 2 depicts a basic diagrammatic configuration of a system including an aerosol generator and
an exposure chamber for exposing cells to aerosolized (or nebulized) substances at the ALI. The
materials are aerosolized (or nebulized) using an aerosol generator (or nebulizer), which is connected
to the exposure chamber. Deposited concentration of NOAA can be determined by incorporating quartz
crystal microbalance (QCM) and/or electron microscopy (EM) grids in the wells without cells (A). The
cells are cultured on membrane inserts and exposed to dry aerosols or nebulized material at the ALI
(B). Cells treated with air only can be used as a negative control (C). Sampling ports can be included
at several check points to obtain aerosol or medium samples. The various components of the exposure
system and their applicability to the assessment of NOAA are described in 5.2.2.2 to 5.2.2.3.
Key
a NM sample g cell culture insert
b aerosol generator or a nebulizer h outlet for sampling medium
c aerosol i well with a QCM or an EM grid
d exhaust j well with cells exposed to NMs at ALI
e air only k well with cells exposed to clean air at ALI
f exposure chamber
NOTE Adapted from Reference [5].
Figure 2 — Example of a set‐up of an in vitro air‐liquid interface exposure system
5.2.2.2 Choice of appropriate aerosol generator
Aerosol generators, in combination with exposure chambers, enable a direct gas-phase exposure of
test systems to aerosols. They generate an aerosol atmosphere that can be used to expose the ALI cell
systems to aerosolized or nebulized particles. Several methods have been used to generate aerosols
but the choice of a particular method depends on the physical characteristics of the material to be
[5]
aerosolized, such as density, viscosity, state of matter and target aerosol size . For example, for liquid or
biological materials, collision nebulizers, jet nebulizers, ultra-sonic atomization or vibrating membrane
generators are often used; whereas, the generation of aerosolized solid materials involves using spray-
[15][22][27]
drying, rotating scrapers, venturi-style powder dispersions or fluidized powder bed methods
[28] [2]
. Detailed information about aerosol generators is given in ISO/TR 19601 .
An aerosol generator is generally connected to the exposure chamber via auxiliary connectors that
provide a means to deliver the generated test material atmosphere to the chamber where the cell
systems are housed.
6 © ISO 2020 – All rights reserved

5.2.2.3 Choice of exposure chambers
The choice of a specific exposure chamber depends on the basic study design and purpose of testing,
including the number of test and control conditions, and sampling (test material, biomarkers, etc.)
requirements. Exposure chambers can be used to expose cells cultured at the ALI to aerosolized NOAA
(and other materials). Several exposure chambers have been described in the literature and include
[29][30][31][32][33]
both commercially available systems and those developed by independent researchers
[34][35][36][37][38][39]
. These chambers vary from one another in factors such as cost, compatibility with
aerosol generators, ease of availability and use, modularity, level of throughput, ability to consistently
[5][29]
deliver aerosols at multiple dilutions, ease of cleaning and exposure duration . However, all
available exposure systems have some general similarities, including a compartment that houses the
cells and equipment to facilitate generation, delivery and/or characterization of aerosol atmosphere.
The configuration and the choice of the exposure system depends on the experimental objectives and
requirements. Of the available systems that have been used to test NOAA, some are more user-friendly
as compared to others that require an in depth understanding of the engineering controls included in
the system.
The different features of the various exposure systems are as follows.
a) Type of exposure system: Exposure chambers can be “closed-box” or “flow-through” systems based
on how the test aerosol is fed into the system and delivered to the cells inside. One example of a
closed-box system is an exposure chamber in which the test material is nebulized inside, where it
forms a dense cloud, and which uses cloud dynamics to uniformly mix and deposit (sedimentation)
the aerosol onto the membrane with the cells. There is no air flow into or out of the device in
[40]
the closed-box type system . On the other hand, in flow-through systems, the test material is
aerosolized and fed into the exposure chamber with air flow. While the aerosol cloud settles onto
the cells, the droplet-depleted air leaves the chamber through the exhaust located on the other end
[41]
of the chamber .
b) Flow alignment: Depending on the type of exposure chamber, the alignment of airflow can either
be horizontal or vertical. In a horizontal or incubator/box-type setup [see Figure 3 a)], the test
atmosphere enters the exposure chamber through an inlet and exits through an outlet leading to
[18]
a horizontal exposure flow above the inserts of the cultures . On the other hand, in a stagnation
point flow set-up [see (Figure 3 b)], the flow is vertical and directed towards the cell cultures,
providing a more precise way of exposing the cells.
a)  Incubator/box‐type setup b) Stagnation point flow set-up
NOTE Adapted from Reference [18].
Figure 3 — Example of set‐ups of an in vitro air‐liquid interface exposure system
c) Enhancement of deposition efficiency:
1) Electrostatic deposition [see Figure 4 a)]: Electrostatic deposition enhancement is a technique
that uses charge to increase the efficiency of NOAA deposition. Although the physiological
relevance of using electrostatic deposition is debatable due to the possible adverse effects
of applying high electrical fields to the cells, this technique has been used to enhance the
[5][18][42]
deposition of NOAA .
2) Droplet deposition [see Figure 4 b)]: Droplet deposition involves suspending or dissolving
the test material in a liquid medium and then generating the droplet aerosol (generally using
a nebulizer) to expose the cells. Although this technique provides a way to attain a high
deposition of test material, it cannot be used to test materials in their dry form (or when their
[18][42]
dry physico-chemical properties need to be maintained) .
3) Thermophoresis [see Figure 4 c)]: Thermophoresis or thermal precipitation involves deposition
of the particles onto the cells along a temperature gradient. The temperature gradient reaches
the range (35 °C to 37 °C) used in normal cell culturing near the cell surface. Thus, there are
no obvious concerns related to the adverse effects on cells and no pre-treatment of the test
[18][42]
material is needed so it retains its physico-chemical characteristics .
a)  Electrostatic deposition b)  Droplet deposition c)  Thermophoresis

a
Electrostatic forces.
b
Gravitational forces.
c
Thermal gradient.
NOTE Adapted from Reference [18].
Figure 4 — Techniques to enhance NOAA deposition at ALI
d) Number and alignment of inserts: The number of test conditions, replicates and appropriate
controls depend on the purpose of testing (e.g. hazard assessment or identification), but, in general,
exposure systems should include clean air control and a reference membrane to determine the
deposited dose (e.g. an insert without cells or a quartz microbalance). The ability to test multiple
concentrations and durations of exposure is equally important to establish the dose- and/or time-
response relationship.
e) Control and characterization of test atmosphere: Depending on the exposure chamber, parameters
such as flow rate, temperature and humidity should be monitored. It is also important to be able to
monitor and characterize aerosol atmosphere and maintain sterile conditions inside the exposure
chamber.
f) Characterization of deposited dose and culture medium:
1) Deposited dose: Determination of the deposited dose is important to interpret the observed
biological response or in other terms to establish a dose-response relationship. Deposition of
NOAA can be generally determined as a function of mass [quartz crystal microbalance (QCM)],
surface area or particle count (electron microscopy grid). A reference membrane insert usually
houses the QCM or the EM grid. Alternately, the material deposited directly on the membrane
can be recovered and analysed using chemical or florescence based techniques. Deposited
dose can also be predicted using in silico models, but they are usually based on a number of
assumptions related to NOAA properties.
2) Sample cell culture medium to measure translocation or release of biomarkers and metabolites
by cells: To establish a dose- and time-dependent biological response, it is important to assess
8 © ISO 2020 – All rights reserved

the relevant biomarkers at different time-points throughout the experiment duration. Several
systems are available that vary in the ease with which the samples can be collected from the
cell systems.
g) Compatibility with different aerosol generators: Modular systems that are compatible with a
variety of aerosol generators provide flexibility in testing different types of materials, obtaining a
specific size and concentration range of particles, and choosing the duration of exposures.
5.3 Considerations for characterizing NOAA tested in vitro studies of airborne
nanomaterials
Thorough characterization of NOAA at various stages of in vitro testing using (e.g. as manufactured,
as aerosolized, as deposited) is critical to determine relationships between the observed biological
outcomes and concentration and properties. Some of the NOAA characteristics that are critical in
this regard include particle size, size distribution, shape, aggregation/agglomeration, solubility
(dissolution), presence of trace impurities (e.g. metals and endotoxins), surface characteristics
(e.g. area and charge), crystalline structure, dustiness, composition and purity. Determination of
physical and chemical properties of NOAA using standardized protocols is important as it decreases
the interlaboratory variability and facilitates comparison of data. Several guidance documents and
standards are available related to characterization, see References [175] and [176]. Those specifically
[2]
related to aerosolized NOAA are given in ISO/TR 19601 . Suspending the nano-objects in simulated
lung fluids can provide an insight into their dissolution rate (biodurability or biopersistence) potential,
agglomeration and aggregation, and kinetics in biological fluids. Several simulated lung fluids have been
[43][44][45]
used to assess the aforementioned NOAA parameters . Information about acellular systems is
[17]
given in ISO/TR 19057 . If the nano-object is suspended in a medium (e.g. in case of nebulization),
thorough characterization of its suspension should be conducted. Detailed information related to the
[46]
characterization of nano-object suspensions is given in ISO/TS 19337 .
In addition to assessing the physical and chemical properties of nano-objects, it is important to
determine the concentration and form of the fraction deposited on the cell surface. Unlike chemicals
that form a homogeneous solution in the medium, nano-objects form a heterogeneous dispersion with
aggregates of different sizes. The deposition of NOAA in the medium is largely dependent on size and
density: with large particles settling by inertia but, as particle size is reduced (to about 0,3 micron), the
deposition is reduced to a minimum. As the particle size is reduced further, the deposition increases
[47]
due to diffusional forces . With limited tools to accurately predict the concentrations of particles
that reach the cell layer, the behaviour of nano-objects in dispersion complicates the dose-response
determination, which is critical for toxicological assessments.
Exposure of cell systems at the ALI bypasses the effects of medium on nano-objects to some extent by
allowing a fairly direct deposition of aerosols onto the cell surface. Although, also within the aerosol,
changes in the nano-object characteristics can occur, for example due to evaporation of the (watery)
vehicle, or by aggregation of the nano-objects within the droplets of the aerosol. The human respiratory
system has a characteristic particle size dependent deposition (or collection) curve. The deposition of
aerosol particles onto the surface of the lungs and penetration though the liquid surface that lines the
lungs depends on several factors, including particle diameter, air flow, surface tension and the presence
of electrostatic, thermal or molecular gradients. Similarly, the size distribution delivered and deposited
in in vitro systems at the ALI will depend on the choice of the exposure equipment (aerosol generator
and exposure chamber), with some systems capable of continuous exposures over a period of time and
others capable of single exposure at a fixed concentration. Characterization of the deposited fraction
can provide information regarding the form and mass of NOAA that the cells are actually exposed to.
Both qualitative (e.g. electron microscopy) and quantitative methods (e.g. quartz crystal microbalance
and gravimetrically determined deposited mass measures) are available to assess the deposited dose.
While choosing the exposure system, factors such as whether the device should simulate the lung
deposition curve as a function of particle diameter or simply have repeatable high deposition efficiencies
as a function of particle diameter will also need to be considered and checked for each NOAA type and
form. Additional features that determine the choice of exposure chambers are given in 5.2.2.3.
5.4 Choice of cell systems
5.4.1 General
The respiratory system can be divided into three main regions: nasopharyngeal, tracheobronchial
and alveolar. These regions are composed of more than 40 types of specialized cells, such as epithelial
cells that form the lining, macrophages that engulf foreign materials, and alveolar cells that secrete
lung surfactant and are involved in gas exchange. Mucus-producing goblet cells and ciliated epithelial
cells form the muco-ciliary system that actively removes foreign materials and microbes from the
respiratory tract. One in vitro model containing all lung cell types is currently not technically feasible;
however, models containing key cell types can be used to predict the effects of inhaled substances on the
lungs. The choice of cell types also depends on the region of the respiratory tract that the test substance
is predicted or known to localize in and on the relevant biological outcome. Several cell-based systems
that have been used to assess the effects of nano-objects are described in 5.4.2 to 5.4.3.
5.4.2 Mono‐culture systems
Mono-culture systems of human-derived cells and cell lines have been extensively used to assess NOAA
[48][49][50][51][52][53]
toxicity under submerged conditions and at ALI . Mono-culture models provide a good
indication of potential overt toxicity of NOAA, and are often used as a first tier for prioritizing the need
for further testing in more complex in vitro systems. Since every cell type is functionally specialized,
the choice of the cell type depends on the expected in vivo target location (within the respiratory tract
or systemically). Table 1 shows some examples of human-based cell types that have been used to test
[54][55][56]
NOAA and a brief description for each . These lung cell types are commercially available and
primary cells can be obtained from the hospitals.
Table 1 — Examples of a few human‐based cell types that have been used to test NOAA
Cell type Type Source Features Endpoints assessed References
Human Primary Tracheal and Upon differentiation they — Apoptosis [54], [57],
bronchial carinal show a pseudostratified [58], [59], [60],
— Oxidative stress
epithelial biopsies epithelial phenotype, [61], [62]
(HBE) (above composed of ciliated,
— DNA fragmentation
cells bifurcation non-ciliated and basal cells.
of the lung
— Genotoxicity
— Inflammatory
response
— Barrier integrity
— Cytotoxicity
16HBE140- Cell line Bronchial 16HBE140- cells have a — DNA fragmentation [57], [63],
epithelium wild-type chloride (Cl) [64], [65]
— Genotoxicity
ion-transport phenotype,
form polar monolayers,
— Inflammatory
have well defined tight
response
junctional complexes,
generate high transepithe-
— Barrier integrity
lial electrical resistance
(TEER), and limit movement
of paracellular markers
and macromolecules. They
express high levels of cystic
fibrosis transmembrane
conductance regulator
(CFTR) mRNA and protein.
10 © ISO 2020 – All rights reserved

Table 1 (continued)
Cell type Type Source Features Endpoints assessed References
Small Primary 1 mm They express intercellular — Cell viability [66], [67], [68],
airway bronchiole adhesion molecule 1 (ICAM- [69], [70], [71]
— Cell proliferation
epithelial area 1), vascular cell adhesion
Cell cycle
cells molecule 1 (VCAM-1),
(SAEC) surfactant protein A (SP-A),
— Reactive oxygen
SP-D, and aquaporin 3.
species (ROS)
— Lipid peroxidation
DNA (e.g. DNA
damage and
methylation)
— Metallothionein
(MT)
Human Cell line Bronchus They express — Cytotoxicity [72], [73],
bronchial vimentin, collagen I, [74], [75], [76],
— Pro-inflammatory
epithelial E-cadherin, ICAM-1, [77], [78],
response
cell line VCAM-1, and clara cell sec- [79], [80],
(BEAS-2B) retary protein (CCSP). This [81], [82], [83],
— Genotoxicity
cell line does not polarize or [84], [85],
form tight junctions. [86], [87],
— Apoptosis
[88], [89]
— Oxidative stress
— Membrane integrity
— Cellular
transformation
— Epithelial-
mesenchymal
transition
Calu-3 Cell line Airway This cell line is used as a — Barrier properties [90], [91], [92],
epithelium model of the airway submu- Injury [93], [94], [95],
cosal glad acinar serous cell, [96], [97]
— Diseases of the
due to the relatively high
bronchial
levels of expression of cystic
epithelium
fibrosis transmembrane
conductance regulator
(CFTR). They form ciliated
and secretory cell popula-
tions, tight monolayers at
ALI, and also express pro
SP-C and mucin.
A549 Cell line Lung This cell line is used to — Oxidative stress [54], [62], [98],
epithelium represent type II pulmonary [99], [100],
— Cytotoxicity
epithelial cells because it [101], [102],
contains lamellar bodies and [103], [104],
— Genotoxicity
microvilli, and has the abili- [105], [106],
ty to express CFTR, SP-A, C, [107], [107],
— Apoptosis
D, and mucin 1 (MUC1). [108], [109],
[110], [111],
— Translocation
[112], [113]
Table 1 (continued)
Cell type Type Source Features Endpoints assessed References
NCI-H292 Cell line Lung These cells express caten- — Cytotoxicity [54], [101],
epithelium ins, ICAM-1, cluster of dif- [114]
— Inflammation
ferentiation (CD) 58, CD44,
and low levels of CFTR, but
— Oxidative stress
do not express selectins
and VCAM-1. They develop
TEER when cultured on
permeable membranes.
NCI-H292 cells have been
used to study paracellular
migration.
Human Cell line Lung This is an immortalized — Cytotoxicity [115], [116]
alveolar epithelium human alveolar type I-like
— Barrier integrity
epithelial cell line. hAELVi cells form
lentivirus tight intercellular junctions.
— Translocation
immortal-
ized cell line
(hAELVi)
THP-1 Cell line Blood These cells were derived — Cytotoxicity [117]
from the peripheral blood
— Inflammation
of a human male with acute
monocytic leukaemia.
— PPAR-γ activation
THP-1 cells have Fc and C3b
receptors and lack surface
and cytoplasmic
immunoglobulins.
5.4.3 Co‐cultures/three‐dimensional systems
Although mono-culture systems have been extensively used to assess the non-specific toxicity of
NOAAs, co-culture systems, with multiple cell types, can more closely mimic an in vivo-like situation
and can be used to study the intercellular interplay between multiple cell types. The cell types listed
in Table 1 have been used in combination with other cell types to assess the impact of NOAA on the
respiratory tract. The choice of cells to include in a co-culture system depends on the purpose of the
[55]
study . For example, a co-culture of endothelial and epithelial cells can be used to simulate the lung-
blood barrier while macrophages and/or dendritic cells can be cultured with other lung cells to study
the role of immune cells. Of note here is that the choice of the aerosol generator and exposure chamber
is only relevant to systems where cells are exposed to test substance at ALI.
Table 2 lists examples of co-culture systems (submerged and at ALI) that have been used in NOAA
toxicity studies along with the endpoints assessed in those studies.
Table 2 — Examples of a few human‐based co‐culture systems that have been used to test NOAA
Co‐culture system Endpoints tested Test material References
Co-culture of SAECs and — Cytotoxicity — Multi-walled carbon nanotubes [118], [119],
human microvascular (MWCNTs) [120]
— Inflammation
endothelial cells
— Printer-emitted nanoparticles
— Oxidative stress
— Uptake
— Morphology
— Angiogenesis
12 © ISO 2020 – All rights reserved

Table 2 (continued)
Co‐culture system Endpoints tested Test material References
Co-culture of hAELVi and — Barrier integrity — Silver nanoparticles (Ag NPs) [115]
THP-1
— Cytotoxicity
— Translocation
Calu-3, macrophages (THP-1), — Translocation — Polystyrene nanobeads [94]
and endothelial cells
(HPMEC-ST1.6R cell line)
Triple co-culture of A549 — Cytotoxicity — Ag NPs [118], [121],
cells, human peripheral blood [122], [123],
— Pro- and anti- — Gold (Au) NPs
monocyte derived dendritic [124], [125],
Inflammatory
cells (MDDC), and monocyte [126]
— MWCNTs
response
derived macrophages (MDM)
— Oxidative stress
Triple co-culture of — Cytotoxicity — Paint NPs [127]
16HBE14o-, THP-1, and
— Inflammation — Titanium dioxide (TiO ) NPs
human lung microvascular 2
endothelial cells (HLMVEC)
— Barrier integrity — Ag NPs
— Silicon dioxide (SiO ) NPs
Triple co-culture of — Cytotoxicity — MWCNTs [128]
6HBE14o-, MDDC
— Pro-inflammatory — Single-walled CNTs (SWCNTs)
and MDMs cultured at ALI
response
— Oxidative stress
Triple co-culture of A549 — Cytotoxicity: — SiO [129], [130]
epithelial cells, THP-1, and
— Proliferation — MWCNTs
endothelial cells (EA.hy 926
or HUVEC)
— Inflammation
— Barrier integrity
— Oxidative stress
Triple co-culture of NCI-H441 — Cytotoxicity — SiO NPs [131]
alveolar epithelium cell line,
— Barrier integrity
ISO-HAS1 human microvas-
cular endothelium cell line,
— Pro-inflammation
and THP-1 cells
— Oxidative stress
— Uptake
Tetra-culture of A549 cell — Oxidative stress — SiO -Rhodamine NPs [26]
line, THP-1, mast cells
— Barrier integrity
(HMC-1), and endothelial
cel
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