Comment on Shvedova et al. (2016), “gender differences in murine pulmonary responses elicited by cellulose nanocrystals”
Shatkin and Oberdörster Particle and Fibre Toxicology
Comment on Shvedova et al. (2016), “gender differences in murine pulmonary responses elicited by cellulose nanocrystals”
Jo Anne Shatkin 2 10
Günter Oberdörster 1 9
0 Department of Physiology & Pharmacology, School of Medicine/WVU , Morgantown WV , USA
1 Engineering and Controls Technology Branch, NIOSH/CDC , Morgantown, WV , USA
2 Exposure Assessment Branch, NIOSH/CDC , Morgantown, WV , USA
3 Forest Product Laboratory, USDA Forest Service , Madison, WI , USA
4 Department Pathology, University of Pittsburgh , Pittsburgh, PA , USA
5 Department of Chemistry, University of Pittsburgh , Pittsburgh, PA , USA
6 Environmental and Radiation Health Sciences Directorate, Health Canada , Ottawa, Ontario K1A 0K9 , Canada
7 Department of Environmental & Occupational Health, University of Pittsburgh , Pittsburgh, PA , USA
8 Free Radical Center, University of Pittsburgh , Pittsburgh , USA
9 University of Rochester, School of Medicine and Dentistry , Rochester, NY , USA
10 Vireo Advisors LLC , Boston, MA , USA
A recent publication in “Particle and Fibre Toxicology” reported on the gender differences in pulmonary toxicity from oro-pharyngeal aspiration of a high dose of cellulose nanocrystals. The study is timely given the growing interest in diverse commercial applications of cellulose nanomaterials, and the need for studies addressing pulmonary toxicity. The results from this study are interesting and can be strengthened with a discussion of how differences in the weights of female and male C57BL/6 mice was accounted for. Without such a discussion, the observed differences could be partially explained by the lower body weights of females, resulting in higher doses than males when standardized to body weight or lung volume. Further, few conclusions can be drawn about the pulmonary toxicity of cellulose nanocrystals given the study design: examination of a single high dose of cellulose nanocrystals, administered as a bolus, without positive or negative controls or low dose comparisons, and at an unphysiological and high dose rate. Simulating the bolus type delivery by inhalation would require a highly unrealistic exposure concentration in the g/m3 range of extremely short duration. A discussion of these limitations is missing in the paper; further speculative comparisons of cellulose nanocrystals toxicity to asbestos and carbon nanotubes in the abstract are both unwarranted and can be misleading, these materials were neither mentioned in the manuscript, nor evaluated in the study.
Cellulose nanocrystals; Cellulose nanomaterials; Inhalation; Gender differences; Pulmonary toxicity
In their recent publication, “Gender differences in murine
pulmonary responses elicited by cellulose nanocrystals”,
Shvedova et al.,  exposed C57BL/6 mice by pharyngeal
aspiration to suspensions of cellulose nanocrystals (CNCs)
(40 μg/mouse/day; cumulative dose of 240 μg/mouse).
The authors employed a variety of biochemical, cellular,
histopathological and physiological measures to compare
responses observed in the lungs of male and female mice.
As strong advocates for proactive approaches to assessing
the safety of nanomaterials, we would be most interested
in these findings, however the study design limits the
ability to relate the results to effects from CNC exposure
under realistic conditions.
1Vireo Advisors LLC, Boston, MA, USA
Full list of author information is available at the end of the article
The authors state that the “primary goal of this study
was to determine whether gender affects pulmonary
function, global mRNA expression, and cytokine/chemokine
inflammatory responses in the lung of C57BL/6 mice”.
The findings of observed gender differences, with females
showing a higher pulmonary toxicity is interesting, and as
the authors point out, such gender differences in
respiratory diseases have been reported in previous
studies. However, the observed “gender differences…” would
be strengthened with a discussion of how differences in
the weights of female and male C57BL/6 mice was
accounted for. At 7–8 weeks, when Shvedova et al. began
their acute exposures, female C57BL/6 mice have an
average weight of 18 g while male mice have an average weight
of 23 g , more than a 20 % difference, which might
explain the greater responses in female mice because of the
higher dose per unit BW in females. The authors state the
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C57BL/6 mice used in their study weighed 20.0 ± 1.9 g,
but a discussion of the weight distributions of males and
females used in their study was not provided, it certainly
would strengthen the interpretation of results. The
observed gender differences might simply be explained by
lower body weights of females, resulting in higher doses
than males when standardized with body weight.
As mentioned, the results of the study are difficult to
interpret in terms of human health impact, given its overall
design. Specifically, the exposure method (pharyngeal
aspiration of bolus doses) and examination of effects from a
single concentration (240 μg/mouse cumulative exposure),
equivalent to a very high deposited dose in humans,
requires closer examination. Shvedova et al. estimated that
the CNC dose administered to mice is equivalent to a
human worker exposed to the Occupational Safety &
Health Administration (OSHA) limit for 42 days.
However, it is scientifically not justifiable in terms of effects to
equate a deposited bolus dose (exposure duration is a
fraction of a minute) with the same dose achieved after an
exposure for many days in the lung. A more realistic
comparison of the dose in mice to the dose deposited in
workers’ lungs has to consider the following: Key is that
there is a difference in effects and underlying mechanisms
induced by very high vs. very low dose rates . Effects
induced by high bolus-type delivery can be used for hazard
identification, provided that dose–response data are
established to determine a slope . Unfortunately,
though, results from such studies cannot be used for risk
characterization (establishing limit values).
The principle of our approach for mouse-human
extrapolation modeling involves the following steps: We
used the Multiple-Path Particle Dosimetry (MPPD)
model (Version 3.04) to determine the deposited fraction
inhaled by a 20 g mouse of an aerosol with mass median
aerodynamic diameter (MMAD) of 0.6 μm and
geometric standard deviation (GSD) of 2.0 and aerosol density
of 1. Respiratory parameters (tidal volume and breathing
frequency) were allometrically adjusted to body weight
which is essential when running the MPPD model. The
model derived deposition fraction in the alveolar region
of the mouse lung gave a value of 5.1 %.
As a next step, an estimate of the deposited dose in
humans over an 8 h (hr) workplace exposure was
performed, at a concentration of 5 mg/m3, which is the OSHA
occupational limit for respirable cellulose dust. The
deposition fraction in the alveolar region of the human lung,
using the MPPD model with the MMAD and GSD given
above, turned out to be 8.1 %. The 8-h deposited dose in
the alveolar region under light physical exercise breathing
conditions was calculated as 3,985 μg/day at 5 mg/m3
exposure concentration. This is equivalent to 6.3 ng/cm2 of
the alveolar surface area (634,620 cm2 at functional residual
capacity [FRC]) in the human lung. The equivalent
deposited dose in the mouse by inhalation would then be
3.3 μg/mouse for a one day (8 h) exposure (mouse alveolar
surface area at FRC of 526 cm2 x 6.3 ng/cm ). This is 12
times less than the 40 μg/mouse delivered in the study. In
addition, as mentioned above, the impact of an 8-h
inhalation exposure vs. a less than a minute bolus delivery has to
Finally, in order to determine a mouse equivalent
inhalation exposure concentration that results in the same
deposited lung dose as human workers deposit when
exposed to the OSHA limit of 5 mg/m3 for 8 h, we used
the following correlation:
Deposited Dose = Minute Ventilation x Expos.
Concentration x Depos. Fraction x Expos. Duration
(The deposited dose over 8 h is 3.3 μg/mouse; the
MPPD derived deposition fraction is 0.051 [see above];
body weight allometrically adjusted tidal volume and
breathing frequency for a 20 g mouse are 0.148 ml and
252/min, respectively; and exposure duration is 8 h).
Rearranging and solving the above equation for Exposure
Concentration gives a value of 3.6 mg/m3, which is in a
similar range as the OSHA exposure limit for workers.
However to simulate the bolus type delivery used in
the mouse study, the dose of 3.3 μg/mouse would have
to be inhaled in one minute - rather than 8 h - at an
exposure concentration of 1.73 g/m3. Nobody would claim
this Exposure Concentration to be realistic; and yet, that
is exactly the equivalent to bolus-type dosing in terms of
the dose delivered to the respiratory tract. Unfortunately,
this has been done - and continues to be done, and
accepted without question - in numerous other studies.
(Additional issues of unequal distribution between
aspiration and inhalation are not considered here).
Conclusions from this derivation of a human/mouse
equivalent dose in the alveolar region are: (i) Shvedova
et al. exceeded the estimated human daily deposited
dose—at the Permissible Exposure Limit allowable by
OSHA - by a factor of 12 when dosing the mice. (ii)
simulating the bolus type delivery with inhalation would
require a highly unrealistic exposure concentration in
the g/m3 range of extremely short duration; (iii) effects
and induction of underlying mechanisms are due to the
high, unrealistic and unphysiological exposure
conditions which have to be interpreted with great caution
. Part of any study must be a critical assessment of
the relevance of administered doses in animal (and
in vitro) experimental studies in order to avoid
erroneous conclusions. For example, the reported significant
gender differences in this study may be simply a result
of differences in male/female body size if doses have not
been adjusted; or are they due to the study design of
only one very high dose? Again, determining the slope
of a dose–response relationship would be essential to
answer these and other questions. We encourage further
discussion of the importance of dosing, which would
include more details of our dosimetric calculations.
In order to comment on the possible human toxicity
of CNCs, the authors should have investigated several
doses in order to demonstrate response related to dose
of CNC exposure. This is especially important given that
high-dose effects in in vivo studies are inherently
difficult to interpret. It is well documented that
dosedependent transitions in the principle mechanism of
toxicity occur at high exposures ; for example, high
doses - amplified by very high dose rates - may result in
non-linearity of responses, effects occurring from
saturated receptor pathways (for both activating and
detoxifying interactions), and inflammation due to
conditions of overwhelming defenses  that are not
representative of effects from realistic dose exposures.
The lack of negative and positive controls of known agents,
together with the lack of different doses to characterize
dose–response relationships, limits the ability to conclude
there are substance-specific effects rather than as result of
inflammation due to simple foreign particle introduction
resulting from a bolus, high-dose exposure; similar outcomes
are known to occur from high doses of any poorly soluble
dust. The study design incorporates interesting measures of
gene and protein expression following exposure, however the
issue is not discussed regarding whether these high dose
responses relate specifically to CNC, or might similarly occur
due to common respiratory triggers (such as other fibrous
and non-fibrous particle types) at these exposure levels. The
lack of low dose testing similarly limits interpretation of the
gene and cytokine responses.
We question, further, why the abstract includes an
unexpected statement comparing CNC to both carbon
nanotubes and asbestos, never to be mentioned again in the
paper. A comparison to asbestos as a positive benchmark
control was not part of the study design, neither was a
negative benchmark included. Adding such benchmarks
combined with a dose–response approach - would have
enhanced the study beyond a simple design by allowing to
rank CNC against well-characterized benchmarks.
Without this, referring to asbestos solely in the abstract is
unwarranted and unjustified.
The sulfated CNCs examined by Shvedova et al. were
158 nm long and are not classifiable as World Health
Organization (WHO) fibers . Additionally, they are
1–2 orders of magnitude shorter than fibers we would
expect to induce mechanisms that lead to toxicity under
the asbestos fiber toxicity paradigm, where fibers longer
than 15–20 μm are critical . We are not suggesting
that inhalation of high doses of CNCs would not cause
the inflammatory response observed by Shvedova et al.,
however the toxicity observed is better expressed in
terms of the very high lung burden, rather than
comparing it in the abstract to asbestos.
In summary, readers of “Particle and Fibre Toxicology”
should recognize that the study outlined by Shvedova et
al. was an investigation into gender differences of
pulmonary toxicity from bolus-type lung exposure at a high dose
of a poorly soluble dust. The conclusions that can be
drawn about the pulmonary toxicity of CNCs - that very
high doses of CNCs cause inflammation - are common to
even benign fibrous and non-fibrous particles.
Authors’ response to: comment on Shvedova et al. (2016), “gender differences in murine pulmonary
responses elicited by cellulose nanocrystals”
Anna A. Shvedova1,3*, Elena R. Kisin1, Naveena Yanamala1, Mariana T. Farcas1, Autumn L. Menas1,
Andrew Williams4, Philip M. Fournier5, Jeffrey S. Reynolds2, Dmitriy W. Gutkin6, Alexander Star5, Richard S. Reiner7,
Sabina Halappanavar4 and Valerian E. Kagan8,9
We would like to thank Drs. Shatkin and Oberdörster
for their interest and comments on our paper  and we
would like to take this opportunity to clarify some of the
issues raised. One of the specific comments is focused
on the relevance of our original findings to differences
in the total body weights between the female and male
mice. Our study determined the respiratory toxicological
endpoints after several pharyngeal aspiration exposures
to cellulose nanocrystals (CNC) in male and female mice
with a specific focus on the comparison of the relative
responses associated with gender differences. Significant
differences in the responses to respirable CNC with a
higher pulmonary toxicity in female mice were
described. However, Shatkin and Oberdörster suggest
that these differences might be due to the lower body
weights of female mice, resulting in higher relative doses
vs males. Accumulating evidence suggests that gender
can have a profound effect on incidence and severity of
a variety of pulmonary diseases [8–10]. It is well known
that changes in the lung volume/mass, rather than body
weight, define the respiratory abnormalities. Additionally,
it has been established that the lung volume/mass of male
and female mice of the same age are not different, in spite
of the differences in the body weights [11, 12]. As per
Environmental Health Criteria 239 , the tissue dose
which is the amount distributed to and present in a
specific tissue of interest, in this case – the lung, would be in
fact, the same for male and female mice. In our study, only
the pulmonary responses were compared between male
and female mice: inflammation and damage, TGF-β, and
collagen, oxidative stress and pulmonary functions as well
as the global mRNA expression were measured in lung.
Thus, the comment on the employed doses with respect
to the total weight differences between males and females
is without merit. We maintain that the stronger responses
to CNC documented in female mice were due to the
gender associated differences in the pulmonary reactivity,
rather than to 14 % variance in the total body weight
between the female and male mice. We thank Shatkin and
Oberdörster for raising this issue in their letter , and
the opportunity to expand our discussions. Further the
comment by Shatkin and Oberdörster was useful as it
allowed us to explain an important point: that in spite of
the slight differences in the total body weight between
female and male mice – there was essentially no gender
differences between the lungs of the animals either in
terms of their weight/area or functions.
Shatkin and Oberdörster expressed concerns that the
dosages employed in this study exceeded the estimated
human daily deposited dose – at the Permissible Exposure
Limit allowable by OSHA - by a factor of 12. It has to be
acknowledged that direct quantitative comparisons
between rodent and human toxicological assessments are
difficult to make. This is due to uncertainties associated
with various methods/tools available for modeling
nanomaterial deposition in the lungs and/or differences in the
physicochemical characteristics inherent to each material
being investigated, as well as those related to each species.
With full understanding of these limitations, the dose
responses in mice can still provide useful information for
meaningful modeling and approximate evaluations
relevant to realistic human exposure scenarios. Several
mathematical models, including the MPPD method preferred
by Shatkin and Oberdörster, have been developed and
used by various groups to improve translation of the
in vivo rodent assessments to corresponding human
equivalent exposures [15–18]. In our study, mice were
exposed over an 18 day period to 6 single doses (once every
3 days) of 40 μg of CNC materials using a pharyngeal
aspiration technique (cumulative dose ~240 μg/mouse) in
lieu of a single bolus dose. Therefore, for estimating
human equivalent exposures to attain similar lung
burdens, we opted to make several assumptions including no
clearance over the 18 day period and the equivalency of
the exposure dose to the dose deposited in the alveolar
region. In our comparisons, we first estimated lung
burden in rodent models, then normalized it to lung burden/
alveolar epithelial surface area to further estimate human
equivalent exposure time period as follows.
Human Alveolar Deposition = Exposure Concentration x
Ventilation/8 h working x Deposition Fraction
OSHA Conc. of allowable exposure to Cellulose =
5 mg/m3 (respirable fraction)
Ventilation / 8 h working (20 L/min * 0.001 m3/L *
60 min/h * 8 h/day) = 9.6 m3/d ()
Deposition fraction for CNC having largest
dimension ~100-300 nm  = ~15 % ([19, 20])
Total alveolar surface area of human lung =
634,620 cm2 at functional residual capacity [FRC]
Mouse alveolar surface area = 526 cm2 at FRC.
Based on this, the lung burden in humans per day can
be estimated as 7200 μg or 11.4 ng/cm2 per day. Thus,
the accumulated alveolar lung burden in humans
ignoring the clearance and other potential factors over 18 days
will be 18*11.4 = ~205 ng/cm2. The T1/2 for alveolar
clearance in humans is ~ 1 year and can be ignored in
these estimates, as the clearance would be insignificant
over the 18 days required to achieve the equivalent
worker lung burden. Assuming that the CNC particles
administered through pharyngeal aspiration deposit
predominantly in the deep lungs (the alveolar region),
the dose employed in mice would result in a lung
burden of ~0.456 μg/cm2 or 456 ng/cm2, i.e. only ~ 2.3
times higher than can be expected from equivalent
exposures per day in humans. Thus we estimated that the
human equivalent lung burden would be achieved in
~42 days (2.3 x 18 days = 41.4 days). It has to be noted
that if the total dust concentrations, instead of respirable
fraction, of OSHA Permissible Exposure Limit (PEL –
15 mg/m3) or NIOSH Recommended Exposure Limit
(REL – 10 mg/m ) were considered, then the respective
human alveolar lung burden would be equivalent to
34 ng/cm2 or 23 ng/cm2, respectively. Over 18 days, this
would result in the accumulation of 612 or 414 ng/cm ,
which are slightly higher than or closer to the average
CNC dose in mice per day employed in our study.
Moreover, the employed exposure regimen and
concentrations over 3-weeks of exposure are further justified as
one considers that humans may be exposed chronically
for longer periods of times.
Can the effects reported in our study be due to
nonlinear responses stemming from receptor
oversaturation pathways and/or conditions of overwhelming the
defenses—lung overload phenomenon? So far, this has
been demonstrated in rats, but not in mice or humans.
Porter et al. [21, 22] demonstrated that a lung burden
of 6 mg/rat of exposure to silica particles (alveolar
surface area ~0.4 m2 of rat lung [23, 24] had not
reached overload and had not decreased the clearance
rate. This is equivalent to 0.9 mg/mouse lung (alveolar
surface area ~0.06 m2 of mouse lung [23, 24]) which is
~4 times higher than the concentrations of CNC (up to
240 μg/mouse) we have investigated in several of our
studies [1, 25, 26]. Importantly, several studies
indicated that the dose dependent effects of nanomaterials
were mostly linear within this dose range..
Shatkin and Oberdörster overlooked one of the most
essential experimental features of our study : the
employment of chronic treatments achieved by scheduled
repeated exposures (twice a week for 3 weeks resulting
in an accumulated dose of 240 μg) to deliver CNC by
pharyngeal aspiration. This regimen achieves the
accumulation of particles in the lung over time, which is closer
to potential occupational exposures than a single daily
bolus dose (as supposed by Shatkin and Oberdörster).
Importantly, this protocol may be one of the best ways to
dose animals in cases when generation of nanomaterial
aerosol for inhalation studies (eg, nano-cellulose
materials) could represent technical difficulties . Several
studies have documented the noninvasive and
reproducible character of particle deposition and clearance from
the mouse lower respiratory tract after pharyngeal
aspiration [28, 29]. Moreover, direct comparisons of bolus
inhalation vs aspiration exposures of SWCNTs further
demonstrated the efficiency of the aspiration technique in
studies of fibrous particles . The repeated exposure
regimens have been employed by others for pharyngeal
aspiration [18, 31–33] and intra-tracheal instillation
exposures [34–37]. Furthermore, the deposition estimates
using MPPD model (v3.0) preferred by Shatkin and
Oberdörster also have several limitations. These include
(a) deposition models for mouse are based on only two
strains (BALBc and B6C3F1)—both of which are models
for either asthmatic or polygenic diseases, (b) calculations
on mouse extrapolation are based on experimental data
for spherical particles, whereas CNC particles with
elongated structures could exhibit different kinetics, (c)
generally low estimates of alveolar surface area (64.5 m2 vs
102 m2 for humans and 0.03 m2 vs 0.06 m2 for mice), and
(d) consideration of endotracheal vs nose-only exposure.
Why did we choose a cumulative dose of 240 μg/mouse?
We agree that determining the slope of a dose–response
relationship is important for the assessment of the
relevance of the mouse doses used in our study to human
exposures. The dose response of bolus adverse effects of
CNC exposure (50 – 200 μg/mouse) in mice by
pharyngeal route has been published previously . Our
study described dose–dependent effects assessed by
several outcomes including inflammation,
cytokine/chemokine release, pulmonary damage, and oxidative stress
markers - protein carbonyls and 4-hydroxynonenal.
Accordingly, the doses selected for repeated exposure
regimen in this study were similar to our low-dose bolus
exposure (40 μg/mouse) given to mice twice a week for
3 weeks thus reaching cumulative dose of 240 μg/mouse
closer to the highest CNC bolus dose.
Shatkin and Oberdörster  further commented on
the lack of positive and/or negative controls in our
study. Our previous work demonstrated that asbestos
administration (employed as a positive control) at the
same lowest CNC dose (50 μg/mouse) demonstrated
lower acute toxicity compared to CNC particles at equal
mass concentration . While detailed comparisons of
pulmonary toxicity and long term effects of cellulose
nanocrystals with asbestos and other fibrous materials
(e.g., CNTs, CNF…) are definitely of great interest, they
were not the topic of this study and we are not
suggesting from our present data that CNC are asbestos-like
with respect to the spectrum of asbestos induced
diseases. We share the common opinion that such studies,
together with the comparison of inhalation studies, are
essential for the further assessment of possible human
toxicity of CNCs.
Finally, Shatkin and Oberdörster  question whether
comparisons of CNC to carbon nanotubes and asbestos
was necessary in the abstract. It is common
knowledge in the field of nanotoxicology that high aspect
ratio materials, particularly carbon nanotubes, can be
potentially pathogenic like asbestos. The similarity in
mechanisms and pathways of toxicity have been
articulated and emphasized in many published papers and
included in their titles or abstracts [39–46] as well as
in studies of nanocellulose materials [25, 47–49].
Thus, while not directly investigated in the current
study, this paradigm has been widely accepted by the
toxicology community, it was not directly investigated in
the current study and we agree that this inference should
have been left out.
In summary, we do not believe that the slight differences
in the total body weights of female and male C57BL/6
mice contributed to the elevated responses in female mice
upon exposure to CNC. Our study was aimed at the
investigation of gender differences of pulmonary toxicity from
repeated pharyngeal aspiration exposures to CNC
materials in mice, rather than exploration of dose–response
effects of CNCs. We believe this study representing a
“proof of principle” or “hypothesis forming” study ,
was an investigation into gender differences of pulmonary
toxicity from repeated pulmonary aspiration exposure to
CNC and needs to be followed up by long term inhalation
studies, as rightfully emphasized by Shatkin and
Oberdörster . Further, the specific gene expression changes
related to carbohydrate/pattern /polysaccharide and
glycosaminoglycan binding and signaling, as detailed in our
paper, support a biological response as a result of CNC
exposure. Taken together, our data provide evidence that
raise doubts concerning the validity of the conclusion
drawn by Shatkin and Oberdörster  that very high
doses of CNCs cause inflammation and that such
pulmonary responses are common to even benign fibrous and
non-fibrous particles. However, we do agree that there is a
critical need for further detailed research aimed at
mechanistic understanding of potential risks of human
exposure to CNCs. Studies in our research group, detailing the
pulmonary effects of CNCs via inhalation exposure route
at relevant exposure limits, are currently underway.
Finally, we would like to thank Shatkin and Oberdörster
for their thought provoking commentary  highlighting
the critical need for continuous research aimed at better
understanding of the potential significance and risk for
human exposures to CNCs.
CNC: Cellulose nanocrystals; FRC: Functional residual capacity; GSD: Geometric
standard deviation; MMAD: Mass median aerodynamic diameter;
MPPD: Multiple-Path Particle Dosimetry; OSHA: Occupational Safety & Health
Administration; WHO: World Health Organization
The authors acknowledge the support of James D. Ede and Kimberly J. Ong,
independent consultants for Vireo Advisors LLC in preparing this letter.
JAS is president of Vireo Advisors LLC, an advisory firm to public and private
organizations, including those seeking to commercialize nanomaterials.
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