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COMMON MECHANISM-BASED STUDY DESIGN FOR INHALATION TOXICITY TESTING OF
POORLY SOLUBLE (NANO-/ULTRAFINE-) PARTICLES: ARE NEW TESTING STRATEGIES
AND INTERPRETATION GUIDELINES NEEDED?
Juergen PAULUHN
•
Bayer HealthCare Research Center, Aprather Weg, 42096 Wuppertal, Germany,
juergen.pauluhn@bayer.com.
Abstract
Current inhalation testing guidelines are focused on the evaluation and assessment of the toxicological
properties of inhaled chemical substances at the portal-of-entry and systemically. With the exception of
poorly soluble particles, these substances show a rapid clearance from the lung. Physical properties
(gas/vapor/liquid/solid) play a role for localized deposition which generally supports a categorization into
systemic, lower, airway, and upper respiratory tract toxicants. Poorly soluble particle display opposite
features, i.e., commonly they do not have any substance-specific toxicity, they are deposited and
accumulated in the lower respiratory tract, and have two competing mode of actions: (a) an acute mode as a
result of surface activity (deposited dose-dependent adsorption of essential alveolar proteins or peptides)
and (b) a chronic mode due to their accumulation and exhaustion of the existing phagocytic capacity of the
lung. Any decrease in clearance will increase the residence time of particles in the lung. Common secondary
squealae include a cumulative dose and particle-residence time related sustained inflammation (alveolitis).
At frank lung-overload conditions intra-alveolar changes may progress to structural changes und remodeling,
such as fibrosis and neoplasia. Accordingly, in inhalation studies with poorly soluble particles, the most
critical hallmark of pulmonary toxicity that needs to be characterized is the transition of lung burdens from the
homeostatic steady-state to various stages of lung overload. As a corollary, the maximum tolerated dose
needs to be defined based on particokinetics and not necessarily particodynamics. Current inhalation testing
guidelines provide limited guidance to stakeholders conducting and assessing such studies, including the
following: particle size measurements, verification/falsification of cumulative pulmonary dose assessments in
a highly compartmentalized target organ, identification of key endpoints integrating alveolitis and subsequent
structural changes in relation to time course changes in the retained dose and how these variables can be
translated from experimental models to humans. However, better approaches are needed to estimate doses
at which homeostatic steady-state lung burdens can be maintained which, in turn, deliver the most meaningful results from repeated exposure inhalation studies. Despite the common mode of action of inhaled
insoluble, spherical particulate matter (PM), a unifying, most appropriate metric conferring pulmonary
biopersistence and toxicity has yet not been demonstrated. Nonetheless, there is compelling evidence from
repeated rat inhalation exposure studies suggesting that the particle displacement volume is the most
prominent unifying denominator linking the pulmonary retained dose with toxicity. This analysis provides
strong evidence that pulmonary toxicity is dependent on the volume-based cumulative lung exposure dose.
Lung toxicity, evidenced by PMN in BAL occurred at lung doses exceeding 10-times the overload threshold.
Furthermore, the conclusion is supported that repeated inhalation studies on rats should utilize an
experimental window of cumulative volume loads of respirable PM in the range of 1 µl PM/lung (no-adverseeffect range); however, not exceeding ≈10 µl PM/lung that would lead to retention half-times increasing ≈1
yr. This can be targeted best by computational toxicology to estimate the dose-dependence and time-course
of PM-lung retention and elimination during the exposure and recovery periods, respectively. Inhalation
studies exceeding the overload threshold volume may lead to meaningless findings difficult to extrapolate to
any real-life scenario. In summary, this analysis supports a volume-based generic mass concentration of 0.5
µl PMrespirable/m³ x agglomerate density, independent on nano- or submicron-sized properties, as a generic
no-adverse effect level in both rats and humans.
Keywords: lung overload, pulmonary toxicity, carbon nanotubes, nanoparticles, agglomerates
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1.
HYPOTHESIS AND MODELING
1.1
Critical Mode of Action
Unlike other laboratory animals and humans, rats appear to be more susceptible to the lung overload-related
effects caused by poorly soluble particulate matter (PM) due to impaired alveolar macrophage (AM)mediated alveolar clearance. Oberdörster [1-3] proposed that the threshold of causing particle-induced
chronic effects is the pulmonary dose that results in a first reduction in macrophage-mediated clearance. It
was suggested that a rats’ macrophage-mediated clearance is impaired at an estimated volumetric loading
of 6% or 1 µl PM/g-lung and more significant impairment occurred at 10 µl/g-lung volumetric loading [4-6].
The half-time of PM for alveolar clearance in rats under non-overloading conditions has been reported to be
in the range of 50-80 days [7,8]. The objective of this paper to further conceptualize the mechanisms
involved in lung overload and how they can be translated to humans [8,9].
A fraction of the inhaled submicronsized particles is deposited and retained, inter alia, in the alveolar region.
Once deposited, they may exert a localized and direct disturbance to their microenvironment. The first interaction appears to depend on the capacity of PMs to adsorb biomolecules at their initial site of deposition.
Accordingly, the binding isotherms of the most avidly binding biomolecules, the PM surface area and/or
porosity, and especially its pulmonary dose, are decisive for the acute effects to expect. This is followed by a
secondary response, viz. the AMs present in the alveolus are attracted by chemotactic stimuli with ensuing
PM uptake and phagocytosis. Due to the given volume of this cell, its fractional loading is limited by the
particle volume and not particle mass or surface area. In this context, the additional agglomeration of already
aggregated PM (nano-)structures may consume an even higher displacement volume in the alveolar
macrophage than their larger bulk crystals. Depending on the degree of PM-loading, modulating factors will
be secreted for attracting both
further phagocytic cells as well
as inflammatory cells. Both cell
types come with an armamentarium of enzymes that attempt
to chemically crack PMs by oxidative and other mechanisms. If
dysregulated or overwhelmed,
the AM may digest itself or
neighboring tissue structures. As
long as it is functional, the AM
accumulates PM with subsequent mechanical clearance out
of the alveolar region. The more
phagocytes are attracted to this
site of the lung, the higher is the
increase in the ‘volume of
distribution (Vd) along with
increased particle load.
Fig 1. Acute and chronic mode of action of retained PMs in the alveolus following repeated exposure.
Accordingly, as depicted in Fig. 1, two modes of action must be considered, the acute, rapidly reversible
effect (lowest curve in Fig. 1), and the accumulating long-term effect (top curve in Fig. 1). Suffice it to say, to
prevent irrelevant acute-on-chronic effects to occur, toxicity studies must be designed by utilizing daily dose
increments that do not acutely exhaust the pulmonary defense system. To the contrary, high-dose instillation
of any large dose by one single bolus may be predominated by acute effects and an aberrant PM clearance.
Although expedient and methodologically simple, such type of pulmonary exposure may have little
resemblance to those exposure patterns leading to accumulative low-dose effects as they may occur at the
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workplace.
1.2
Pharmacokinetic Modeling of Dose and Effect
In rat inhalation toxicity studies the most conservative threshold for toxic effects is considered to be the
cumulative pulmonary dose that results in a reduction in clearance. This is mediated by an increased Vd as a
physiological reflection of the adaptive increase of phagocytic cells relative to the cumulative PM-volume
load. Previous inhalation studies and analyses have shown that such kinetic changes precede the ensuing
inflammatory response [8]. Hence, the critical threshold estimated by pharmacokinetic modeling is
intrinsically more conservative than any empirically determined inflammatory endpoint. Such generic
modeling appears to be feasible because the chronic, retention-dependent kinetics depends more on the
kinetics of the particle-laden AM rather than the properties of the PM itself. However, a major prerequisite is
that the PM is both insoluble and not cytotoxic. Relating the total AM volume (Vd) to the cumulative overloadthreshold dose (which has been defined to be 6%) the overload threshold (Vd-thresh) can then be estimated as
shown below. As can be appreciated from Fig. 2, especially for complex, tangled, and intertwined structures
such as MWCNT the integrated properties of the structure may be a more important variable than nanotubes
itself, apart from the aspect that the rigidity of the tube is also a critical determinant for the agglomerated/
aggregated assemblage structure. The cumulative PM-volume load per lung, at the end of any repeated
inhalation exposure study, can be calculated at the outset of any type of inhalation study taking into
considerations the constraints detailed in Fig. 4.
where: Ci = actual mass concentration of PM
in the vicinity of the breathing zone of rats,
mg/m³; Fa = fractional deposition of PM in the
alveolar region, PMresp, as estimated by
MPPD2 calculations; Ed = number of
exposure days; ρe = effective density of
agglomerated PM in the vicinity of the
breathing zone of rats, g/cm³.
PMresp is calculated using MPPD2 modeling software [10,11] based on the empirically determined mass
median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) obtained during rat noseonly inhalation studies [12-15]. The densities of PMs have to be
Critical orifice cascade
determined by methodologies reflective of the aggregate rather
Impactor: 0.25-0.5 µm stage
than their material density. The relationship of aerodynamic (dae)
and geometric (dgeo) is given in Fig. 2. The estimation of the density
of airborne assemblages can be made from this relationship.
d ae = d geo × δ = 1.5 × 0.1 = 0.47 µm
2µm
dgeo=1.5 µm
Thus, PM-related pulmonary toxicity appears to be contingent upon
the cumulative volumetric lung dose that exceeds the homeostatic
range of particle accumulation in pulmonary phagocytes. Up to this
threshold the normal phagocyte-mediated elimination half-time of
AM does not exceed a t1/2 of 60-80 days [8]. Based on the
relationships detailed above, this threshold has been shown to be
in the range of 1 µl PM-volume/g-lung and can be converted to a
mass-based generic NO(A)EL for any type of repeated rat
inhalation studies, as shown in Fig. 3.
Fig. 2. Assemblage structure of MWCNT with effective density of ρe = 0.1-0.3 g/cm³.
For setting occupational exposure levels (OELs) data from subchronic inhalation studies on rats are an
essential prerequisite. In these types of inhalation studies, using a 6 hour/day, 5-times/week exposure
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regimen for 13 consecutive weeks, the fractional PM volume deposited in the pulmonary region of the lung
per exposure day can be estimated. Nose-only exposed rats have respiratory minute volumes (VE) of
about 0.8 L/min/kgrat. For a 6 hour exposure period at 0.8 L/min-kgrat this is equal to VE = 0.29 m³.
Subtraction of the physiological dead-space reduces the total inhaled volume by 1/3 which yields the
alveolar ventilation volume of 0.19 m³alv. This means, the generic no-observed-adverse-effect-level
(NOAEL) of rats exposed for 13-weeks, or the 2-yr equivalent, can readily be modeled [8,9,15]. In terms of
inhalation chamber concentrations and exposure durations adjustment from alveolar ventilation to normal
ventilation have to be employed. Hence, the generic volumetric overload-threshold is attained when using
daily exposure concentrations at 0.83 and 0.54 µl PMresp/m³ for 3- and 24-months repeated inhalation
exposures, respectively. Volume concentrations can readily be calculated from the effective density of the
aggregate by using appropriate methodologies [16]. However, caution must be exercised when doing so
as assemblages from nanostructures may be morphologically much more complex than those from higherdensity micron-sized crystals. Therefore, especially for the former, the dispersion technique applied may
affect both compaction and effective density of the PM which needs to be taken to consideration.
where: 1 µl is the threshold volume of AM at which 6% of the
Vd of the alveolar macrophage pool is consumed;
f v i is the
fractional volumetric daily exposure dose to attain pulmonary steady-state (study duration-specific
accumulation factor for PM volume). The study duration-dependent fraction ‘i’ can be calculated as
detailed [8] . For 1, 4, 13, and 104 week studies this factor becomes 4.9, 17.5, 40, and 61, respectively.
The pulmonary deposited dose ‘PMresp’, is estimated by MPPD2 calculation, ρ is the effective density of
agglomerated PM, ideally from breathing zone samples of rats, g/cm³.
1.3
Validation of Hypothesis
Fe
3O
4
Fe
3O
4
CB
To
ne
r
M
W
CN
T
Concentration [mg/mł ]
Based on the comparison made in Fig. 3, the empirical (inflammation-based) and predicted (kinetic
volumetric overload threshold-based) NO(A)ELs are in reasonable agreement. As long as differences occurr
they appear to be caused experimental design factors, especially dose-selection and spacing of exposure
concentrations. In all studies statistically significant inflammation was characterized by increased neutrophilic
granulocytes in bronchoalveolar lavage fluid (BAL12
PMN). Along with the objective to further refine animal
NO(A)ELobserved
4 wks
bioassays for the derivation of safe occupational expo10
NO(A)ELpredicted
sure levels (OELs), common mechanistic denominators
of higher- and lower-than-unit density, biopersistent
8
nanosized and submicronsized insoluble PM have been
identified. Based on the results of this analysis, it
6
13 wks
appears as if the potency of PMs to induce
inflammation-related sustained lung injury is largely, if
4
not exclusively, dependent on biokinetic rather than
13 wks
PM-inherent properties. Occupationally, poorly soluble
2
yr
2
particulates are commonly regulated in one class with
13 wks
similar OELs. The outcome of this analysis is not at
0
variance to this approach. The results depicted in Fig. 3
support the use of the aggregate PM-volume rather
than mass or surface area as the key unifying
ρ: 0.2
1.2
2
5 g/cm³
denominator[8].
Fig 3. Comparison of modeled and experimentally derived NOAELs from rat inhalation studies with different
exposure durations.
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1.4
Computational Toxicology
Published evidence suggests that repeated exposure inhalation studies on rats represent the most sensitive
bioassays for inhaled granular biopersistent particulate matter. This analysis suggests that the circumvention
of any overload-like condition to occur may also prevent the occurrence of secondary inflammatory
responses with long-term sequelae as a result of lung overload. The comparative analysis of volumetric lung
burdens with the concomitant decrease in PM-clearance and increased inflammation supports the
conclusion that repeated inhalation studies on rats should utilize an experimental window of cumulative
volume concentrations between 1 to 10 µl PM/lung. This range can be targeted best by the modeling of
particle deposition and lung retention biokinetics. Inhalation studies exceeding that upper-volume limit may
generate meaningless findings difficult to extrapolate to any real-life scenario. Likewise decompensated
overload is expected to occur at high cumulative lung burdens that may also lead to frustrated phagocytosis
and deterioration in lung function. Such phenomena have no physiologic counterpart at the lower end of the
cumulative dose-response curve. Moreover, using modeling procedures for dose selections aiming at well
rationalized retention half-times not exceeding 1 year provide a better means to compare findings across
different laboratories and to characterize the toxic properties of insoluble nano- and submicronsized PM. As
a future goal, this simplified but better targeted and hypothesis-based testing approach may also be
conducive to a reduction of experimental animal studies for OEL setting.
These considerations show convincingly that any replacement of in vivo animal inhalation studies by cell or
tissue in vitro cultures is a difficult and complex exercise. Isolated tissues or cells do not ‘inhale’ which
means even the artifactual acute aspects of PM toxicity (see Fig. 1) cannot be reproduced in vitro in any
simplified manner nor are the comparative dosimetric aspects of active (lung) or passive (in vitro) dosing yet
resolved. Moreover, there are no means to adequately model the aspects of pulmonary particokinetics in
vivo (apart from dissolution
Simulation of accumulation of particles in the lung using
and binding of substrates
I:
the particle/agglomerate volume metric at steady-state
Assumptions:
and multiples thereof.
contained in culture media).
• Granular morphology
• No substance-specific
Therefore, well designed
Predict NOAEL for lung toxicity and MTD based on generic
II:
mode of action
partico-kinetics and specific agglomerate density
• No leachable impurities
and executed repeated inhamodifying this MOA
•
Negligible
dissolution
lation studies on rats appear
Design a 4 week repeated inhalation study on rats to verify/
III:
refute the simulation and the kinetic paramerization
to be yet indispensible for
state-of-the-art risk characBenefits
• MOA-specific study design
• PBPK-based dose-selection terization. Nonetheless, in
• Better study rationale
Hypothesis
no
yes
IV:
vivo approaches bear a high
• Self-validating conduct of
true
study, especially in regard
potential for mechanistic
to target organ dosimetry
• Less animals
research, for instance, to
analyze further the reasons
Generic toxicity & kinetics
Standard testing package needed to
solely dependent on physical
evaluate the specific MOA and related
why for some PMs modeled
particle properties
NOAELs at different study durations
V:
data may not be verified by
animal rat inhalation studies
Generic OEL (0.5 µl PM/m³ x δ)
Substance-specific OEL
(see ‘no path’ in Fig. 4).
Fig 4. Five-step approach amalgamating mechanistic and kinetic information to verify/refute the hypothesis
that a PM exerts its toxicity in a PM-generic or a PM-specific manner.
2.
FACETS OF PARTICLE INHALATION TOXICITY BIOASSAYS
2.1
Globally Harmonized Inhalation Testing
Inhalation toxicity testing, opposite to all other toxicity potency tests, has multiple experimental variables and
the outcome of testing may be more dependent on technical equipment and experimental physics than in
any other discipline of toxicology. Moreover, the data are not generated solely for the purpose of “classification and labeling” but also for risk management reasons. These include definitions of ceiling levels for
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21. – 23. 9. 2011, Brno, Czech Republic, EU
emergency response measures (acute) up to safe exposure levels for workers and consumers. Contemporary international recognized testing guidelines require standardized particle characteristics for inhalation
testing. The particle-size distribution of aerosolized solid or liquid test substances should allow exposure of
all relevant regions of the respiratory tract of the tested animals. If it is not possible to predict, a priori, the
most responsive region of the tract or the most harmful particle-size range that deposits throughout the entire
rodent respiratory tract, then an aerosol bracketing a particle-size mass distribution of MMAD 1 to 3 µm
appears to be most appropriate for repeated PM inhalation exposure studies with rats. The GSD should
ideally be in the range of 1.5 to 3.0 [17, 18].
It must be emphasized that exposure atmospheres generated under such conditions serve, at the first place,
solely the purpose to allocate the substance under investigation to one of the given toxicity classes,
disregarding largely the specific physical properties of the test agent or real-life exposure pattern. This
particular feature, however, is considered to be of ultimate importance for a hazard related classification,
since hazard is heavily dependent on the potential of exposure - which means intensity and duration - and
not necessarily on “intrinsic toxicity” alone. Classification takes into account mainly the inherent relative acute
toxicity as determined in highly standardized and controlled laboratory experiments whereas risk characterization combines inherent toxic properties with exposure variables likely to occur under normal handling and
use of the substance. This ‘risk-related’ approach to assess the potential of inhalation exposure is not
applicable to the default route (oral administration) as it is independent upon the likelihood of exposure.
For some substances, for example, pastes, highly agglomerated powders, or highly viscous materials with
low vapor pressure, heroic measures have to be used to generate airborne particulates of sufficiently high
concentration and respirability whereas for other materials this occurs almost spontaneously. Such
dependence on exposure intensity and duration are not applicable to any other route of exposure.
Inhalation chambers should be versatile so that they can serve multiple purposes, e.g., acute to chronic
toxicity testing. Up-to-date exposure
technology utilizes the ‚directed or
past flow nose-only‘ mode of exposure
(see Fig. 5). Animals are exposed in a
manner
minimizing
„anisokinetic
F
t


sampling
errors“
and
preventing
the
Ct = C0 1 − e− V 
monitoring


rebreathing of the exposure atmosphere. The inhalation chamber
equilibrium should be attained fast
Time [seconds]
exhaust
(see offset of Fig. 5) and temporal as
well
as
spatial
stability
and
homogeneity
must
be
verified.
Numerous artifacts can occur in illvalidated inhalation systems. One of
the most straight-forward validation is
the analysis of exhaled CO2 at a
dummy
exposure
port.
Any
inadequate chamber performance is
readily identified by a mismatch in CO2
[19].
Concentration [Arbitrary Units]
120
Aerosol photometer
Equation fitted to data
100
80
60
40
20
0
0
60
120
180
240
300
360
420
480
540
600
HEPA CAPSUL E F ILT ER
P
7
6
8b
5
2
1
8a
14 cm
3
4
35 cm
Fig 5. Modular directed-flow nose-only system for inhalation exposure of small laboratory rodents
2.2
Dose-Time-Effect Analyses
Inhaled dosages are estimated from the actually determined breathing concentrations and are conveniently
integrated to calculate the inhaled exposure dose over time. This needs to be supplemented by computa-
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21. – 23. 9. 2011, Brno, Czech Republic, EU
tional approaches taking into account the actual PM aerosol size and the breathing patterns of the exposed
animals. Rat bioassays with PMs should be supplemented with analyses of the inhaled PM-lung burden in
order to compare reliably dose-response and dose-time-course effects. Toxicological effects can be probed
utilizing a wide array of endpoints in bronchoalveolar lavage (BAL) and histopathology. Both approaches
provide complementary results and should be utilized in concert. The strength of the former is that any type
of effect can be probed in a quantitative manner; however, not knowing whether all regions of the lung were
adequately accessed by the BAL-fluid. Similarly, the origin of the effect can not readily be deduced from
BAL, especially when the injury pattern is more focal than diffuse. These shortcomings of BAL can readily be
complemented by histopathology.
In regard to PM-induced pulmonary inflammation, the dose-response and time-course analysis of protein
(increased extravasation of plasma into the alveoli due to inflammation) and polymorphonuclear neutrophils
(PMN) in BAL are commonly considered as the most sensitive endpoints to characterize injury.
Fig 6. Comparison of dose-time-effect modeling with empirical data (test substance iron oxide, ρ = 5 g/m³)
These relationships demonstrate that for the anticipated mode of action typical for PMs, the results from
computational modeling can readily be compared with the extent and time course of pulmonary inflammation
(Fig. 6). In terms of inhalation toxicology, the following messages are delivered by the study depicted in Fig.
6: (I) Iron oxide (magnetite) should theoretically be ≈50-times less potent than MWCNT in causing lung
overload-like conditions (Fig. 3). This is because at equal mass lung burdens iron oxide consumes a ≈50times lower intracellular displacement volume in the AM than the low-density MWCNT. (II) The lowest
concentration chosen should be the unequivocal NOAEL of study while the next higher concentration (20
mg/m³) may cause a borderline and transient overload condition. Therefore, a 6-months exposure-free
recovery period after the 4-week PM exposure period was used the verify reversibility. As shown in the right
lower panel of Fig. 6, the experimental data demonstrate borderline elevations in protein which reached the
level of the air-only exposed control at the predicted 3 months time point with full reversibility up to 20 mg/m³.
No reversibility was attained at the next higher exposure levels. Thus, the effect profiles probed by BAL
matched exactly those predicted by the mathematical modeling of lung burdens. (III) Frank kinetic overload
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21. – 23. 9. 2011, Brno, Czech Republic, EU
was attained at 50 and 100 mg/m³. The dynamic kinetic model predicted elimination half-times of t1/2=174
and 282 days, respectively. Following the rules of toxicokinetics, full recovery would be expected to occur
after 870 and 1410 days. Accordingly, the longevity of the laboratory rat is hardly sufficient to prove that full
reversibility occurs even at such a lung overload condition. Thus, the fate predicted in the left panel of Fig. 6
is decisive for the effect(s) observed and quantified right panel of Fig. 6. This example shows that the often
used attributes of PM-induced time-course effects “reversible” or “irreversible” can only be correctly assigned
when knowing the associated descriptors of particokinetics.
3.
CONCLUSIONS
The concatenated interplay of particle physics, animal and human biokinetics, dose metrics, dosimetry, and
the resultant integrated toxicological outcome demonstrate that contemporary inhalation toxicology requires
interdisciplinary and integrated approaches to better characterize and understand particle-induced lung
toxicity. Attributes such as ‘irreversibility’, ‘supertoxicity’ and others are meaningless as long as they are
disconnected to a specific testing regimen, a specific dose and mode of administration (one single
cumulative live time dose administered as bolus by intratracheal instillation or state-of-the-art inhalation
toxicology to multiples of OEL yet not inducing frank kinetic overload of the lung).
Therefore, the question raised in the title of this paper “are new testing strategies and interpretation
guidelines needed” can be answered with yes, indeed. However, the emphasis is directed to the logic of
testing and not necessarily the testing methodology itself. This exercise demonstrate that the currently
available guidance given by OECD inhalation guidelines [16,17] is sufficiently detailed to design and execute
state-of-the-art inhalation studies with both submicron and nanosized particulate matter. However, what is
urgently needed is to utilize the limited resources of costly inhalation toxicology judiciously and consistent
with animal welfare considerations. PMs should not be tested in any indiscriminant and non-hypothesisbased manner just to fulfill generic regulatory needs. As long as it can be demonstrated that the PM under
consideration has a common mode of action, toxicity, and kinetics, computational toxicology can close
suitably ostensive data gaps. However, the future will show whether ‘simulated’ inhalation studies with
minimal but hypothesis-based experimental validation, e.g., 4-week inhalation toxicity study only, will
successfully be gaining access to the regulatory system of registration, evaluation, and authorisation of PMs.
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