Drug and Chemical Toxicology, 2012; 1–10, Early Online

© 2012 Informa Healthcare USA, Inc.

ISSN 0148-0545 print/ISSN 1525-6014 online
DOI: 10.3109/01480545.2011.648327

REVIEW ARTICLE

Regulatory toxicology considerations for the development of

inhaled pharmaceuticals
Keith Owen*
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12

Drug Safety R&D, Pfizer Global R&D, Sandwich, Kent, United Kingdom

Abstract
The preclinical safety studies required to support the development of inhaled drugs are generally the same as for
other routes of administration. Repeat-dose toxicology studies should be conducted by inhalation to ensure the
characterization of both the local (i.e., respiratory) and systemic toxicity, although some studies (e.g., reproductive)
can be performed by utilizing alternative routes, when it is paramount to maximize systemic exposure. Respiratory
tract changes in preclinical species can include irritancy of the larynx and nasal cavity, particularly in rodents. Such
changes are not necessarily predictive of a risk to humans because of the exquisite sensitivity of the rodent larynx
and the lack of exposure to the nasal cavity after oro-inhalation of drugs in the clinical setting. The design of poorly
soluble molecules to limit systemic exposure places greater emphasis on the elimination of drugs from the lungs by
macrophages. Consequently, an increase in macrophage numbers is often noted, and in the absence of any other
changes, this is generally considered to be a nonadverse, physiological response to an inhaled particulate. Other
For personal use only.

changes in the lung, which can include an inflammatory response and/or epithelial hyperplasia, resulting from
irritancy or particulate overload, are a safety concern and are not monitorable in humans. For such changes, safety
margins can be calculated in terms of the drug deposited per unit weight of lung. These factors should be taken into
account when designing preclinical studies or programs for inhaled drugs.
Keywords: Preclinical, inhalation, risk assessment, pharmaceutical, lung, pathology, toxicokinetics, contamination

Introduction metabolism, which can restrict the efficacy of oral or

Lower respiratory tract infections and chronic obstruc-
tive pulmonary disease are major causes of death
worldwide, which, taken together, exceed the two lead-
ing causes: coronary heart disease and stroke (World
Heath Organization, 2008). In the UK, 1 in 7 people are
affected by some form of chronic lung disease, most
commonly chronic obstructive pulmonary disease
and asthma (British Heart Foundation, 2011). For
treatment of respiratory disease, the preferred route of
drug administration is by inhalation. Inhaled admin-
istration ensures direct exposure to the target tissues
and allows a rapid onset of action. It also permits the
use of small doses, which can aid in limiting systemic
exposure and, consequently, reduce the likelihood of
undesirable side effects. This strategy also avoids the
complicating factors of plasma binding and first-pass
parenteral preparations of a drug. Indeed, less than
5% of an oral or intravenous (i.v.) dose even reaches
the lungs, with the remaining drug either being not
absorbed, eliminated harmlessly, or contributing to
unwanted side effects (Leach, 2007). In attempts to
limit systemic exposure, drugs destined for inhalation
administration are often designed to be metabolically
labile and possess low aqueous solubility and oral
bioavailability, high potency and plasma protein bind-
ing, high tissue affinity, and slow dissociation from the
target receptor.
The largest use of inhaled therapies is in the arena
of asthma and chronic obstructive pulmonary disease,
where inhaled beta-agonists, muscarinic antagonists,
corticosteroids, and combinations of these drugs are the
first line of treatment. Drugs to treat cystic fibrosis and

*Current address: RespiVert Ltd., Imperial College, London, United Kingdom

Address for Correspondence: Keith Owen, RespiVert Ltd., Imperial College BioIncubator, Level 1, Bessemer Building, Imperial College,
Prince Consort Road, London SW7 2BP, United Kingdom; Fax: +44 207 594 1039; E-mail: keith@respivert.com
(Received 20 July 2011; revised 21 October 2011; accepted 14 November 2011)

1
 2 K. Owen

Table 1. Current inhaled pharmaceutical agents. effects in the respiratory tract, in addition to defining the
Use Class of drug Examples systemic toxicity of the drug. In some cases, it may be
Asthma/COPD SABA Salbutamol necessary to supplement the inhaled dose with an oral or
LABA Salmeterol parenteral dose to attain systemic exposures high enough
Formoterol to induce toxicity (DeGeorge et al., 1997), as was the case
Indacaterol during the development of salmeterol (Owen et al., 2010).
LAMA Ipratropium When studying the carcinogenicity of an inhaled drug,
Tiotropium at least one of the two rodent studies should utilize the
ICS Beclomethasone inhaled route of administration (DeGeorge et al., 1997).
Fluticasone Although the oral route can be used as an alternative,
Mometasone it would not be suitable if proliferative or preneoplastic
Ciclesonide changes (e.g., hyperplasia) had been noted in the respi-
LABA/ICS Salmeterol/ ratory tract in chronic inhaled toxicity studies, there was
combination fluticasone no distribution to respiratory tract tissues, or if the meta-
Formoterol/ bolic profile after oral administration was significantly
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12

budesonide different from that seen after inhalation (DeGeorge et al.,

SABA/LAMA Salbutamol/
1997).
combination ipratropium
Although it is feasible to perform in vivo genetic (e.g.,
Mast cell stabilizer Cromolyn sodium
micronucleus test), safety pharmacology (e.g., cardiovas-
Methylxanthine Theophylline
cular, neurobehavioral, and respiratory), and reproduc-
Bacterial infections Antibiotics Tobramycin
tive toxicology studies by the inhaled route, it is often
Gentamycin
acceptable to conduct these studies using alternative
Influenza Antiviral Zanamivir
Cystic fibrosis Mucolytics rDNAase
routes, because it is paramount to maximize systemic
Mannitol
exposure. The oral route may seem an attractive alterna-
Saline
tive to inhalation, because 80–90% of an inhaled dose can
Pulmonary arterial Prostacyclin analogs Treprostinil
be swallowed (Wilson, 1987; Bennett et al., 1999); how-
ever, inhaled drugs to treat respiratory diseases tend to
For personal use only.

hypertension Iloprost
Anesthetics Halogenated Isoflurane be designed to be of low solubility and oral bioavailabil-
hydrocarbons Halothane ity. Therefore, to maximize exposure, these studies are
COPD, chronic obstructive pulmonary disease; SABA, short- often performed using the i.v. (or subcutaneous) route
acting β-agonists; LABA, long-acting β-agonist; LAMA, long- of administration; conversely, no such issue would exist
acting muscarinic antagonist; ICS, inhaled corticosteroids; for inhaled drugs designed for the treatment of systemic
rDNA, recombinant DNA.
diseases.

respiratory infections are also well established for use by
this route of administration (see Table 1).
Newer strategies have evolved that target the delivery
of systemically active pharmaceuticals through the lungs,
and this has led to inhaled insulin preparations and
drugs in development to treat pain and migraine among
other conditions (Rubkin, 2010; Wadher et al., 2011).
Drugs designed for the treatments of such conditions
are usually soluble and bioavailable, therefore allowing
rapid systemic drug delivery through deep lung inhala-
tion. Once inhaled, the drug is quickly distributed into
the bloodstream, providing a fast onset of action that is
comparable to i.v. administration, but with greater ease,
patient comfort, and convenience.
The package of preclinical safety studies needed to
support the administration of inhaled drugs to humans
in clinical trials is generally the same as that for other
routes of administration, with development plans usu-
ally following the recommendations outlined in the rel-
evant International Conference on Harmonization (ICH)
guidelines (e.g., ICH, 2009, 2011). Where possible, repeat-
dose toxicology studies should employ the inhaled route
of administration to mimic the intended clinical route of
administration and to ascertain any potential for adverse
Method of administration
The basic concept of administration of drugs to labora-
tory animals by inhalation from a dynamic system is
quite simple. A test aerosol is generated into a stream of
air, which is drawn through a chamber by an exhaust/
pump system. The animals can either be exposed within
large chambers (i.e., whole body) or the drug can be
delivered, by significantly smaller chambers, direct to
the breathing zone (Drew, 1990; Wong, 2007). The use
of whole body exposure systems in the pharmaceutical
industry has diminished as a result of the large quanti-
ties of test material required and the potential for cross-
contamination and ingestion through grooming. Rodent
and lagomorph studies usually employ a snout-only
exposure system, whereas nonrodent studies are gener-
ally performed using an oro-nasal mask (Wong, 2007).
Administration through an oro-pharyngeal tube can
also be used for large animal species when the supply
of test article is very limited, a short exposure time is
required, or when dose-limiting nasal irritancy has been
encountered.
Using the dosing systems available, inhalable atmo-
spheres can be successfully generated from metered
dose inhalers, dry powder blends, and nebulized

 Drug and Chemical Toxicology


solutions or suspensions. Currently, the most com-
mon development path for small molecules to treat
respiratory diseases involves dry powder inhalers. For
such a formulation, test atmospheres can easily be
generated using well-established systems, such as the
Wright dust feed mechanism or the rotating brush gen-
erator. Aerosol generation for large biopharmaceutical
molecules is more difficult, because they tend to be
moisture sensitive in the dry form and unstable in the
liquid form. The difficulty is compounded by the need
for dry powder formulations to often be compressed
into a solid cake to allow successful aerosol generation,
whereas concentric jet atomizers and ultrasonic nebu-
lizers have the potential to damage protein-based drugs
through high-energy shearing or vibration forces. Many
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12
new methods of particle engineering are therefore
being developed for inhaled biopharmaceuticals, such
as microcrystallization, spray drying, and supercriti-
cal fluid technology (Shoyele and Cawthorne, 2006).
In these circumstances, specially developed devices
are sometimes needed for clinical use; however, they
are generally unsuitable for use in toxicology studies
because of the requirement for a high concentration
and/or exposure period, leading to the need to adapt or
construct entirely new devices for the toxicology work
(Hardy, 2008).
The deliberate generation of an aerosol and the wide
For personal use only.
range of doses, combined with the very low limits of quan-
tification (often in the pg/mL range) required to detect
the drug in biological fluids at very low doses, means that
cross-contamination between dose groups (including
controls) can occasionally occur and would be apparent
during toxicokinetic assessments. However, a variety of
procedures to limit transfer of drug between groups by
equipment and clothing, along with the use of individu-
ally ventilated cages, and the use of cabinets with good
air extraction, which are maintained at a slightly negative
pressure, can be employed to avoid excessive contami-
nation and potential invalidation of a study. The use of
naïve controls, analysis of plasma for metabolites, and
measurement of control chamber concentrations can all
be used to provide reassurance that the control animals
have not received the test material in error.
Intratracheal instillation is not a method that should
be used routinely to characterize the toxicity of an
inhaled drug. Although it may be used to deposit a pre-
cise amount of compound directly into the lungs, the
method is nonphysiological, involving invasive delivery
and anesthetized animals, whereas the distribution of
the test compound throughout the lungs may not be
uniform and may not replicate the same pulmonary
distribution after inhalation. Further, the dose and/or
dose rate are usually substantially greater than would be
observed after inhalation and the upper respiratory tract
is bypassed (Driscoll et al., 2000). This method, however,
can be useful in providing information on the relative
toxicity between compounds or over a range of doses
(Wong, 2007; Pauluhn, 2008).
© 2012 Informa Healthcare USA, Inc.
Regulatory toxicology for inhaled drugs 3
Study design
Although inhalation toxicity studies present challenges
that are unique compared to those performed by other
routes of administration, many of the factors influ-
encing toxicity, such as biological (e.g., animal strain,
age, and health) and environmental (e.g., housing,
temperature, and humidity), are universal. Dosing by
inhalation is generally more complex than other routes
of administration, requiring considerable preliminary
work to produce suitable formulations (e.g., powdered
blends, metered dose inhalers, or nebulized solutions/
suspensions) and reproducible test atmospheres, in
addition to allowing the animals to acclimatize to
the dosing apparatus. Chamber concentrations and
particle size are regularly determined during the dos-
ing phase of a study using an appropriate analytical
method, whereas total particulate concentrations are
determined gravimetrically. These extra procedures
ultimately make the studies more labor intensive and
therefore more expensive.
The design principles for inhaled toxicology studies
are the same as for any other route of administration
and should include an evaluation of both systemic and
local (i.e., respiratory tract) effects of the drug. Acute
studies are generally not performed and regulatory
requirements for the assessment of extremely high
doses can be obtained from dose-escalating or dose-
range–finding studies, which are performed to aid in
the selection of the high doses for subsequent subacute
and chronic repeat-dose studies. Studies should include
both sexes and should be performed in one rodent and
one nonrodent species. Exposure should occur daily,
along with the examination of animals for clinical signs
of toxicity. Periodic examinations should include body
weight, food consumption, clinical pathology (e.g.,
hematology, blood biochemistry, and urinalysis), oph-
thalmoscopy, electrocardiography (e.g., nonrodent),
and toxicokinetics.
Information on systemic exposure of animals during
repeated-dose toxicity studies is essential for the inter-
pretation of the results (ICH, 1995; EMEA, 2005). The
principles involved for determining the toxicokinetics
of a drug during inhaled studies are the same as for
any other route of administration, except that in some
circumstances, extremely low doses (e.g., <10 μg/kg)
may be utilized and the dose is estimated rather than
known. The first sample is often taken after the end of
the exposure period to avoid the potential for sample
contamination, whereas the systemic exposure is often
normalized to take into account the daily variability of
delivered dose and so to give a value more represen-
tative of the overall achieved doses over the course
of the study. Determination of lung concentrations,
and therefore an indication of the percent deposition,
can be performed using homogenized tissues and
suitable “cold methods,” such has high-performance
liquid chromatography/tandem mass spectrometry mass
spectrometry. Single-dose radiolabel studies can be
 4 K. Owen
performed to study the ADME (absorption, distribu-
tion, metabolism, and excretion) of an inhaled drug,
but to avoid the substantial cost of the synthesis of
relatively large quantities of radiolabeled material and
to be able to accurately quantify administered doses,
some pharmaceutical companies use surrogate oral
and/or i.v. routes of administration (Webber, 2005).
At the end of the treatment period, a full necropsy
should be performed and organ weights and presence
of gross lesions should be recorded. A full microscopic
examination should be performed, including a detailed
examination of the respiratory tract. Usually, all lung
lobes (including main bronchi) are examined along
with transverse sections of four levels of the nasal cavity/
nasopharynx (sectioned at the level of the posterior part
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12
of upper incisors, incisive papilla, second palatine crest,
and first molar teeth) to allow adequate examination of
the squamous, transitional (nonciliated respiratory),
respiratory (ciliated respiratory), and olfactory epithe-
lium, and the draining lymphatic tissue. If nasal irritancy
is expected, it may be advantageous to take six sections
or more to allow for a more detailed examination of the
tissue (Morgan, 1991). Three sections of the larynx (e.g.,
base of epiglottis, ventral pouch, and cricoid cartilage)
and at least two levels of the trachea, including one lon-
gitudinal section through the carina of the bifurcation of
the extrapulmonary bronchi and one transverse section,
For personal use only.
along with the bronchial lymph node, should also be
examined.
In some cases, it may be desirable to use broncho-
alveolar lavage (BAL) analysis to complement histo-
pathologic examination of the lung. This can be used
to identify early indicators of biochemical changes in
circumstances where it is desirable to investigate the
mechanism of the toxicity. The fact that these indicators
can be quantified also provides a means of developing
a dose-response curve for the inhalation toxicity of the
drug of interest and may provide a “no effect” level of
exposure for regulatory purposes. Obviously, time-
course studies in rodents would require additional
animals, and if lung lavage is performed, the unlavaged
lobe should be sectioned for histopathology at multiple
levels (Henderson, 2005). The total and differential cell
count in BAL fluid is a good measure of lung inflamma-
tion. The presence of neutrophils is a sensitive indicator
of an inflammatory response, whereas an increase in
lymphocytes or eosinophils suggests an immune-based
response. There are also a number of early biochemical
mediators of pulmonary inflammation, which include
tumour necrosis factor-alpha and interleukin-1, which
are cytokines released from the resident macrophages
that promote the adherence of circulating inflamma-
tory cells to the endothelium, whereas an increased
protein content can suggest an increased permeability
of the alveolar/capillary barrier. Other markers of lung
toxicity include lactate dehydrogenase, a cytoplasmic
enzyme whose presence extracellularly indicates cell
death, beta-glucuronidase activity, which is a measure
 Drug and Chemical Toxicology
of activated macrophages, alkaline phosphatase, which
is associated with type II cell secretions, and glutathione
peroxidase and reductase, which increase as a protec-
tive mechanism against lipid peroxidation (Henderson,
1984, 2005; OECD, 2009a).
Dose calculation
One major difference from other routes of administra-
tion is that the exact dose delivered by inhalation is not
known, but is estimated. For oral and parenteral routes of
administration, a discrete amount of material is adminis-
tered from a defined formulation (e.g., by gavage, tablet,
capsule, or injection), whereas for inhalation, delivered
dose depends on a number of variables, such as exposure
concentration, particle size, exposure time, and breath-
ing patterns. Regional deposition within the respiratory
tract can also be taken into account.
The delivered dose (mg/kg) achieved in toxicology
studies can be calculated according to the following
formula:
RMV × C × D × F
Dose =
BW
where RMV = respiratory minute volume (L/minute),
C = airstream concentration of the drug (mg/L), D = dura-
tion of daily exposure (minutes), BW = body weight (kg),
and F = inhaled fraction (%).
Of these parameters, airstream concentration, body
weight, and exposure time will be known. Some labo-
ratories have the capability to determine RMV during a
study; however, it is usually an estimate based on a func-
tion of body weight according to the formula developed
by Bide et al. (2000) or by the Association of Inhalation
Toxicologists (Alexander et al., 2008). The latter method
is gaining popularity because it utilized RMV measure-
ments from laboratory animal species only, whereas
Bide et al. utilized 18 animal species ranging from mice
to giraffes. For simplicity, the inhaled fraction is often
assumed to be 100%.
Dry powder blends of drug and lactose (with or
without ternary agents) are currently the preferred for-
mulation for the development of drugs to treat pulmo-
nary diseases. For practical (e.g., reproducibility and
overloading of equipment) and scientific (e.g., avoiding
excessive lung deposition, increased particle size result-
ing from aggregation, and obstruction at the proximal
nose as a result of dust overload) reasons, the maximum
achievable doses are often limited so as not to exceed a
total particulate concentration of 2 mg/L (Whalan and
Redden, 1994). Under such circumstances, high dose
levels in the region of 40–50 and 20–25 mg/kg can be
achieved when using a 1-hour exposure and a 40% (w/w)
dry power/lactose blend in rats and dogs, respectively.
Achieving these doses is dependent on a variety of factors
in addition to the blend strength and exposure period,
such as the efficiency of the apparatus, chamber extract
flow rate, generator speed, and surface area of the packed

material. An alternative strategy is to define the limit dose
as the maximum dose achievable while ensuring a mass
median aerodynamic diameter (MMAD) of <4 μm (SOT,
1992); in general, this method gives lower doses, because
the MMAD will increase with increasing concentration as
a result of aggregation of drug:drug and/or drug:vehicle.
There is no pharmaceutical regulatory guidance detail-
ing what constitutes a maximum feasible or a limit dose
for inhaled studies, and both approaches described have
been accepted by regulatory authorities. The setting of
limit doses is further complicated by the fact that expo-
sures can be extended, if required, with rats and dogs
occasionally being successfully subject to exposure peri-
ods of 6 and 3 hours, respectively. The value in admin-
istering doses of up to 300 mg/kg to a rat by inhalation
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12
is debateable, although in the case of nebulized studies
with poorly soluble drugs, prolonged exposures can be
necessary to provide sufficient lung margins to allow a
robust evaluation of local tolerance.
The achievable doses for solutions will primarily
depend on the solubility of the drug substance, but also
on exposure period, efficacy of the apparatus, nebulizer
airflow input and output, and the chamber aerosol extrac-
tion rate. A strategy can be employed during the develop-
ment of a dry powder formulation to perform the initial
toxicology and clinical studies with a nebulized solution,
because this can result in a rapid transition from selec-
For personal use only.
tion of a drug candidate to the clinic. This strategy avoids
the prolonged dry powder formulation work required
before toxicology can commence and the drug require-
ments and doses (e.g., <1 mg/kg for a 1-hour exposure)
are relatively low as a result of the relatively insoluble
nature of the material, thus minimizing the time needed
for chemical synthesis. However, this strategy will not
necessarily characterize any potential for local irritancy
with the final dry powder formulation and so can lead
to overall longer times to significant clinical trials (e.g.,
phase IIb) while additional studies are performed to
address this gap. In some cases, there is also a need to
change salt form because of difficulties in finding stable
crystalline forms suitable for formulation as a dry powder.
Doses achieved for metered dose inhalers are generally
influenced by the same generic system limitations as for
other formulations, but are further affected by how many
actuations are possible simultaneously (e.g., six), the rate
at which the canisters can be discharged (e.g., 10/min-
ute), and the shot strength (e.g., 50–300 μg); these factors
usually result in doses of less than 1 mg/kg in rodents.
Deposition and clearance
A substantial proportion of an orally inhaled dose
is deposited in the oropharynx and swallowed, thus
becoming subject to oral absorption. How much drug
is absorbed through the gastrointestinal tract would
depend on the proportion swallowed and the oral bio-
availability of the drug. For salbumatol, it has been
estimated that 60–80% is swallowed, with only 10–20%
reaching the lungs after administration from a meter
© 2012 Informa Healthcare USA, Inc.
Regulatory toxicology for inhaled drugs 5
dose inhaler metered dose inhaler (Lipworth, 1996),
although recent improvements in the design of delivery
devices has seen pulmonary deposition increase to up
to 40%, in some cases. The potential for limiting systemic
exposure has seen the targeting of drugs with a low oral
bioavailability (e.g., fluticasone propionate has an oral
bioavailability of <1%) (Issar et al., 2005).
Deposition in the human respiratory tract depends on
physical forces and particle size and whether the drug is
given by nasal inhalation or oro-inhalation. Larger drug
particles (5–30 μm) given by nasal administration will
not penetrate the deep lung, but will primarily deposit in
the nasopharyngeal region by impaction. Subsequent to
administration by either nasal or oral inhalation, those
particles in the range of 1–5 μm will deposit along the
tracheobronchial tract by the process of sedimentation,
with particles of 1 μm and less reaching the alveoli and
being deposited by diffusion (Inchiosa, 2006).
Species differences in regional particle deposition
do exist, with obligate nose-breathing rodents show-
ing higher nasal and tracheobronchial deposition than
pulmonary deposition, compared to that observed in
humans. There is experimental evidence to suggest that
inhaled particles of a MMAD between 1 and 4 μm will
deposit within all regions of the rat respiratory tract, but
within this size range, nasopharyngeal and tracheobron-
chial deposition increase as particle size increases, with
pulmonary deposition remaining relatively constant.
Once particles are larger than 4 μm, deposition is pri-
marily in the nasopharyngeal region (Raabe et al., 1988;
Schlesinger, 1985), with pulmonary deposition dropping
off rapidly so that exposure at 6 μm is small and at 10 μm
negligible. For dogs and monkeys, the deposition profiles
are much more like those observed for humans. Thus,
for inhalation toxicology studies, an aerosol bracket-
ing a MMAD of 1–4 (for acutes) or 1–3 μm (for repeated
administration) is recommended, with a geometric stan-
dard deviation (GSD) in the range of 1.5–3.0 (SOT, 1992;
OECD, 2009b). Some organizations use 7 μm as a cutoff
for exposure to the lungs, and indeed if the MMAD was
>7 μm, then the study would be considered to be invalid
with little or no pulmonary exposure occurring and thus
one of the major aims of an inhaled study (i.e., detection
of local adverse effects) not being achieved.
Clearance of particles from the respiratory tract occurs
by a combination of mechanisms. Particulate deposited
in the nasopharynx region may be removed by coughing
or sneezing, or is swallowed. Clearance from the tra-
cheobronchial region is primarily along the mucociliary
escalator system to a point where the particles can be
expectorated or swallowed. Although material that is
swallowed is removed from the respiratory tract, it is liable
to be absorbed into the systemic circulation, depending
on its bioavailability/solubility. Another important clear-
ance mechanism for those materials reaching the tra-
cheobronchial tree or alveoli is by the phagocytic activity
of macrophages. These particle-laden macrophages then
can move up the mucociliarly escalator or be removed
 6 K. Owen
through the lymphatic circulation. The lytic activity of the
macrophages can also aid in the dissolution of the mate-
rial into products that can be absorbed into the systemic
circulation. Soluble material deposited in the lung can,
of course, be absorbed into the systemic circulation; this
absorption through the respiratory epithelium increases
with increasing lipid solubility (Witschi and Last, 2001;
Inchiosa, 2006).
In addition to physical clearance of drug from the
lung, it is important to consider metabolic mechanisms.
Many cell types within the respiratory tract express cyto-
chromes P450 (CYP) and phase II enzymes (for review,
see Dahl, 1995). Concentrations of these enzyme systems
vary between different species; for example, the human
lung has relatively low CYP levels compared to the liver,
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12
whereas, despite variation in specific isoenzymes, the
total levels in rats are similar between the two tissues
(Turton and Hooson, 1998). In addition, a number of
reportedly lung-specific CYPs, such as CYP2S, 2F, and
4B1, exist (Somers et al., 2007). Generally, the highest
levels of CYP isoenzymes appear to be located in the
olfactory epithelium of the Bowman’s gland in the nasal
cavity, in Clara cells, and, to a lesser extent, type II cells
of the lung (Dahl, 1995; Dormons and Van Bree, 1995).
Given the metabolic capability of the lung, production
of reactive metabolites in lung cells could contribute to
lung-specific toxicities (Dahl and Gerde, 1994).
For personal use only.
Local irritancy
The predominant pathologic findings in the respiratory
tract of animals exposed to drugs by inhalation tend to be
those related to irritation and may be located in the nasal
cavity and larynx or, to a lesser extent, in the lungs. There
are a range of “irritant” mechanisms, which include the
stimulation of sensory nerves or rodent-specific mucosal
defense mechanisms, increased protein extravasation
into the lumen of the airways, neurogenic inflammation,
and disruption of the air-blood barrier (Pauluhn, 2008).
One of the most common findings noted during rat
inhaled studies is that of squamous metaplasia of the
larynx (Lewis, 1991; Miller and Renne, 1996). This is a
simple adaptive tissue response characterized by the
replacement of the delicate ciliated cuboidal respira-
tory epithelium with a more durable squamous cell
epithelium. It has been proposed that four features (e.g.,
anatomy, airflow, epithelial, and particulate characteris-
tics) predispose the rat to develop squamous metaplasia
on inhalation of drugs (Lewis, 1981). The larynx in the rat
is nearly linear to the nasal turbinates. This, combined
with the rats being obligate nose breathers, means that
there is a near-direct impaction of inhaled drugs on the
anterior surface of the larynx. In humans, the larynx is
more sharply angled (almost 90 degrees) to the oronasal
cavity, leading to a significant reduction in the amount of
impaction on the larynx.
Minor changes on the surface of the rodent larynx,
namely, a loss of cilia from the surface epithelium and a
slight flattening of the epithelium lining the base of the
 Drug and Chemical Toxicology
epiglottis, can sometimes be noted in response to rela-
tively innocuous inhaled pharmaceuticals. It has been
suggested that epithelial alteration is the most appropri-
ate term for this type of subtle alteration, because full
squamous metaplasia is not present (Kaufmann et al.,
2009). Subsequent to an initial insult with material of
a more irritant nature, inflammation with edema and
a suppurative inflammatory infiltrate in the lumenal
epithelium and submucosa can be noted throughout
the larynx and this may lead to necrosis and ulceration,
usually around the base of the epiglottis. If no further
exposure occurs, the normal mucosal morphology of
the larynx will be restored by epithelial cells migrating
in from either side of the ulcer, then proliferating. If the
larynx is repeatedly exposed to an irritant, the inflamma-
tory response and loss of cilia will eventually stimulate
squamous metaplasia of the mixed ciliated and noncili-
ated (i.e., transitional) epithelium. The epithelium can
eventually become hyperplastic and -keratotic, being
covered by a thick stratum corneum (Renne and Gideon,
2006). Studies have demonstrated partial or complete
regression of laryngeal squamous metaplasia and hyper-
plasia after recovery periods (Osimitz et al., 2007), with
complete recovery taking between 6 and 13 weeks after a
13-week exposure period (Renne et al., 2007). There has
been no report of such changes progressing to more seri-
ous cellular changes (Osimitz et al., 2007).
Another common finding in rodents subject to inhala-
tion exposure to pharmaceutical agents is that of irritancy
in the nasal cavity. This is not surprising, because the
nasal chambers are the first structures of the respiratory
tract to be subjected to inhaled insults. As with the lar-
ynx, atrophy, degeneration, erosion, ulceration, hyper-
plasia, and metaplasia of the respiratory and olefactory
epithelium can all be induced by irritant pharmaceu-
ticals. Goblet cell proliferation in the respiratory epi-
thelium is a nonspecific adaptive reaction occasionally
noted, whereas rhinitis and, in severe cases, pronounced
inflammatory exudate may also be noted in the nasal
chambers in severe cases of irritancy (Gopinpath et al.,
1987). Eosinophilic globular inclusions in epithelial cells
have been reported on a few occasions, and although
their significance is unknown, it may be that these reflect
a defense response (Buckley et al., 1985; Gopinpath et al.,
1987).
Irritant changes in the lungs of animals receiving
inhaled pharmaceuticals are less frequent than those
in either the larynx or nasal cavity, but take a variety of
forms. In the bronchial tree, in cases of minor irritation,
the mucin-secreting cells of the bronchiolar epithelium,
which are predominantly of a serous type, are replaced
by goblet cells, and the mucin-secreting cells extend into
the terminal bronchioles where they replace Clara cells.
Clara cells themselves can be affected with the charac-
teristic apical “bleb” being absent and a more basophilic
appearance being noted, representing either an atro-
phic or regenerative change. More significant irritation
can cause ulceration of the bronchiolar epithelium,

accompanied by fibrinous inflammatory exudate in the
lumen. The exudate can become organized with infiltra-
tion of fibroblasts, leading to a narrowing of the airways
(Gopinath et al., 1987). In the lung, if the initial damage
is not severe, a period of epithelialization follows, dur-
ing which type II pneumocytes proliferate and line the
alveolar wall in an attempt to repair any damage. In cases
of marked diffuse alveolar damage, pulmonary edema,
congestion, and hemorrhage may be noted after injury
to type I pneumocytes. Subsequent to severe acute dam-
age, exudate from the leaking capillaries will induce an
inflammatory reaction with infiltration of fibrocytes,
fibroblasts, cell debris, and macrophages, which results
in fibrosis (Greaves, 2007). Although more chronic lung
changes can be induced by xenobiotics, these are rarely
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12
observed with inhaled pharmaceuticals, because drugs
or doses that are severely irritant would result in either a
reduction in dose as the compound progresses through
the drug development cycle or discontinuation of the
project before commencement of longer term studies as
a result of small safety margins. A thorough review of the
many pulmonary changes that can be seen in laboratory
animals after inhalation of irritant material is contained
in the publications by Gopinath et al. (1987) and Greaves
(2007).
Macrophage accumulation and lung overload
For personal use only.
One consequence of designing drugs that have a very
low solubility, to avoid systemic absorption, is significant
amounts of undissolved drug accumulating in the alveoli.
One of the normal clearance mechanisms is for these par-
ticles to be engulfed by macrophages and then cleared to
the exterior on the mucociliary escalator or by migration
to the pulmonary interstitium and, subsequently, the
local lymph nodes (Snipes, 1995; Miller, 2000). Evidence
of such normal clearance of dust is frequently visible
in lung sections from control animals as aggregates of
foamy macrophages, typically located at the bronchiolar-
alveolar junction, with a lack of any other associated
changes (Lee et al., 1986). Often, an increase in the inci-
dence of this finding is noted in treated groups; however,
such changes are considered not adverse and are known
to be fully reversible (Warheit et al., 1997; Driscoll et al.,
1996). Occasionally, such changes are accompanied by
an increase in lung weight (Elder et al., 2005). If the rate
of deposition exceeds the maximum rate of clearance
and/or dissolution, the insoluble drug accumulates in
the macrophages and interstitium until it causes impair-
ment of macrophage clearance function and therefore a
further increase in the rate of accumulation, leading to
lung overload (Morrow, 1992; Snipes, 1995). The induc-
tion of these interrelated events has been postulated to
be related to the volume of material phagocytized by the
alveolar macrophage pool (Morrow, 1992). The thresh-
old for inert particles causing load overload has been
reported to be approximately 1 mg/g of lung (Morrow,
1986). A progressive neutrophilic inflammatory reaction
then occurs in response to the increasing particle load.
© 2012 Informa Healthcare USA, Inc.
Regulatory toxicology for inhaled drugs 7
The elaboration of cytokines by macrophages, and prob-
ably other cell types, recruits other inflammatory cells,
resulting in local tissue damage with regenerative hyper-
plasia (e.g., bronchioloalveolar epithelial cells and type II
pneumocytes) and fibrosis (Driscoll et al., 1996). Under
these conditions, rats are more susceptible than either
mice or hamsters to progression to lesions of epithelial
metaplasia (i.e., bronchiolization), persistent alveolar
cell hyperplasia, and septal fibrosis, and such adverse
changes can eventually progress to pulmonary neopla-
sia of various types in rats, but not in humans or other
toxicology species (ILSI, 2000, Bermudez et al., 2002).
An increased cellularity of the local lymph nodes is often
noted and is considered to represent a secondary change
in response to the increased trafficking of macrophages
as they clear insoluble material. This phenomenon does
not occur with soluble drugs or vehicles (e.g., lactose)
because they do not need to be cleared by macrophages.
Risk assessment
As for other routes of administration, safety margins for
behavioral changes or target organ toxicity after the inha-
lation of a drug should be expressed in terms of multiples
of the systemic exposure at the maximum recommended
therapeutic dose in humans. Safety margins for target
organ toxicities (e.g., hepatotoxicity and nongenotoxic
carcinogenicity) are generally based on systemic expo-
sures of the drug in terms of plasma area under the curve,
whereas for acute effects (e.g., tachycardia and convul-
sions), the margins are often based on maximum plasma
concentrations. In the absence of toxicokinetic data,
exposure margins should be expressed in terms of body
surface area (mg/m2).
In terms of upper respiratory tract changes (e.g.,
squamous metaplasia), the rodent larynx is extremely
sensitive to insult from inhaled particles and the
changes are not usually predictive of irritancy in
humans (Gopinath et al., 1987; Osimitz et al., 2007).
Lesions resulting from irritancy in the nasal cavity are
largely considered irrelevant to a drug administered to
humans by oro-inhalation, because little or no exposure
would occur in the clinical setting (Dudley et al., 1989;
Owen et al., 2010). Additionally, it should be noted that
irritant effects on both these regions of the upper respi-
ratory tract would be easily monitorable in humans
receiving such a drug by inhalation. Also, whereas such
lesions are often noted in rodents, their presence in
dogs or monkeys is relatively rare (Renne et al., 2007).
If required, safety margins can be calculated for nasal
changes using standard nasal surface area values and
particle sizes, which would be determined in the toxi-
cology study.
Adverse pulmonary changes, which can include
cellular infiltration, epithelial degeneration, necrosis,
hyperplasia, and fibrosis, are considered to be a safety
concern because they are adverse and nonmonitorable
in humans and so require appropriate safety margins.
In calculating safety margins for lung changes, the drug
 8 K. Owen
deposited per unit weight of lung is usually calculated,
although margins could also be expressed in terms of
lung surface area or total administered dose. In gen-
eral, it is assumed that approximately 10 and 25% of a
dose is deposited in the lungs of rats and dogs, respec-
tively (Snipes, 1989). In humans, the deposited dose is
often assumed to be the proportion of the dose with a
fine particle mass <4.7 μm, which often translates to
between 10 and 30%. Although this approach is widely
accepted by regulatory authorities, the U.S. Food and
Drug Administration (FDA) is relatively conserva-
tive and usually assumes 100% deposition in humans
because of a lack of suitable biomarkers of lung injury
and because the drug is generally being introduced into
an already compromised lung. The FDA also requests
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12
that lung safety margins should be a minimum of 10-fold
for rodents and 5- to 6-fold for nonrodents. Though the
preclinical deposition values can be refined using “real”
data generated in the toxicology studies (see study design
section above), it is unclear whether data from humans
obtained by gamma scintigraphy, positron emission
tomography, single-photon emission computed tomog-
raphy (Newman et al., 2003), or direct (Nave et al., 2010)
or indirect pharmacokinetic methods (Thorsson et al.,
1994) would influence the view of the FDA because
their deposition assumption is based on philosophi-
cal safety concerns, rather than experimentally derived
For personal use only.
data (Forbes et al., 2011). Macrophage accumulation in
response to drug deposition without any accompanying
inflammatory or degenerative or regenerative changes is
generally considered to be nonadverse (Lee et al., 1986;
Snipes, 1995). There have been, however, some instances
where, because of such findings, the FDA has placed a
drug on clinical hold until there are data that show the
changes do not become more marked on longer dosing.
These circumstances must not be confused with phar-
macologically driven macrophage accumulation, which
is not related to the removal of insoluble drug (e.g., that
resulting from the administration of corticosteroids)
e.g., that resulting from the administration of corticos-
teroids (Okazaki et al., 1992; FDA, 2000, 2006), which
probably occurs because of their effects on surfactant
turnover (Young and Silbajoris, 1986). In general, pul-
monary effects, such as inflammation, have been rare
after inhalation of biologics, and where they have been
observed, they have tended to be where the animal pro-
tein differs considerably from the human protein, and so
the reaction may not be predictive of effects in humans
(Wolff, 1998).
Finally, there are some examples where the conditions
of snout-only exposure can exacerbate the potential for
specific toxicities in rats. Antimuscarinic drugs have the
ability to dry secretions and inhibit ciliary activity and
mucociliary transport (Greaves, 2007). Prolonged expo-
sure of animals to such drugs can cause an increase in
respiratory tract irritancy or an increase in the severity
of macrophage accumulation as a result of inhibited
clearance of the drug. This has been noted as a dramatic
 Drug and Chemical Toxicology
difference between the irritancy of the same drug when
given as a nebulized solution or dry powder, or when a pre-
viously innocuous drug is given in combination with an
antimuscarinic agent (unpublished data). Such changes
would have little relevance to the human situation where
the drug is inhaled in a single short burst, and doses are
limited to avoid clinical signs, such as dry mouth. Some
beta2-adrenergic agonists (e.g., formoterol) and alterna-
tive propellants (e.g., 1,1,1,2-tetrafluoroethane) have
caused testicular toxicity in rats when administered by
snout-only exposure, whereas administration by other
routes (i.e., oral or i.v.) or by whole body (i.e., inhalation)
exposure either avoided the toxicity or greatly increased
the threshold dose required (Brock et al., 1996; Owen,
2009). The mechanism for these changes is unknown,
although restraint and thermal stress has been sug-
gested to play a role (Lee et al., 1993; Dodd et al., 1999).
Regardless of the cause, the snout-only conditions are
not relevant to the human situation.
To summarise, though general principles for deter-
mining drug safety are the same for all routes of admin-
istration, there are some peculiarities resulting from
administration of drugs to animals by inhalation (e.g.,
dose estimation, prolonged exposure periods, lack of
sensitive biomarkers of pulmonary toxicity, and use of
lung deposition factors). These should be considered
in the design of preclinical studies and the interpreta-
tion of findings.
Acknowledgments
The author thanks Tom Brodie for proofreading the
manuscript for this article and for providing valuable
comments and suggestions, particularly regarding the
pathologic descriptions.
Declaration of interest
The author reports no conflicts of interest. The author
alone is responsible for the content and writing of this
paper.
References
Alexander, D. J., Collins, C. J., Coombs, D. W., Gilkeson, I. S.,
Hardy, C. J., Healey, G., et al. (2008). Association of Inhalation
Toxicologists (AIT) working party recommendation for standard
delivered dose calculation and expression in non-clinical aerosol
inhalation toxicology studies with pharmaceuticals. Inhal Toxicol
20:1179–1189.
Bennett, J. A., Harrison, T. W., Tattersfield, A. E. (1999). The contribution
of the swallowed fraction of an inhaled dose of salmeterol to it
systemic effects. Eur Respir J 13:445–448.
Bermudez, E., Mangum, J. B., Asgharian, B., Wong, B. A., Reverdy, E.
E., Janszen, D. B., et al. (2002). Long-term pulmonary responses
of three laboratory rodent species to subchronic inhalation of
pigmentary titanium dioxide particles. Toxicol Sci 70:86–97.
Bide, R. W., Armour, S. J., Yee, S. (2000). Allometric respiration/body
mass data for animals to be used for estimates of inhalation of
toxicology to young adult humans. J Appl Toxicol 20:273–290.

British Heart Foundation. (2011). Facts about respiratory diseases.
Available at: www.lunguk.org/media-and-campaigning/media-
centre/lung-stats-and-facts/factsaboutrespiratorydisease.htm.
Accessed April 2011. 15th April 2011.
Brock, W. J., Trochimowicz, H. J., Farr, C. H., Millischer, R-J., Rusch,
G. M. (1996). Acute, subchronic, and developmental toxicity and
genotoxicity of 1,1,1-trifluoroethane (HFC-143a). Toxicol Sci
31:200–209.
Buckley, L. A., Morgan, K. T., Swenberg, J. A., James, R. A., Hamm,
T. E., Jr., Barrow, C. S. (1985). The toxicity of dimethylamine in
F-344 rats and B6C3F1 mice following 1-year inhalation exposure.
Fundam Appl Toxicol 5:341–352.
Dahl, A. R. (1995). Metabolic charateristics of the respiratory tract.
In: Concepts in inhalation toxicology, 2nd ed. (pp. 141–160).
Washington, DC: Taylor and Francis.
Dahl, A. R., Gerde, P. (1994). Uptake and metabolism of toxicants
in the respiratory tract. Environ Health Perspect 102(Suppl
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12
11):67–70.
DeGeorge, J. J., Ho Ahn, C., Andrews, P. A., Brower, M. E., Choi,
Y. S., Chun, M. Y., et al. (1997). Considerations for toxicology
studies of respiratory drug products. Regul Toxicol Pharmacol
25:189–193.
Dodd, D. E., Leahy, F. H., Feldman, M. L., English, J. H., Vinegar, A.
(1999). Reproductive toxicity screen of trifluoroiodomethane
(CF3I) in Sprague-Dawley rats. Inhal Toxicol 11:1041–1055.
Dormons, J. A. M. A., Van Bree, L. (1995). Function and response of
type II cells to inhaled toxicants. Inhal Toxicol 7:319–342.
Drew, R. T. (1990). Basic principles of inhalation exposure. J Am Coll
Toxicol 9:389–396.
Driscoll, K. E., Carter, J. N. M., Howard, B. W., et al. (1996). Pulmonary
inflammatory chemokine and mutagenic responses in rats after
sub-chronic inhalation of carbon black. Toxicol Appl Pharmacol
For personal use only.
136:372–380.
Driscoll, K. E., Costa, D. L., Hatch, G., Henderson, R., Oberdorster, G.,
Salem, H., et al. (2000). Intratracheal instillation as an exposure
technique for the evaluation of respiratory tract toxicity: uses and
limitations. Toxicol Sci 55:24–35.
Dudley, R. E., Patterso, S. E., Machotka, S. V., Kesterson, J. W. (1989).
One month inhalation study of tolobuterol hydrochloride in rats
and dogs. Fundam Appl Toxicol 13:694–701.
Elder, A., Gelein, R., Finkelstein, J. N., Driscoll, K. E., Harkema, J.,
Oberdörster, G. (2005). Effects of subchronically inhaled carbon
black in three species. 1. Retention kinetics, lung inflammation,
and histopathology. Toxicol Sci 88:614–629.
EMEA. (2005). Guideline on the evaluation of control samples in
nonclinical safety studies: checking for contamination with the
test substance. CPMP/SWP/1094/04.
Forbes, B., Asgharian, B., Dailey, L. A., Ferguson, D., Gerde, P.,
Gumbleton, M., et al. (2011). Challenges in inhaled product
development and opportunities for open innovation. Adv Drug
Deliv Rev 63:69–87.
Gopinath, C., Prentice, D. E., Lewis, D. J. (1987). The respiratory
system. In: Gresham, G. A. (Ed.), Atlas of experimental toxicological
pathology. Current histopathology (vol. 13; pp. 22–40). Boston,
Massachusetts, USA: MTP Press.
Greaves, P. (2007). Respiratory tract. In: Histopathology of preclinical
toxicity studies: interpretation and relevance in drug safety
evaluation, 3rd ed. (pp. 215–269). New York: Elsevier.
Hardy, C. (2008). Non-clinical safety assessment of inhaled
biopharmaceuticals. Dev Life Sci 8:16–18.
Henderson, R. F. (1984). Use of bronchoalveolar lavage to detect lung
damage. Environ Health Perspect 56:115–129.
Henderson, R. F. (2005). Use of bronchoalveolar lavage to detect
respiratory tract toxicity of inhaled material. Exp Toxicol Path
57:155–159.
Inchiosa, M. A. (2006). Toxicokinetics: deposition, absorption,
distribution, and excretion. In: Salem, H., Katz, S. A. (Ed.),
Inhalation toxicology, 2nd ed. (pp. 405–439). New York: Taylor and
Francis.
© 2012 Informa Healthcare USA, Inc.
Regulatory toxicology for inhaled drugs 9
International Conference on Harmonization (ICH). (1995). Topic
S3A. Note for guidance on toxicokinetics: a guidance for assessing
systemic exposure in toxicology studies. CPMP/ICH/384/95.
Geneva, Switzerland: ICH.
International Conference on Harmonization (ICH). (2009). Topic M3
(R2). Non-clinical safety studies for the conduct of human clinical
trials and marketing authorization for pharmaceuticals. CPMP/
ICH/286/95. Geneva, Switzerland: ICH.
International Conference on Harmonization (ICH). (2011). Topic
S6 (R1). Preclinical safety evaluation of biotechnology-derived
pharmaceuticals. CHMP/ICH/731268/1998. Geneva, Switzerland:
ICH.
International Life Sciences Institute (ILSI). (2000). The relevance of the
rat lung response to particle overload for human risk assessment:
a workshop consensus report. Inhal Toxicol 12:1–17.
Issar, M., Mobley, C., Khan, P., Hochhaus, G. (2004). Pharmacokinetics
and pharmacodynamics of drug delivery to the lungs. In: Hickey,
A. J. (Ed.), Pharmaceutical inhalation aerosol technology, 2nd ed
(pp. 198–233). New York: Marcel Dekker.
Kaufmann, W., Bader, R., Ernst, H., Harada, T., Hardisty, J., Kittel, B.,
et al. (2009). 1st International ESTP Expert Workshop: “Larynx
squamous metaplasia”. A re-consideration of morphology and
diagnostic approaches in rodent studies and its relevance for
human risk assessment. Exp Toxicol Pathol 61:591–603.
Leach, C. L. (2007). Inhalation aspects of therapeutic aerosols. Toxicol
Pathol 35:23–26.
Lee, K. P., Henry, N. W., Trochimowicz, H. J., Reinhardt, C. F. (1986).
Pulmonary responses to impaired lung clearance following
excessive TiO2 dust deposition. Environ Res 41:144–167.
Lee, K. P., Frame, S. R., Sykes, G. P., Valentine, R. (1993). Testicular
degeneration and spermatid retention in young male rats. Toxicol
Pathol 21:292–302.
Lewis, D. J. (1981). Factors affecting the distribution of tobacco smoke-
induced lesions in the rodent larynx. Toxicol Lett 9:189–194.
Lewis, D. J. (1991). Morphological assessment of pathological changes
within the rat larynx. Toxicol Pathol 19:352–357.
Lipworth, B. J. (1996). Pharmacokinetics of inhaled drugs. Br J Clin
Pharm 42:697–705.
Miller, F. J. (2000). Dosimetry of particles in laboratory animals and
humans in relationship to issues surrounding lung overload and
human risk assessment. Inhal Toxicol 12:19–57.
Miller, R. A., Renne, R. A. (1996). Effects of xenobiotics on the larynx
of the rat, mouse, and hamster. In Jones, T. C., Dungworth, D.
L., Mohr, U. (Eds.), Respiratory system, 2nd ed., (pp. 51–57). ILSI
monographs on pathology of laboratory animals. New York:
Springer-Verlag.
Morgan, K. T. (1991). Approaches to the identification and recording
of nasal lesions in toxicology studies. Toxicol Pathol 19:337–351.
Morrow, P. E. (1986). The setting of particulate exposure levels for
chronic inhalation toxicity studies. J Am Coll Toxicol 5:533–544.
Morrow, P. E. (1992). Dust overloading of the lungs: update and
appraisal. Toxicol Appl Pharmacol 113:1–12.
Nave, R., Watz, H., Hoffman, H., Boss, H., Magnussen, H. (2010).
Deposition and metabolism of inhaled ciclesonide in the human
lung. Eur Respir J 36:1113–1119.
Newman, S. P., Pitcairn, G. R., Hirst, P. H., Rankin, L. (2003).
Radionuclide imaging technologies and their use in evaluating
asthma drug deposition in the lungs. Adv Drug Deliv Rev
55:851–867.
Okazaki, S., Yamazaki, K., Tanmura, T., Hoshiya, T., Anabuki, K.,
Tanaka, H., et al. (1992). [A 13-week subcutaneous toxicity study
of prednisolone farnesylate (PNF) in rats]. [Article in Japanese] J
Toxicol Sci 17:1–48.
Organization for Economic Co-operation and Development (OECD).
(2009a). OECD guidance document on histopathology for inhalation
toxicity studies, supporting TG 412 (subacute inhalation toxicity:
28 day) and TG 413 (subchronic inhalation toxicity: 90 day).
Draft version, 28 September 2009. Available at: www.oecd.org/
dataoecd/45/2/43822718.pdf. AccessedAugust 2011. 3rd August 2011.
 10 K. Owen
Organization for Economic Co-operation and Development (OECD).
(2009b). OECD guidelines for the testing of chemicals, section
4: health effects. Guideline 412. Subacute inhalation toxicity:
28-day study. Available at: www.oecd-ilibrary.org/environment/
oecd-guidelines-for-the-testing-of-chemicals-section-4-health-
effects_20745788. Accessed September 2011. 1st Sept 2011.
Osimitz, T. G., Droege, W., Finch, J. M. (2007). Toxicologic significance
of histologic change in the larynx of the rat following inhalation
exposure: a critical review. Toxicol Appl Pharmacol 225:229–237.
Owen, K. (2009). Preclinical toxicology for inhaled drugs—practicalities
and implications. J Aerosol Med Pulm Drug Deliv 22:304.
Owen, K., Beck, S. L., Damment, S. J. P. (2010). The preclinical
toxicology of salmeterol hydroxynaphthoate. Hum Exp Toxicol
29:393–407.
Pauluhn, J. (2008). Inhalation toxicology: methodological and
regulatory challenges. Exp Toxicol Pathol 60:111–124.
Raabe, O. G., Al-Bayati, M. A., Teague, S. V., Rasolt, A. (1988). Regional
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Calgary on 06/17/12
deposition of inhaled monodisperse coarse and fine aerosol
particles in small laboratory animals. Ann Occup Hyg 32:53–63.
Renne, R. A., Gideon, K. M. (2006). Types and patterns of response in
the larynx following inhalation. Toxicol Pathol 34:281–285.
Renne, R. A., Gideon, K. M., Harbo, S. J., Staska, L. M., Grumbein, S.
L. (2007). Upper respiratory tract lesions in inhalation toxicology.
Toxicol Pathol 35:163–169.
Rubkin, B. K. (2010). Air and soul: the science and application of
aerosol therapy. Respir Care 55:911–921.
Schlesinger, R. B. (1985). Comparative deposition of inhaled aerosols
in experimental animals and humans: a review. J Toxicol Environ
Health 15:197–214.
Shoyele, S. A., Cawthorne, S. (2006). Particle engineering tech­
niques for inhaled biopharmaceuticals. Adv Drug Deliv Rev
58:1009–1029.
For personal use only.
Snipes, M. B. (1989). Long-term retention and clearance of particles
inhaled by mammalian species. Crit Rev Toxicol 20:175–211.
Snipes, M. B. (1995). Pulmonary retention of particles and fibers.
In: Concepts in inhalation toxicology, 2nd ed. (pp. 193–227).
Washington, DC: Taylor and Francis.
Society of Toxicology (SOT). (1992). Technical committee of the
Inhalation Speciality Section. Society of Toxicology (SOT)
recommendations for the conduct of acute inhalation limit tests.
Fundam Appl Toxicol 18:321–327.
Somers, G. I., Lindsay, N., Lowdon, B. A., Jones, A. E., Freathy, C., Ho,
S., et al. (2007). A comparison of the expression and metabolizing
activities of phase I and II enzymes in freshly isolated human lung
 Drug and Chemical Toxicology
parenchymal cells and cryopreserved human hepatocytes. Drug
Metab Dispos 35:1797–1805.
Thorsson, L., Edsbacker, S., Conradson, T-B. (1994). Lung desposition
of budesonide from Turbohaler is twice that from a pressurized
metered dose inhaler P-MDI. Eur Respir J 7:1839–1844.
Turton, J., Hooson, J. (1998). The respiratory system. In: Target organ
pathology: a basic text (pp. 347–351). Washington, DC: Taylor and
Francis.
U.S. FDA. (2000). NDA 021-077. Drug approval package: Advair diskus
(fluticasone propionate and salmeterol xinafoate) inhalation
powder—FDA review. Approval date: 24 August 2000. Available
at: www.accessdata.fda.gov/drugsatfda_docs/nda/2000/21077_
AdvairDiskus.cfm. Accessed 5th June 2011.
U.S. FDA. (2006). NDA 021–247. Drug approval package: Aerospan
(flunisolide HFA, 80 mcg) inhalation aerosol—FDA review.
Approval date: 27 January 2006. Available at:www.accessdata.
fda.gov/drugsatfda_docs/nda/2006/021247_aerospan_toc.cfm.
Accessed 5th June 2011.
Wadher, K., Kalsait, R., Umekar, M. (2011). Pulmonary insulin delivery:
challenges and current status. J Pharmaceut Sci Res 3:1052–1059.
Warheit, D. B., Hansen, J. F., Yuen, I. S., Kelly, D. P., Snajdr, S. I., Hartsky,
M. A. (1997). Inhalation of high concentrations of low toxicity dusts
in rats results in impaired pulmonary clearance mechanisms and
persistent inflammation. Toxicol Appl Pharmacol 145:10–22.
Webber, C. (2005). Inhaled-dose ADME studies. Dev Life Sci 6:10–13.
Whalan, J. E., Redden, J. C. (1994). Interim policy for particle size
and limit concentration issues in inhalation toxicity sudies.
U.S. EPA. Available at: cfpub.epa.gov/ncea/cfm/recordisplay.
cfm?deid = 186068. Accessed September 2011. 2nd Sept 2011.
Wilson, A. (1987). Aerosol dynamics and delivery systems. In: Jenne,
J. W., Murphy, S. (Eds.), Drug therapy for asthma: research and
clinical practice (pp. 389–412). New York: Marcel Dekker.
Witschi, H., Last, J. A. (2001). Toxic responses of the respiratory system.
In: Klassen, C. (Ed.), Casarett and Doull’s toxicology, 6th ed. (pp.
515–534). New York: McGraw-Hill Medical Publishing Division.
Wolff, R. K. (1998). Safety of inhaled proteins for therapeutic use. J
Aerosol Med 11:197–219.
Wong, B. A. (2007). Inhalation exposure systems: design, methods,
and operation. Toxicol Pathol 35:3–14.
World Heath Organization. (2008). Factsheet 310: top 10 causes
of death. Available at: www.who.int/mediacentre/factsheets/
fs310_2008.pdf. AccessedApril 2011. 14th April 2011.
Young, S. L., Silbajoris, R. (1986). Dexamethasone increases adult rat
lung surfactant lipids. J Appl Physiol 60:1665–1672.