ISO/TS 5387:2023

TECHNICAL ISO/TS
SPECIFICATION 5387
First edition
2023-10
Nanotechnologies — Lung burden
mass measurement of nanomaterials
for inhalation toxicity tests
Nanotechnologies — Mesure de la masse de la charge pulmonaire des
nanomatériaux pour les études de toxicité par inhalation
Reference number
© ISO 2023
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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 . 4
5 Use of lung burden measurements for risk assessment of nanomaterials .4
6 Inhalation exposure and tissue sampling to determine lung burden .5
6.1 Inhalation exposure . 5
6.2 Lung burden evaluation in single or multiple lobes . 5
6.3 Post-exposure observation points . 6
7 Available methods for lung burden measurements . 7
7.1 General . 7
7.2 Carbon nanomaterials . 8
7.3 Metal-based nanomaterials . 8
7.4 Polymeric nanomaterials and others . 9
8 Application of lung burden data to toxicokinetics of nanomaterials .9
8.1 General . 9
8.2 Sampling points . 9
8.3 Particle lung clearance and retention kinetics . 10
8.3.1 General . 10
8.3.2 One-compartment first-order clearance model . 10
8.3.3 Two-compartment first-order model . 11
Annex A (informative) Option A for test scheme for 28-d and 90-d studies — Gases,
vapours, liquid aerosols, and fast dissolution solid aerosols.15
Annex B (informative) Option B for test schemes for 28-d and 90-d studies — Poorly
soluble aerosols .16
Annex C (informative) Lung burden measurement methods .17
Bibliography .21
iii
Foreword
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iv
Introduction
Inhalation is a primary route of exposure to aerosolized nanomaterials and therefore appropriate
inhalation toxicity tests are required to address risk assessment needs for these materials. For this
reason, the Organisation for Economic Cooperation and Development (OECD) recently updated its
inhalation toxicity test guidelines 412 (subacute) and 413 (subchronic) to make them applicable to
[1][2]
nanomaterials. These revised test guidelines require post-exposure lung burden measurements
to be undertaken when a range-finding study or other relevant information suggests that inhaled test
nanomaterials are poorly soluble with low dissolution rate and likely to be retained in the lung. The
measurements of lung burden inform on pulmonary deposition and retention of nanomaterials in the
lung. At least three lung burden measurements are needed to evaluate clearance kinetics.
This document gives information on how to derive clearance kinetic parameter values using lung
[1] [2]
burden measurement data. This document complements OECD TG 412 and OECD TG 413 . As
References [1], [2] and [3] only provide limited information on methods for lung burden measurement
for nanomaterials or the derivation of lung clearance kinetics, this document provides useful supporting
[1] [2]
information for conducting inhalation studies based on OECD TG 412 and OECD TG 413 .
v
TECHNICAL SPECIFICATION ISO/TS 5387:2023(E)
Nanotechnologies — Lung burden mass measurement of
nanomaterials for inhalation toxicity tests
1 Scope
The document provides information on the measurement of nanomaterial mass in tissue after inhalation
exposure, which can inform on lung clearance behaviour and translocation.
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 in the ISO 80004 series 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
aerodynamic diameter
diameter of a spherical particle with a density of 1 000 kg/m that has the same settling velocity as the
particle under consideration
Note 1 to entry: Aerodynamic diameter is related to the inertial properties of aerosol (3.2) particles and is
generally used to describe particles larger than approximately 100 nm.
[4]
[SOURCE: ISO/TR 27628:2007, 2.2 ]
3.2
aerosol
metastable suspension of solid or liquid particles in a gas
[4]
[SOURCE: ISO/TR 27628:2007, 2.3 ]
3.3
mass median aerodynamic diameter
MMAD
calculated aerodynamic diameter (3.1) which divides the particles of a measured aerosol (3.2)
distribution in half based on the mass of the particles where fifty percent of the particles by mass will
be larger than the median diameter and fifty per cent of the particles will be smaller than the median
[11]
[SOURCE: EPA IRIS Glossary ]
3.4
manufactured nanomaterial
nanomaterial (3.8) intentionally produced for commercial purposes to have selected properties or
composition
[SOURCE: ISO 80004-1:2023, 3.1.9, modified — "for commercial purposes" has been added to the
definition.]
3.5
mixture
mixture composed of two or more substances in which they do not react
Note 1 to entry: A solution is a mixture as well.
[8]
[SOURCE: GHS, 2011 ]
3.6
mobility
propensity for an aerosol (3.2) particle to move in response to an external influence, such as an
electrostatic field, thermal field or by diffusion
[4]
[SOURCE: ISO/TR 27628:2007, 2.9 , modified — the domain "" has been removed.]
3.7
nanofibre
nano-object with two similar external dimensions in the nanoscale and the third dimension significantly
larger
Note 1 to entry: A nanofibre 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 80004-1:2023, 3.3.5, modified — Notes 1 and 2 to entry have been added.]
3.8
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 80004-1:2023, 3.1.4, modified — Note 1 to entry has been replaced.]
3.9
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
(3.7) or nanoplate may be preferred to the term nanoparticle.
Note 2 to entry: Ultrafine particles may be nanoparticles.
[SOURCE: ISO 80004-1:2023, 3.3.4, modified — "where the lengths of the longest and the shortest axes
of the nano-object do not differ significantly" has been added to the definition and Note 2 to entry has
been added.]
3.10
nanotube
hollow nanofibre (3.7)
[SOURCE: ISO 80004-1:2015, 3.3.8]
3.11
single-walled carbon nanotube
SWCNT
SWCNT single-walled carbon nanotube 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.
3.12
multi-wall carbon nanotube
MWCNT
MWCNT multi-walled carbon nanotube 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-walled carbon nanotubes (3.11) nesting
each other, and would be cylindrical for small diameters but tends to have a polygonal cross-section as the
diameter increases.
3.13
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.
[5]
[SOURCE: ISO 26824:2013, 3.1.1 ]
3.14
poorly soluble particle
inhaled test particles that are likely to be retained in the lung
[1]
[SOURCE: OECD TG 412, paragraph 2 ]
3.15
primary particle
original source particle of agglomerates or aggregates 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.
[5]
[SOURCE: ISO 26824:2022, 3.1.4 ]
3.16
secondary particle
particle formed through chemical reactions in the gas phase (gas to particle conversion)
[4]
[SOURCE: ISO/TR 27628:2007, 2.17 ]
4 Abbreviated terms
AAS Atomic absorption spectrometry
AgNP Silver nanoparticles
AuNP Gold nanoparticles
BALF Bronchoalveolar lavage fluid
CoO Cobalt oxide
CuO Copper oxide
DEMC Differential electrical mobility classifier
DEMS Differential electrical mobility spectrometer
ECA Elemental carbon analysis
GD Guidance document
GHS Globally harmonized system
HPLC High performance liquid chromatography
ICP-MS Inductively coupled plasma mass spectrometry
LALN Lung-associated lymph node
MMAD Mass median aerodynamic diameter
NDIR Non-dispersive infrared
OECD Organisation for Economic Cooperation and Development
PEO Post-exposure observation
sp-ICP-MS Single particle ICP-MS
TG Test guideline
TiO Titanium dioxide
UV-Vis Ultraviolet-visible spectrometry
WPMN Working Party on Manufactured Nanomaterials
ZnO Zinc oxide
5 Use of lung burden measurements for risk assessment of nanomaterials
The concept of lung overload hypothesis was first proposed in Reference [13]. The determination of lung
[14]
burden of inhaled nanomaterials is therefore of great relevance to assess a possible lung overload.
[15] [13]
Morrow has also proposed that a continuously increasing prolongation of particle lung clearance
occurs when the retained lung burden exceeds a certain threshold. Decreased clearance capacity of
alveolar macrophages will lead to inflammatory reactions and to an increase in the translocation of the
[16]
inhaled particles to interstitium and lung-associated lymph nodes .
Lung burden data can be used for the risk assessment of poorly soluble with low dissolution rate
particles (e.g. as obtained from tests according to References [1] and [2]). When pulmonary effects are
driving the human health risk assessment, risk assessors need to evaluate whether the occurrence of
the pulmonary effects is better characterized by administered exposure concentration or by retained
dose in the lungs. The human equivalent dose and lifetime human exposure may be calculated for risk
estimation. Applications of such principles are available in literature, e.g. References [17] and [18].
Another value of lung burden data is the possibility of reading across hazard data from studies using
[19]
the same material with different primary particle sizes. The same external concentrations can result
in differences in retained dose. Conversely, different external concentrations can result in the same
[20]
retained dose for different particle sizes .
6 Inhalation exposure and tissue sampling to determine lung burden
6.1 Inhalation exposure
For inhalation exposure of nanomaterials, nanomaterials are frequently generated in situ or powdered
forms of nanomaterials are dispersed and generated and delivered into the inhalation chamber. The
[21]
generation of nanomaterials aerosol for inhalation toxicity testing is described in ISO/TR 19601 and
[22] [10]
ISO 10801 , and monitoring of such aerosols in the inhalation chamber is described in ISO 10808 .
References [10], [21] and [22] also provide methods of aerosol concentration monitoring and
physicochemical characterization as well as OECD test guidelines.
6.2 Lung burden evaluation in single or multiple lobes
For inhalation toxicity testing of nanomaterials, please refer to References [1] and [2]. Depending on
the type of nanomaterial, the study director can use data from a range-finding study to determine the
appropriate post-exposure duration as well as the optimal number and timing of sampling intervals
for a repeated exposure inhalation study. Although the TGs require using one lung (right lung) for lung
burden measurement and the other lung (left lung) for histopathological evaluation, recent studies with
AgNPs and AuNPs demonstrated that nanoparticles deposit in the rat lung lobes evenly, thus, any lobe
[23][24]
can be used for lung burden measurement. As shown in Figure 1, the right lung lobe consisted of
[23][25]
four lobes. Soluble nanoparticles with high dissolution rate such as silver nanoparticles as well as
[24]
poorly soluble with low dissolution rate particles such as gold nanoparticles were evenly deposited
in rat lungs after subacute (5 d) inhalation exposure. Using any lobe for lung burden measurements
opens the opportunity to use the remaining lobes for other measurements, such as histopathological
tissue preparation and BALF assay, in the same rat. Such an approach can maximize the number of
endpoints measured and has the potential to reduce the number of animals used in testing. Although
fibrous or plate forms of nanomaterials such as carbon nanotubes and graphenes were not tested and
proven for even deposition throughout the lung lobes, some lung burden and thereafter lung clearance
kinetic study has been conducted for carbon nanotubes.
A recent study given in Reference [26] on the lung deposition and retention of multi-walled carbon
nanotubes (MWCNTs) [where the mass median aerodynamic diameter (MMAD) is 1,015 μm] after
28 d of inhalation and for 28 d post-exposure showed that the lung clearance kinetics of MWCNTs can
[26]
be effectively evaluated using one lobe from the right lung. The BAL fluid was collected from the
right lung after occluding the post-caval lobe and left lung. The left lung was then used to evaluate
[24]
the histopathology and the post-caval lobe to evaluate the lung burden. In another recent study,
quantitative analyses of lung burdens on various shapes of carbon nanomaterials including printex-90
3 3 3 3
carbon black (50 mg/m ), nanomaterial NM-401 (0,5 mg/m and 1,5 mg/m ), NM-403 (1,5 mg/m ),
and MWCNT-7 (1,5 mg/m ) nanotubes were conducted after 28-d inhalation exposure. Their MMAD
was 940 nm for printex-90 carbon black, 790 nm for NM-401, 1 940 nm for NM-403 and 1 780 nm for
[27]
MWCNT. The middle right lobe was separated and used for lung burden analysis successfully .
Key
1 trachea
2 left bronchus
3 superior lobe
4 middle lobe
5 inferior lobe
6 post-caval lobe
7 left lung
SOURCE: Reference [3]. Reproduced with the permission of the authors.
Figure 1 — Rodent trachea and lungs
6.3 Post-exposure observation points
Although References [1] and [2] prescribe only one mandatory sampling point [post-exposure
observation (PEO)-1)], it is recommended to conduct two additional sampling points (PEO-2 and PEO-
3) right after the termination of exposure (PEO-1, post-exposure observation) to conduct toxicokinetic
or particokinetics studies. The concept of “particokinetics” is introduced to address the dynamic
biological behaviour of ENMs at the molecular level (including gravitational sedimentation, dispersion,
aggregation and interaction with biomolecules in suspending media), cellular level (including cellular
uptake, transport, biotransformation and elimination) and whole-organism level (including absorption,
[28]-[32]
distribution, metabolism and excretion in vivo). In addition, lung burden measurement at
exposure d-1 (6-h exposure) can provide information about the solubility of test nanomaterials and the
retention trend after the designed exposure period, because lung retention time and biopersistence
increases as particles are poorly soluble.
Additional satellite groups can be added to the main study to evaluate recovery, persistence, delayed
occurrence of toxicity or lung burden for a post-treatment period of an appropriate length. Designs of
main studies with satellite groups are shown in Annexes A and B. The study director should modify the
design of a study based on the physicomaterial characteristics and kinetics of a test chemical to achieve
the most robust data.
All satellite groups are exposed concurrently with the experimental animals in the main study and at
the same concentration levels and there should be concurrent air or vehicle controls as needed. The
scheduling and design of satellite groups depend on whether the test chemical is a solid aerosol and is
likely to result in lung retention following Annexes A and B. If the test chemical is likely to result in lung
retention, the main study is conducted as described in option B in Annex B; otherwise, the main study
is conducted as described in option A in Annex A (used for test chemicals as gas, vapour, aerosol or a
mixture thereof). Satellite groups can be included to evaluate recovery in option A in Annex A; option B
in Annex B (used when testing chemicals that are likely to be retained in the lungs) provides for satellite
groups for the evaluation of recovery and/or for lung burden measurements. Satellite recovery groups
at PEO-2 consist of five males and five females per concentration in option A and option B in Annexes A
and B, respectively.
These recovery groups are exposed concurrently with the experimental animals in the main study
and at the same concentration levels, and there should be concurrent air or vehicle controls as needed.
When testing poorly soluble with low dissolution rate solid aerosols that are likely to be retained in the
lungs, one or two additional satellite groups of five males per concentration may be added to measure
lung burden at different post-exposure time points (see option B in Annex B). These additional lung
burden measurements (i.e. PEO-2 and/or PEO-3) may be added to the design when the study director
would like to understand the post-exposure clearance kinetics of the test substance. Since three-time
points are generally required to provide information on clearance kinetics, lung burden measurements
are performed within 24 h after exposure termination (PEO-1) and at two additional PEOs (PEO-
2 and PEO-3). However, the use of two-time points may provide sufficient information under some
circumstances, such as when the main objective is to identify whether clearance is very slow. Lung
burden measurements are preferably performed in males, which have a higher minute volume than
females and may thus have greater lung burdens. OECD guidelines provide following options.
— The study director can choose to schedule PEO-3 before the recovery group (PEO-2) (if included), if
considered more appropriate.
— If the use of two post-exposure time points is considered sufficient, lung burden measurements can
be performed at PEO-1 (main study) and at PEO-2 (recovery group) only, if the timing for evaluation
of recovery and lung clearance can be aligned to one another. The satellite group at PEO-3 can then
be omitted from the study.
— The study director can choose to perform lung burden measurements at PEO-1 (main study) and at
PEO-3 (satellite group) and to use both sexes of the recovery groups (PEO-2) for BALF analysis.
Sometimes, for the lung clearance kinetic study, three PEOs can be insufficient to derive the toxicokinetic
parameters.
7 Available methods for lung burden measurements
7.1 General
The lung burden of nanomaterials can be evaluated by various methods. In simple terms, these can be
divided into:
a) measurement without digestion of lung tissue, such as radioisotope labelling and direct imaging of
particles in intact tissue samples, and
b) measurement after digestion of lung tissues.
Annex C summarises the literature on lung burden analysis of nanomaterials. For measuring lung burden
after digestion of lung tissue, acids, alkali or protease enzymes are generally used to digest lung tissue
but digesting agents should be selected based on the physicochemical properties of nanomaterials.
Then, the extracted nanomaterials can be quantified by specific methods or instruments to determine
their elemental composition. In this document, the available methods for lung burden measurement
are separately described by the types of nanomaterials such as carbon nanomaterials, metal-based
nanomaterials, polymeric nanomaterials and others.
7.2 Carbon nanomaterials
As carbon nanomaterials such as carbon black, nanodiamond, graphene, carbon nanotube, and carbon
nanofibre are not dissolved in buffers that are used for the lysis of lung tissues, these materials
can be extracted from lung tissue by the acids, alkalis, and protease enzymes. However, acids (e.g.
hydrochloric acid, nitric acid and sulfuric acid) and alkalis can change the physicochemical properties
[33][34]
by inducing defects and oxidation/reduction. The modified physicochemical properties of carbon
nanomaterials by the treatment of acids and alkalis can induce inaccuracy in instrumental analyses.
Recently, the digestion of lung tissue using the proteinase K enzyme has been proposed as an alternative
[33][35]
method to digest lung tissues without damaging the structure of carbon nanomaterials. The
collected carbon nanomaterials can be quantified by the elemental carbon analysis (ECA) with high
[36][37] [38]
performance liquid chromatography (HPLC), non-dispersive infrared (NDIR) analysis, near
[39] [26][35]
infrared fluorescence imaging, ultraviolet-visible (UV-Vis) spectrophotometer and standard
[40]
morphometric point counting methods of histology slices. The ECA is performed using an organic
carbon (OC)/ elemental carbon (EC) analyzer based on NIOSH Manual of Analytical Methods (NMAM)
[41]
5040. The UV-Vis spectrophotometer analysis uses a wavelength of the near-infrared region such
as 750 nm because biological materials such as haemoglobin and proteins show minimal absorbance
[35]
in this region. Notably, the concentration of carbon nanomaterials measured by an organic carbon
(OC/EC analysis is an absolute value, while that of UV-Vis spectrophotometer and NDIR analysis is a
relative value calculated from the standard curve fit). In addition, the labelling of carbon nanomaterials
99m 86
such as Technetium-99 m ( Tc) and yttrium-86 ( Y) radioisotopes can be a method for lung burden
[42][43]
analysis. However, it should be noted that the labelled particles have different physicochemical
properties from the pristine particles and the labelling materials can be OC, carbonate (CC) and EC.
7.3 Metal-based nanomaterials
When metal-based nanomaterials are labelled with radioisotopes or have imageable properties such
as superparamagnetic iron oxide nanoparticles (SPION), the lung burden can be directly measured
from the excised lung without digestion processes. Nanomaterials labelled with radioisotopes such as
105 195 59 48
Ag, Au, Fe O , and V-radiolabelled titanium dioxide (TiO ) particles were successfully tested
2 3 2
[28][44][45][46]
for lung burden analysis. In addition, nanomaterials having imageable properties such as
[47]
SPION particles were quantified by magnetic particle imaging. However, the labelling method using
radioisotopes has the same limitations as described in 7.2. The imaging method also has limitations in
that only relative quantification is possible.
Lung burden measurement of metal-based nanomaterials can be divided into two steps:
sample preparation and quantification. If a sample preparation procedure is sufficient to dissolve both
lung tissue and the nanomaterial of interest (e.g. use of hydrochloric acid, nitric acid and sulfuric acid),
the digestate can filtered or centrifuged then analyzed using an appropriate spectrometry technique
such as atomic absorption spectrometry (AAS) or inductively coupled plasma mass spectrometry
(ICP-MS). The quantification of nanomaterials by these methods was implemented for cerium oxide,
[48]-[54]
titanium dioxide and aluminum oxide. The selection of acids and their combinations should
depend on whether the digestion buffer can dissolve nanomaterials. The use of alkalis such as solvable®
can be applied to digest lung tissue. While some nanomaterials such as copper oxide (CuO), cobalt
oxide (CoO), and zinc oxide (ZnO) are dissolved during acid-assisted sample digestion procedures,
other nanomaterials such as Au and TiO are not dissolved. Thus, samples with these nanomaterials
require pre-treatment to dissolve any tissue followed by digestion to fully dissolve the nanomaterial.
Digestates can be filtered or centrifuged followed by analysis using AAS or ICP techniques. AAS was
[55] [56] [57]
used for aluminum oxide , silver and gold nanoparticles .
When the concentration of nanomaterials is measured by an ICP instrument from samples of acid
digestion or metal-dissolved supernatant after digesting fluid treatment, it is difficult to differentiate
whether the measured concentration of metals is from the particulate form of nanomaterials, dissolved
ions of nanomaterials or elements from lung tissue. To overcome this limitation, the extraction of
metal-based nanomaterials as a particulate form is needed. In recent studies, the use of protease
enzymes showed a good efficacy to lysis lung tissue. Although poorly-soluble with low dissolution rate
nanomaterials do not dissolve in contact with the protease enzymes, the potential for dissolution of
nanomaterials during the tissue digestion process should be evaluated before lysing the lung tissue. If
nanomaterials dissolve in alkalis or by protease enzymes, the use of acids is highly recommended.
The dissolved metal ions can be quantified by an ICP instrument and the measured concentrations
can be converted to the concentration of nanomaterials based on the chemical composition. For
nanomaterials composing elements that are abundant in the tissue, the subtraction of measured
concentration with vehicle control is needed and perfusion is highly recommended to exclude the effect
of components in blood. As some metals such as silica are evaporated during the acid digestion process,
[58]
the availability of metals to this application should be tested before the experiment. The collection
of intact nanomaterials from the lung tissue is thus a good way to measure lung burden because this
method can distinguish the origin of the detected elements. When particulate forms of nanomaterials
are collected, the quantification can be performed by measuring metal elements using an instrument
such as ICP-MS or measuring particulate form nanomaterials using instruments such as single-particle
[59]
(sp)-ICP-MS, UV-Vis spectrophotometer and fluorimeter .
7.4 Polymeric nanomaterials and others
Polymeric nanomaterials such as polystyrene and polypropylene are resistant to acids, alkalis and
[60]
protease enzymes. Therefore, any tissue digestion methods excluding organic solvents can be used to
collect polymeric nanomaterials. Then, the measurement method for the collected nanomaterials should
be decided based on the nanomaterial-specific properties. For example, when polymeric nanomaterials
are labelled with dyes or fluorophores, the standard curve fit with absorbance or fluorescence can be
used for quantification. However, the current levels of technology for the quantification of non-labelled
polymeric nanomaterials should be improved. Any other nanomaterials that were not discussed above
can follow the same procedures such as
a) collection of nanomaterials, and
b) measurement of concentration.
When nanomaterials are dissolved or destructed during the process of collecting nanomaterials
from the lung, the measurement method for the dissolved molecules should be selected based on the
physicochemical properties of nanomaterials.
8 Application of lung burden data to toxicokinetics of nanomaterials
8.1 General
Lung burden measurements performed during repeated exposure studies in rats provide a metric of
retained dose and can be helpful in understanding the toxicity of poorly soluble particles with low
dissolution rates. However, each retained lung burden can have a different kinetic history due to burden-
specific changes in clearance. Lung burden data can be used for the risk assessment of poorly soluble
with low dissolution rate particles (e.g. as obtained from tests according to References [1] and [2]).
When pulmonary effects are driving the human health risk assessment, risk assessors need to evaluate
whether the occurrence of the pulmonary effects are better characterized by exposure concentration
or by retained dose in the lungs. The human equivalent dose and lifetime human exposure may be
[17][18]
calculated for risk estimation .
8.2 Sampling points
Although lung burden measurement is mandatory at only one post-exposure observation period in
option B (at PEO-1), more lung burden measurements may be needed to provide information on clearance
kinetics and persistence/progression response, especially for poorly soluble with low dissolution rate
particles or some soluble particles with high dissolution rate. It is also advantageous to have some
more sampling points such as exposure d-1 (6-h exposure) to estimate daily retention and additional
sampling points during PEOs to fit better for the retention curve. The daily retention obtained from 6-h
exposure will help to estimate the clearance tendency of deposited nanomaterials and the additional
sampling points would be helpful to find better inflection points for some nanomaterials having two-
phase clearance kinetics.
8.3 Particle lung clearance and retention kinetics
8.3.1 General
The clearance of particles from the alveolar or pulmonary region of the lungs has been usually regarded
as a first-order process, which implies a constant proportion of particle is eliminated per unit time.
[13][61]
This model has been used mainly because it provides a kinetically suitable description of lung
[62]
clearance and a relatively simple dosimetric approach. The fraction of organ concentration per
initial organ concentration at PEO-1 was used for estimating retention and clearance kinetics, applying
an appropriate first-order clearance model.
8.3.2 One-compartment first-order clearance model
The first-order model is defined by Formula (1). The retention half-time (T ) is derived using λ and
1/2
natural log (2) as shown in Formula (2).
Mt()=−Ptexp()λ (1)
where
M(t)=M(0)exp(−λt);
M(0) as the lung burden at t = 0 d;
M(t)/M(0) as the retention fraction, i.e. the lung burden at the time of a fraction of the initial
lung burden;
P is the fraction of lung burden cleared (1,0 for one-compartment model);
λ is the clearance rate per day for one-compartment model;
t is the time, in d.
ln 2
() 0,693
T = ≈ (2)
12/
λλ
The example of first-order model of clearance is poorly soluble with low dissolution rate gold
nanoparticles (AuNP) as shown in Figure 2.
Key
X retention fraction for AuNP
Y time, in d
1 AuNP
[32]
Single AuNP in the Y axis signifies that the group treated with AuNP only, not combined with AgNP .
Figure 2 — Lung retention fraction of AuNP at 1-d, 7-d, and 28 p 28-d inhalation exposure to
rats
Table 1 — Retention kinetics of AuNP
First order model
AuNPs
Elimination rate
T λ
1/2
−1
d d
81,5 0,008 5
SOURCE: Reference [32]. Reproduced with the permission of the authors.
Rats were exposed subacutely (28 d) to an aerosol of gold nanoparticles (10,8 nm) at a mass
3 3
concentration of 17,7 µg/m ± 1,7 µg/m and lung burdens were determined at 1-d, 7-d and 28 d post-
exposure. The data are presented in Figure 1 and the estimated T and elimination rate in Table 1.
1/2
Most poorly soluble particles with low dissolution rate follow the first-order model.
8.3.3 Two-compartment first-order model
Some nanoparticles which are soluble with high dissolution rate may form poorly soluble with low
[25][46][32]
dissolution rate secondary nanoparticles after reacting soluble ions with biomolecules. The
two-phase model or two-exponential time-decay function used computer programming based on
Formula (3), prior to which the retention fractions were converted to logarithmic variables. The
retention half-time (T ) was derived using λ λ , and natural log (2) as shown in Formula (4).
1/2 1, 2
Mt =−Ptexpeλλ+−Ptxp (3)
() () ()
11 22
where
M(t)=M(0)exp(−λt);
P is the fraction of lung burden cleared by fast phase;
λ is the fast clearance rate per day for two-compartment model;
P is the fraction of lung burden cleared by slow phase;
λ is the slow clearance rate per day for two-compartment model;
t is the time, in d.
ln()2
0,693
T = ≈ (4)
12/
λλ
An example of the two-compartment first-order model is silver nanoparticles (AgNP). The lung burdens
of Ag from AgNPs were measured on PEOs of 1-d, 7-d, and 28-d to obtain quantitative mass concentrations
per lung. Lung burden measurement suggested that Ag from AgNPs was cleared through two different
modes: fast and slow clearance. The fast clearance component was concentration-dependent with half-
−1 −1
times ranging from two days to four days and clearance rates of 0,35 d to 0,17 d from low to high
concentrations. The slow clearance had half-times of 100-d, 57-d, and 76-d and clearance rates of 0,009
−1 −1 −1
d , 0,012 d , and 0,007 d for the high, moderate, and low concentration exposure (see Figure 3 and
Table 2). The fast clearance component which was concentration-dependent can be dependent on the
dissolution of AgNPs and the slow clearance would be due to slow clearance of the low dissolution AgNPs
secondary particles originating from silver ions reacting with biogenic anions. These secondary AgNPs
can be cleared by mechanisms other than dissolution such as mucociliary escalation, translocation to
[25][46][32]
the lymphatic system or other organs .
3 3
a) High concentration (115,6 µg/m ± 30,5 µg/m )
3 3
b) Moderate concentration (81,5 µg/m ± 11,4 µg/m )
3 3
c) Low concentration (31,2 µg/m ± 8,5 µg/m )
Key
X time, in d
Y retained fraction, in log
1 y = P exp(−d t)+P exp(−d t)
1 1 2 2
2 y = P exp(−d t); slow clearance
2 2
3 y = P exp(−d t); fast clearance
1 1
SOURCE: Reference [25]. Reproduced with the permission of the authors.
Figure 3 — Clearance of kinetics of Ag after 28-d of AgNP exposure and post-exposure
Table 2 — Clearance kinetics of Ag
Concentration Fast clearance Slow clearance
T Rate T Rate
1/2 1/2
−1 −1
d d d d
High 4,04 0,171 100,46 0,007
Moderate 3,23 0,21 56,82 0,012
Low 1,98 0,35 76,17 0,009
SOURCE: Reference [25]. Reproduced with the permission of the authors.
The clearance T of soluble nanomaterials with high dissolution rate is influenced by the exposure
1/2
concentration in both slow and fast clearance. Lower concentration cleared faster than higher
concentration. Compared to AgNPs, the T of lung clearance of poorly soluble with low dissolution
1/2
rate nanomaterials ranged 60-d to 90-d, as seen in TiO and gold nanoparticles. If T is much
2 1/2
[63][32]
longer than these T , it can be lung overload effects. Overload lung burdens by poorly soluble
1/2
with low dissolution rate nanomaterials have been shown to induce chronic lung pathology such as
[64]
fibrosis and lung cancer. The determination of change in lung burden over time with relevant T
1/2
are important parameters for the risk assessment to ascertain if chronic pulmonary effects are due
to the physicochemical properties of the nanomaterials studied or due to overload of a low toxicity
nanoparticles.
Annex A
(informative)
Option A
...