ISO/TR 23463:2022

TECHNICAL ISO/TR
REPORT 23463
First edition
2022-05
Nanotechnologies — Characterization
of carbon nanotube and carbon
nanofibre aerosols to be used in
inhalation toxicity tests
Nanotechnologies — Caractérisation des aérosols de nanotubes
de carbone et de nanofibres de carbone à utiliser dans les tests de
toxicité par inhalation
Reference number
© ISO 2022
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 8
5 Considerations in CNT and CNF inhalation studies . 8
5.1 General . 8
5.2 Workplace exposure scenario . 8
5.3 Existing inhalation toxicity testing guidelines . 9
6 Physicochemical parameters related to the toxicity of CNTs and CNFs .9
6.1 General . 9
6.2 Aerodynamic properties of aerosols for deposition of fibres . 9
6.3 Size and shape (including length, width, aspect ratio, state of aggregation/
agglomeration, and rigidity) . 10
6.4 Specific surface area . 11
6.5 Crystalline structure and defects. 11
6.6 Surface chemistry, functionalization, surface charge, impurities, and radical
generation/scavenging potential . 11
6.7 Biodurability. 12
7 Issues for the characterization of CNT and CNF aerosols .12
7.1 General .12
7.2 Characterization of physicochemical properties of CNT and CNF prior to aerosol
generation . 13
7.2.1 General .13
7.2.2 Size and size distribution . 13
7.2.3 Shape (rigidity and agglomeration/aggregation) . 14
7.2.4 Surface area . 14
7.2.5 Crystalline structures . 14
7.2.6 Surface chemistry, functionalization, surface charge, and radical
generation/scavenging potential . 14
7.2.7 Composition, purity, and impurities . 14
7.2.8 Biodurability (in vivo and in vitro tests) . 15
7.3 CNT and CNF aerosol characterization (sampling and measurement) .15
7.3.1 General .15
7.3.2 Size and size distribution of CNT and CNF aerosols . 16
7.3.3 The shape of CNT and CNF aerosols . 18
7.3.4 Crystalline structure and defects . 18
7.3.5 Surface chemistry. 18
7.3.6 Composition analysis . 19
7.3.7 Fibre density . 19
7.3.8 Concentration . 19
7.4 Direct and indirect measurement . 20
7.4.1 Direct measurement . 20
7.4.2 Indirect measurement . 21
Annex A (informative) Physicochemical properties of CNT associated with biological
activity .22
Annex B (informative) CNT and CNF aerosol monitoring instruments .23
Bibliography .26
iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
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different types of ISO documents should be noted. This document was drafted in accordance with the
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Attention is drawn to the possibility that some of the elements of this document may be the subject of
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This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
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iv
Introduction
Inhalation is the primary route of exposure to aerosolised carbon nanotubes (CNTs) and carbon
nanofibres (CNFs). Exposure to CNTs or CNFs can occur in consumer settings as well as in occupational
settings. Occupational exposure to CNTs or CNFs can occur at all phases of the manufacturing, handling,
[1,2]
and formulation of the material into final products . Consumers are potentially exposed to CNTs
or CNFs released as products of degradation, weathering, or mechanical processes (e.g. grinding or
[3,4]
polishing) from consumer products that contain CNT or CNF embedded into a matrix .
Similar to other nanomaterials, the physicochemical properties of CNTs or CNFs are greatly diverse
in terms of diameter, length, shape, arrangement of carbon atoms, surface chemistry, defects, and
impurities. Their different physicochemical characteristics are responsible for different functional
properties such as mechanical, electrical, optical, and thermal properties. Many previous inhalation
toxicity studies of CNT and CNF aerosols reported various hazards from acute inflammation to
carcinogenicity and the toxicological responses to CNT and CNF aerosols vary depending on their
[5]
physicochemical characteristics .
Among the various physicochemical characteristics, morphological factors such as length and rigidity
[6,7]
have been suggested as key parameters related to the toxicity of CNT and CNF aerosols . CNT and CNF
[8]
aerosols can consist of individual primary fibres in the nanoscale and aggregated or agglomerated
[9]
structures, including those with diameters larger than 100 nm . Among various types of CNT and CNF,
the asbestos-like pathogenicity has been observed only in long (>5 μm) and rigid fibres, but not in short
[6]
or tangled CNT . Thus, a better understanding of the characteristics of generated CNT or CNF aerosols
in relation to toxicity end points is key for risk assessment and safer-by-design approaches.
The framework for material characterization for inhalation studies consists of (1) characterization
of as-produced (pristine) or supplied material, (2) characterization of administered material, (3)
[10]
characterization of material following administration, and (4) human exposure characterization .
This document focuses on the first two characterization needs, which include physicochemical
properties (e.g. size, size distribution, aggregation/agglomeration, and shape) and measurement of
concentration (e.g. mass, number, surface area, and volume). These parameters can be measured by
direct (online) or indirect (off-line) methods and each technique needs specific sampling procedures.
However, the limited technologies in the generation and characterization of nanofibres make it difficult
to perform inhalation toxicity studies, although the inhalation exposure to CNT and CNF is highly likely
[9,11] [8]
in the workplace , and research facilities , where they are in use. In this regard, this document
provides the current status of CNT and CNF aerosol characterization used in the inhalation toxicity
tests as well as the physicochemical properties of CNTs and CNFs and their relationship with toxicity
end points.
This document complements the work of other international organizations including the Organization
for Economic Co-operation and Development (OECD) which has published guidelines and guidance
[12,13]
on the performance of inhalation toxicity studies . ISO 10808 describes the characterization of
nanoparticles in inhalation exposure chambers for inhalation toxicity testing. This document is different
from ISO 10808 and focuses on different types of nanomaterials (nanotubes and nanofibres opposed
to nanoparticles) because many characterization methods and important physicochemical parameters
related to the toxicity of CNT and CNF are different from those of nanoparticles. Recommendations and
guidelines to assist investigators in making appropriate choices for the characterization of CNT and
CNF aerosols to be studied are presented in this document.
v
TECHNICAL REPORT ISO/TR 23463:2022(E)
Nanotechnologies — Characterization of carbon nanotube
and carbon nanofibre aerosols to be used in inhalation
toxicity tests
1 Scope
This document reviews characterization of CNT and CNF aerosols for inhalation exposure studies. The
document also provides useful information on appropriate characterization of CNT and CNF, which is
required to evaluate and understand the inhalation toxicity of CNT and CNF aerosols. This document
neither provides guidance on aerosol characterization for other carbon nanomaterials, nor provides
guidance for characterization of carbon nanotube and nanofibre aerosols in the workplace or ambient
air.
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 80004 (all parts), Nanotechnologies — Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given ISO 80004 (all parts), 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
carbon nanotube
nanotube composed of carbon
Note 1 to entry: Carbon nanotubes usually consist of curved graphene layers, including single-wall carbon
nanotubes and multiwall carbon nanotubes.
[SOURCE: ISO/TS 80004-3:2020, 3.3.3]
3.2
multiwall carbon nanotube
MWCNT
multi-walled carbon nanotube (3.1) composed of nested, concentric or near-concentric graphene sheets
with interlayer distances similar to those of graphite
Note 1 to entry: The structure is normally considered to be many single-wall carbon nanotubes nesting each
other, and would be cylindrical for small diameters but tends to have a polygonal cross-section as the diameter
increases.
[SOURCE: ISO/TS 80004-3:2020, 3.3.6]
3.3
single-wall carbon nanotube
SWCNT
carbon nanotube (3.1) consisting of a single cylindrical graphene layer
Note 1 to entry: The structure can be visualized as a graphene sheet rolled into a cylindrical honeycomb
structure.
[SOURCE: ISO/TS 80004-3:2020, 3.3.4]
3.4
carbon nanofibre
CNF
nanofibre (3.5) composed of carbon
[SOURCE: ISO/TS 80004-3:2020, 3.3.1]
3.5
nanofibre
nano-object (3.28) with two similar external dimensions in the nanoscale and the third dimension
significantly larger
Note 1 to entry: A nanofibre (3.5) can be flexible or rigid.
Note 2 to entry: The two similar external dimensions are considered to differ in size by less than three times and
the significantly larger external dimension is considered to differ from the other two by more than three times.
Note 3 to entry: The largest external dimension is not necessarily in the nanoscale.
[SOURCE: ISO/TS 80004-2:2015, 4.5]
3.6
aerosol
metastable suspension of solid or liquid particles in a gas
[SOURCE: ISO TR 27628:2007, 2.3]
3.7
inhalation chamber system
system prepared to expose experimental animals to an inhaled test substance of predetermined
duration and dose by either nose-only or whole-body method
Note 1 to entry: This system consists of chamber, head-only and nose-only.
Note 2 to entry: The term “nose-only” includes head-only, nose-only, or snout-only.
[18] [12] [13]
Note 3 to entry: [SOURCE: OECD TG 403 , 412 , 413 ]
3.8
nanoparticle generation system
device to make nanoparticle aerosol with controlled size distribution and concentration
[SOURCE: ISO 10808:2010, 3.3]
3.9
aspect ratio
ratio of length to width of a particle
[SOURCE: ISO 10312:2019, 3.8]
3.10
rigidity
inability to be to bent or forced out of shape or ability of a material to resist deformation
Note 1 to entry: This term applies to CNT or CNF.
Note 2 to entry: Asbestos fibres and MWNT-7 are examples of rigid structures.
3.11
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area is
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, or otherwise combined former primary particles.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO 26824:2013, 1.3]
3.12
agglomerate
collection of weakly or medium strongly bound particles 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 80004-2:2015, 3.4]
3.13
biodurability
ability of a material to resist dissolution (3.14) and mechanical disintegration from chemical and
physical clearance mechanisms
[SOURCE: ISO/TR 19057:2017, 3.3]
3.14
dissolution
process of obtaining a solution containing the analyte of interest
Note 1 to entry: Dissolution is the act of dissolving and the resulting species may be molecular or ionic.
[SOURCE: ISO/TR 19057:2017, 3.6]
3.15
aerodynamic diameter
−3
diameter of a sphere of 1 g cm density with the same terminal settling velocity in calm air as the
particle, under the prevailing conditions of temperature, pressure and relative humidity
Note 1 to entry: The particle aerodynamic diameter depends on the size, density and shape of the particle.
Note 2 to entry: Aerodynamic diameter is related to the inertial properties of aerosol particles.
[SOURCE: ISO 4225:2020, 3.1.5.13]
3.16
differential mobility analysing system
DMAS
system to measure the size distribution of submicrometer aerosol particles consisting of a DEMC (3.19),
a particle charge conditioner, flow meters, a particle detector, interconnecting plumbing, a computer,
and suitable software
[SOURCE: ISO 15900: 2020, 3.12]
3.17
geometric mean diameter
GMD
measure of the central tendency of particle size distribution using the logarithm of particle diameters
Note 1 to entry: The GMD is normally computed from particle counts and when noted may be based on surface
area or particle volume with appropriate weighting, as:
n
ΔNdln()
∑ ii
i=m
ln(GMD)=
N
where
d is the midpoint diameter for the size channel, i
i
N is the total concentration
∆N is the concentration within the size channel, i
i
m is the first channel
n is the last channel
[SOURCE: ISO 10808:2010, 3.5]
3.18
geometric standard deviation
GSD
measure of the width or spread of particle sizes, computed for the DMAS (3.16) by
n
Ndln −ln()GMD
[]
∑ ii
i=m
ln(GSD)=
N−1
[SOURCE: ISO 10808:2010, 3.6]
3.19
differential electrical mobility classifier
DEMC
classifier that is able to select aerosol particle sizes from a distribution that enters it and pass only
selected sizes to the exit
Note 1 to entry: A DEMC is sometimes called a Differential Electrical Mobility Spectrometer (DEMS). A DEMC
classified aerosol particle sizes by balancing the electrical force on each particle in an electrical field with its
aerodynamic drag force. Classified particles have different sizes due to their number of electrical charges and
a narrow range of electrical mobility determined by the operating conditions and physical dimensions of the
DEMC.
[SOURCE: ISO 10801: 2010, 3.2]
3.20
count median diameter
CMD
diameter equal to GMD (3.17) for particle counts assuming a logarithmic normal distribution
Note 1 to entry: The general form of the relationship as described in ISO 9276-5:2005 is
()rp− s
CMDx==x e
50,,rp50
where
e is the base of natural logarithms, e = 2,718 28;
p is the dimensionality (type of quantity) of a distribution
p = 0 is the number,
p = 1 is the length,
p = 2 is the area, and
p = 3 is the volume or mass;
r is the dimensionality (type of quantity) of a distribution, where
r = 0 is the number,
r = 1 is the length,
r = 2 is the area, and
r = 3 is the volume or mass;
s is the standard deviation of the density distribution
x  is the median particle size of a cumulative distribution of dimensionality, r.
50, r
[SOURCE: ISO 10808:2010, 3.7]
3.21
mass median aerodynamic diameter
MMAD
calculated aerodynamic diameter which divides the particles of an aerosol in half based on mass of the
particles
Note 1 to entry: Fifty percent of the particles by mass will be larger than the median diameter and 50 per cent of
the particles will be smaller than the median.
[SOURCE: EPA IRIS Glossary; ISO 15779:2011, 3.30]
3.22
mobility diameter
diameter of a spherical particle that has the same mobility as the particle under consideration
Note 1 to entry: Mobility diameter is generally used to describe particles smaller than approximately 500 nm,
and is independent of the density of the particle
[SOURCE: ISO/TR 27628:2007, 2.10]
3.23
particle density
ratio obtained by dividing the mass of a sample of aggregate particles by the volume, including both
permeable and impermeable pores within the particles (but not including the voids between the
particles)
Note 1 to entry: It is expressed as mass per unit volume, i.e. kilograms per cubic meter (kg/m )
[SOURCE: ISO 20290-1, 3.2]
3.24
specific surface area
surface area per unit mass of a particle or material
[SOURCE: ISO/TR 27628:2007, 2.19]
3.25
respirable fraction
mass fraction of inhaled particles which penetrate to the unciliated airways
[SOURCE: ISO 7708:1995, 2.11]
3.26
inhalable fraction
fraction of total airborne particles of given particle size inhaled through the nose and mouth
Note 1 to entry: Adapted from ISO 7708:1995, 2.3.
Note 2 to entry: The fractions specified in 3.3 to 3.8, as defined at specific particle size (characterized by
thermodynamic and aerodynamic diameters), are independent of the basis of measurement, e.g. mass, area or
particle count.
Note 3 to entry: A significant portion of the inhaled particles may be exhaled, but since these are smaller particles
their effect on the mass deposited may be minimal.
[SOURCE: ISO 13138:2012, 3.3]
3.27
nanomaterial
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
Note 2 to entry: See also engineered nanomaterial, manufactured nanomaterial and incidental nanomaterial.
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.28
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each
other.
[SOURCE: ISO/TS 80004-2:2015, 2.2]
3.29
nanoparticle
nano-object (3.28) 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
(3.5) or nanoplate (3.30) may be preferred to the term nanoparticle.
Note 2 to entry: Ultrafine particles may be nanoparticles.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
3.30
nanoplate
nano-object (3.28) with one external dimension in the nanoscale and the two other external dimensions
significantly larger
Note 1 to entry: The larger external dimensions are not necessarily in the nanoscale.
[SOURCE: ISO/TS 80004-2:2015, 4.6]
3.31
nanotube
hollow nanofibre (3.5)
[SOURCE: ISO/TS 80004-2:2015, 4.8]
3.32
particle
minute piece of matter with defined physical boundaries
Note 1 to entry: A physical boundary can also be described as an interface.
Note 2 to entry: A particle can move as a unit.
Note 3 to entry: This general definition applies to particle nano-objects.
[SOURCE: ISO 26824:2013, 1.1]
3.33
primary particle
original source particle (3.32) of agglomerates (3.12) or aggregates (3.11) or mixtures of the two
Note 1 to entry: Constituent particles of agglomerates or aggregates at a certain actual state may be primary
particles, but often the constituents are aggregates.
Note 2 to entry: Agglomerates and aggregates are also termed secondary particles.
[SOURCE: ISO 26824:2013, 1.4]
3.34
hazard
source with a potential to cause injury and ill health
Note 1 to entry: Hazards can include sources with the potential to cause harm or hazardous situations, or
circumstances with the potential for exposure leading to injury and ill health.
[SOURCE: ISO 45001:2018, 3.19]
4 Abbreviated terms
APM aerosol particle mass analyser
APS aerodynamic particle sizer
CMD count median diameter
CML count median length
DCFH-DA 2'-7'dichlorofluorescein diacetate
DMAS differential mobility analysing system
EDX energy dispersive X-ray analyser
EM electron microscopy
GC-MS gas chromatography–mass spectrometry
ICP-MS inductively coupled plasma mass spectrometry
NOAA nano-objects and their aggregates and agglomerates
OECD organization for economic co-operation and development
OPC optical particle counter
SEM scanning electron microscope
TEM transmission electron microscope
TEOM tapered element oscillating microbalance
TG test guideline
TGA thermogravimetric analysis
XRD X-ray diffraction
5 Considerations in CNT and CNF inhalation studies
5.1 General
Inhalation toxicity studies are important for hazard evaluation and as a first step to understanding the
potential health risks to workers and the general population if exposed to aerosolised CNTs and CNFs.
In designing an inhalation study for CNTs and CNFs as well as the interpretation of results obtained
from the inhalation study, physicochemical characterization of CNT and nanofibres before aerosol
generation and in aerosols as generated is critical for the evaluation of the potential health risks of
CNTs and CNFs following inhalation exposure.
5.2 Workplace exposure scenario
When conducting an inhalation toxicity study for CNTs and CNFs, actual workplace exposure scenarios
need to be considered. The generation of CNT and CNF aerosols needs to simulate actual workplace CNT
and CNF exposures in terms of concentration (mass if known), shape, size, size distribution, frequency
of exposure, and handling and manufacturing conditions of the CNTs and CNFs. However, the maximum
concentration needs to be less than 5 mg (total mass)/L for aerosols and particle aerosol higher than
[12,13]
2 mg (total mass)/L can only be attempted if a respirable particle size can be maintained . However,
achieving 2 mg/l (2 g/m ) of CNT and CNF for such low-density material is practically not feasible for
subacute and subchronic studies. Most of the inhalation studies were performed at a dose level less
3 [34,35]
than 5 mg/m , which produced inflammation and carcinogenic effect on the lung . For high dose
levels, the high amount of fibres deposited in the alveoli can produce lung overload in rats (mostly used
experimental animals in inhalation testing), which is believed to trigger the cascade of events leading
to stasis of clearance. Consequently, high enough CNT doses and lung overload can trigger sustained
[36]
pulmonary inflammation . Because nanofibres have a high surface area or number to mass ratio, the
conventional loading guidelines to avoid lung overload can not be applied to CNTs and CNFs.
The starting materials for the generation of CNT and CNF aerosols can be a powder, a liquid of well
or poorly suspended CNTs or CNFs, or a solid-state material. The generation of CNT and CNF aerosols
is difficult because of their hydrophobic nature and agglomeration propensity, although it can be
[37]
improved by the use of dispersion media such as phospholipids . Thus, the diversity and difficulties
in the generation of fibres can limit the simulation in an inhalation toxicity study with respect to the
actual workplace exposures. Various methods of CNT and CNF generation could be adopted as described
[38]
in ISO/TR 19601 .
5.3 Existing inhalation toxicity testing guidelines
The generation of regulatory hazard data are based on existing inhalation test guidelines (TGs)
published by the OECD or equivalent national and international bodies. The OECD TGs for an inhalation
[12] [13]
toxicity study include TGs 436, 412 , 413 , and Guidance Document (GD) 39. Among them, TGs 412
and 413 were recently revised to include the testing of nanomaterials. These TGs highlighted that the
[12,13]
MMAD needs to be less than 2 µm with a GSD up to 3 . Also, GD 39 has been revised to include
issues specific to nanomaterials testing.
6 Physicochemical parameters related to the toxicity of CNTs and CNFs
6.1 General
Because the toxicity of nanomaterials is closely related with their physicochemical properties, a
thorough investigation of the relationship between the physicochemical properties and toxicity
end points is important for a better understanding of the mechanism of toxicity and safer-by-design
approaches of CNTs and CNFs (see Annex A). Furthermore, this information can be very useful for risk
assessment and regulatory purposes as inhalation toxicity studies for every CNT and CNF cannot be
performed. Furthermore, it should be noted that physicochemical properties can modulate each other.
For example, the state of aggregation/agglomeration can be influenced by various factors including
surface charge, surface chemistry, and defects. Furthermore, the surface charge can be influenced
by various factors including surface chemistry and impurities. Besides the intrinsic properties of
CNTs and CNFs, various extrinsic factors, such as dispersion media and biological fluids where fibres
are in contact with, can also modulate the physicochemical properties of CNTs and CNFs. Since the
physicochemical properties can be changed by the aerosol generation processes, the measurement of
[10]
physicochemical parameters of CNTs and CNFs before and after the generation of aerosols is needed .
The information about the correlation between aerosol properties and toxicity end points are limited
because of the complexity and difficulties of inhalation studies. As an alternative study, the direct
administration of CNFs and CNFs into the lung, pleural space, and peritoneal space, or in vitro studies
have been extensively reported and demonstrated several physicochemical parameters related to
the toxicity end points. Thus, the information from these alternative studies can be very useful to
understand the results of inhalation toxicity studies and their relationship with the physicochemical
properties of the generated aerosols.
6.2 Aerodynamic properties of aerosols for deposition of fibres
A characterization of the aerodynamic properties of aerosols is critical to understand the pulmonary
deposition and penetration, and resultant toxicity of CNTs and CNFs because the deposition dose of
CNT and CNF aerosols is the biologically effective dose. The aerosols can deposit in the lung by various
respiratory deposition mechanisms such as inertial impaction, Brownian diffusion, gravitational
sedimentation, interception, and electrostatic effects. The deposition of particles in the respiratory
tract is influenced by several factors including their size, shape, and density.
The size of the aerosolised CNTs and CNFs in an inhalation study is important for pulmonary deposition
and penetration in the alveoli where the clearance is relatively limited. On the basis of information
from other aerosols, it is known that the size of airborne particles from 10 nm to 100 nm has about
[10]
20 % to 50 % deposition rate in alveoli . Whereas, as the size of the aerosolised particles increases
from about 100 nm, more particles can deposit in the airways where the clearance is efficient via a
[39,40]
mucociliary clearance mechanism . The range of aerosol sizes for deposition in the alveoli are
variable depending on the agents (e.g. size, shape, and density) as well as hosts (e.g. sex, strain, species,
and disease state). A first evaluation of the pulmonary deposition of the inhaled nanomaterials is
usually undertaken using in silico lung deposition estimation models. Various models are available
to estimate the total and regional lung deposition of aerosolised nanomaterials. Examples include the
[41]
Human Respiratory Tract Model (HRTM) and the Multiple-Path Particle Dosimetry Model (MPPD)
[42]
. However, these models have been developed for roundish particles, not fibres, thus careful
attention is needed when applying them to fibres and plates. For the application of models to estimate
deposition efficiency, there are also been some issues in the reporting of the specific input parameter
values used within the model, which are necessary to reproduce results. These values are frequently
missing, incomplete or unclear, especially in older publications, although the importance of reporting
these values is increasingly being recognized. Uncertainties in the measurements of aerosol parameters
used as input to deposition models, and their implications for deposition efficiency calculations and
[43]
ultimately dose are also generally ignored .
The shape of aerosols including the state of aggregation/agglomeration can influence the pulmonary
[44]
deposition and clearance . Although the aerosols are highly agglomerated, the CNTs and CNFs
[44]
deposited in the lungs can deagglomerate, which can produce fibre pathogenicity . In contrast,
aerosols composed of singlet fibres can re-agglomerate in the lungs, which can exhibit a changed
[45]
clearance and extrapulmonary translocation . Therefore, deagglomeration or re-agglomeration
of aerosols in contact with biological fluids can be considered in the characterization of their
physicochemical properties.
The density or specific gravity of CNTs and CNFs is important because this property is one of the main
factors that influence the aerodynamic behaviour and deposition fractions in the lungs. The aerodynamic
properties for deposition of CNT and CNF aerosols larger than 0,3 μm are especially influenced by the
[44]
density, whereas particles less than 100 nm are not influenced by the density . Because the density
is highly correlated with the volume, the volume per unit mass increases as the density decreases.
This can induce volumetric overload in cells, especially in phagocytic cells. Nanomaterials that exceed
6 % of normal cell volume can induce volumetric overload, which subsequently impairs cell function;
[44]
therefore, the density of CNTs and CNFs can influence the toxicity of nanofibres . The density is a key
parameter in estimating the deposition of nanomaterials in the lung using in silico estimation models
such as MPPD.
6.3 Size and shape (including length, width, aspect ratio, state of aggregation/
agglomeration, and rigidity)
Size and shape are key factors in the fibre pathogenicity, but these factors are closely related to each
other. For example, as the diameter of CNT and CNF increases, the rigidity increases, and the increased
[7,46]
rigidity can provide better dispersion and reduce aggregation/agglomeration . Comparative
toxicity studies of MWCNTs with different thickness showed that rigid thin MWCNTs have higher
toxicity in vivo and in vitro compared to rigid thick CNTs because thin fibres have a higher potential for
[6,46,47]
disruption of membrane integrity . There is a threshold thickness of MWCNTs to produce piercing
and frustrated phagocytosis to mesothelial cells in vitro as rigid thick (diameter: 150 nm) MWCNTs
[46]
showed much less toxicity than that of rigid thin (diameter: 50 nm) MWCNT . Furthermore, threshold
rigidity values for asbestos-like pathogenicity of MWCNTs in a mouse pleural inflammation model were
[6]
proposed as a bending ratio of 0,66 and static bending persistence length of 0,87 . The length and
width of fibres are important for the pathogenicity of CNTs and CNFs because the respective factors are
critical for frustrated phagocytosis and cell penetration. The lower threshold length of fibres, which can
[48,49]
result in frustrated phagocytosis, has been suggested to be 4 μm to 5 μm . Intraperitoneal injection
of CNTs demonstrated that the long fibres are more potent to produce toxicity and mesothelioma than
[50-52]
short fibres . Furthermore, the range of the width of fibres could also influence cell penetration. It
has been reported that thin fibres with some ranges (e.g. 9,4 nm and 50 nm) are more pathogenic than
[46,47]
thick fibres with some ranges (e.g. 70 nm and 150 nm) .
The aggregation/agglomeration status of CNT and CNF is a result of the complex physicochemical
properties (e.g. diameter, surface charge, defects, and hydrophobicity) and condition of the liquid
medium (e.g. pH, salt, and dispersant) when CNTs and CNFs are dispersed from a liquid suspension.
Unlike nanoparticles, it is more difficult to differentiate aggregation, agglomeration, and tangled forms
of nanofibres. In addition, an evaluation of the impact of the aggregation/agglomeration status on
the toxicity end points has been limited because the aggregation/agglomeration state can vary with
experimental conditions. According to current knowledge, the agglomerated and/or tangled CNTs
can be less toxic than well-dispersed and/or rigid CNTs. If the diameter of the CNTs is too large to be
phagocytosed by reticuloendothelial cells, the CNTs are less potent to produce frustrated phagocytosis
[46]
and inflammasome activation . In addition, if the diameter of tangled CNTs is small enough to
allow for phagocytosis, the toxic potential is also less than well-dispersed CNTs because complete
[53]
phagocytosis is possible . Although some studies have reported that the intratracheal instillation of
agglomerated CNTs can produce severe pulmonary toxicity, such as granulomatous inflammation with
[54]
discrete granulomas often surrounded by hypertrophied epithelioid macrophages , it could not be
[55]
reproduced in an inhalation toxicity study in terms of inhalability and deposition in the lung .
The length and diameter can also be expressed as an aspect ratio (length/diameter), and an aspect ratio
more than 3:1 is well known as a parameter for the asbestos-like toxicity such as granulomas, fibrosis,
[34,56,57]
and cancer . When occurring together, a high aspect ratio (>3:1), thin diameter (<50 nm), long
length (>5 μm), high rigidity (>0,66 bending ratio and >0,87 static bending persistence length), minimal
aggregation/agglomeration, and high durability are believed to be major pathogenic factors of CNT and
CNF.
6.4 Specific surface area
The specific surface areas or surface-to-mass ratios of CNTs and CNFs are relatively high compared
to metal-based nanomaterials due to the low density/mass. Because the specific surface area value
originates mainly from size and shape, the impact of surface area on the toxic potential of CNTs and
CNFs remains unclear. On the other hand, this value is an important dose metric for comparison
between nanomaterials. However, the surface area metric can not be pertinent for CNTs and CNFs,
[58,59]
although the surface area has been suggested as a dose metric for nanoparticles . For example, a
CNT which has been classified by IARC as highly toxic has a low surface-to-mass ratio, while many of
[6,45]
MWCNTs e
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